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Towards Routine Diagnosis of Gastrointestinal
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Infections by Molecular Technology in Australian
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Laboratories
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Shane Byrne
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Departments of Microbiology and Molecular Pathology,
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Sullivan Nicolaides Pathology, Taringa, Australia
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Fellowship Part III Submission
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Shane Byrne, Departments of Microbiology and Molecular Pathology, Sullivan Nicolaides
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Pathology, 134 Whitmore Street, Taringa, Queensland, 4068, Australia.
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[email protected]
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Table of Contents
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Abstract ................................................................................................................................................... 4
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Introduction ............................................................................................................................................ 5
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Current Techniques for Diagnosis of Gastrointestinal Infections ........................................................... 8
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Microscopy .......................................................................................................................................... 8
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Culture ................................................................................................................................................ 8
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Immunodiagnostic Assays for Enteropathogen Antigens ................................................................... 9
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Parasite Enteropathogen Antigen Detection ................................................................................ 10
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Bacterial Enteropathogen Antigen Detection ............................................................................... 11
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Viral Enteropathogen Antigen Detection...................................................................................... 12
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Immunodiagnostic Assays for Enteropathogen Antibody Production ............................................. 13
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Immunodiagnostic Assays Specifically for Bacterial Toxins .............................................................. 15
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Comparison of Immunodiagnostic Assays to Molecular Assays ....................................................... 15
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Current Molecular Approaches to Diagnosis of Gastrointestinal Infections ........................................ 19
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Bacterial Pathogens .......................................................................................................................... 20
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Enterobacteriaceae ....................................................................................................................... 22
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Vibrionaceae ................................................................................................................................. 25
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Aeromonadaceae .......................................................................................................................... 25
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Curved and Spiral Shaped Gram Negative Rods ........................................................................... 26
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Clostridia ....................................................................................................................................... 27
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Listeria monocytogenes ................................................................................................................ 28
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Viral Pathogens ................................................................................................................................. 30
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Rotavirus ....................................................................................................................................... 30
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Adenovirus .................................................................................................................................... 31
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Norovirus and Sapovirus ............................................................................................................... 31
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Astrovirus ...................................................................................................................................... 32
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Human Parechovirus (HPeV)/Enteroviruses ................................................................................. 32
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Parasitic Pathogens ........................................................................................................................... 33
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Amoebae ....................................................................................................................................... 34
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Flagellates ..................................................................................................................................... 34
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Coccidia ......................................................................................................................................... 35
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Ciliates ........................................................................................................................................... 37
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Blastocystis hominis ...................................................................................................................... 37
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Microsporidia ................................................................................................................................ 37
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Helminths ...................................................................................................................................... 38
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Current Commercially Produced Multi Analyte Molecular Products ................................................... 40
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Luminex xTAG GPP ............................................................................................................................ 41
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Seegene ‘Seeplex Diarrhea ACE’ Detection Kits ............................................................................... 44
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Savyon NanoCHIP Gastrointestinal Panel ......................................................................................... 45
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AusDiagnostics EasyPlex FaecalProfile-10 ........................................................................................ 45
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Genetic Signatures ‘EasyScreen Enteric’ Kits (Parasite, Bacteria, Viral) ........................................... 47
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Idaho Technology/BioFire – FilmArray GI Panel ............................................................................... 48
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Potentially Adaptable ‘High’ Multi Analyte Molecular Technologies ................................................... 52
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Life Technologies Open Array Plate and QuantStudio 12K Flex ....................................................... 54
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Fluidigm Genetic Analysis System with 96.96 Dynamic Array IFC and BiomarkHD .......................... 56
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Seqenom MassARRAY Genetic Analysis System in 384 well PCR Plate format ................................ 56
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Healthcare Cost Implications in the Australian Context ....................................................................... 60
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Nucleic Acid Extraction from Stool ....................................................................................................... 64
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Composition of the Stool Matrix and Inhibition ............................................................................... 64
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Processing of Stool ............................................................................................................................ 64
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Nucleic Acid Extraction Chemistry .................................................................................................... 65
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Commercially Available Kits and Modifications ................................................................................ 66
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Recovery of RNA from Stool Samples ............................................................................................... 67
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Control of Molecular Gastrointestinal Infection Testing ...................................................................... 68
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Positive Controls ............................................................................................................................... 68
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Negative Controls.............................................................................................................................. 69
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No Template Controls ....................................................................................................................... 69
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Co-Extracted Seeded Internal Controls............................................................................................. 70
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Control of Reverse Transcription for RNA Targets............................................................................ 71
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Comparative Control to Traditional Methodology ........................................................................... 71
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Potential Workflow Replacing Conventional Testing ........................................................................... 73
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Discussion.............................................................................................................................................. 75
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Figures and Tables ................................................................................................................................ 79
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Bibliography .......................................................................................................................................... 88
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Abstract
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Routine diagnosis of gastrointestinal tract infectious disease remains a significant
component of the diagnostic pathology laboratory and the core techniques employed
have remained relatively unchanged for many years. The provision of traditional
gastrointestinal disease diagnosis remains labour intensive and final results for
conventional bacterial culture techniques have a time to result of 2-5 days.
Microscopic diagnosis of parasitological disease generally has inferior sensitivity
when compared to immunoassay and molecular based testing. In addition, the high
specificity of molecular techniques has also proven superior for organisms that
resemble each other closely or are indistinguishable by visual examination such as
certain Entamoeba.
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The combination of reduced time to result and improvements in sensitivity and
specificity suggest that diagnosis of gastrointestinal disease is arguably the next
most ready component for progressing to routine molecular based testing. The
organisms currently known to cause gastrointestinal disease are relatively well
characterised and this enables the collation of a definitive number of organisms for
specific molecular detection. This number of organisms is not overly large in
comparison to recent advances in the diagnostic density formats of molecular
techniques. Three systems that provide cost effective PCR testing formats that can
cover sufficient organisms are discussed. These systems realise the potential of
becoming genuine replacements for conventional techniques. As therapy is often
empiric or not recommended, antimicrobial resistance testing is not as linked to the
diagnostic protocol to the extent it is when characterising organisms from extraintestinal disease. This allows the application of molecular methods focused on
detection of organisms without the absolute need to elucidate antimicrobial
resistance.
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Several commercial multi analyte enteropathogen kits have also recently become
available and the coverage of these kits is compared to enteropathogens recovered
in our laboratory from 2007-2011. Available performance evaluations and
specifications are also presented. The funding model for Australian laboratories is
discussed. Current remuneration for molecular testing is currently insufficient to
cover the costs required to utilise commercial enteropathogen molecular testing
covering a large range of enteropathogen targets.
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Processing of stool for nucleic acid recovery is a key component of using molecular
techniques. The use of currently available methods and instrumentation supporting
the processing of this complex sample type are discussed. The multiple layers of
process control used in molecular techniques are presented and these are compared
to the level of control of conventional techniques.
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A workflow describing the use of molecular screening using cost effective multi
analyte technology in parallel with a minimised bacterial culture technique is
presented as a possible solution. This protocol combines the benefits of routine
molecular testing with the ability to maintain provision of isolates for public health
outbreak tracing and antimicrobial resistance surveillance/testing as required.
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Introduction
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Routine diagnosis of lower gastrointestinal tract infectious disease remains a
significant component of the diagnostic pathology laboratory, accounting for 15% of
non-urine samples received for processing in our laboratory. The core techniques
employed have remained relatively unchanged for many years. These techniques
are also labour intensive when compared to processing of other microbiology sample
types. Dependent on the specific testing request from the patients’ clinician,
conventional diagnostic workup of a stool specimen typically involves some or all of
the following techniques. Recording a macroscopic description, performing culture
for bacterial pathogens on a variety of solid media with or without prior broth based
selective enrichment. Preparing wet mounts and faecal parasite concentration slides
to examine stool microscopically. Use of ELISA or lateral flow immunoassays and/or
specific stains for amoebic, flagellate, coccidial or microsporidial parasites. And
often the use of ELISA. lateral flow or latex agglutination assays for viruses and
bacterial toxins. Antibody testing of whole blood/serum/plasma for host response to
enteropathogens is also utilised where appropriate.
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Diagnosis and identification of bacterial infections using culture, biochemical and
serological assays is considered the gold standard but the process is both costly and
time consuming. Often final diagnosis of a bacterial aetiology is not available until
several days post collection of a stool sample and often after the infection has run its
course. Reducing the time to diagnosis assists patient management and allows
timely action to be taken to limit further transmission in the community. Typical
bacterial culture techniques require 2-5 days dependent on the protocols used.
Culture also requires significant labour for screening culture plates and in follow up
identification techniques. Although treatment for bacterial infections is often not
recommended, performing antimicrobial sensitivity testing may be valuable for
monitoring the development and spread of antimicrobial resistance. This is
particularly important if the isolate is recovered from a patient who may have
acquired the symptoms during or shortly after international travel. Such isolates
could potentially harbour emerging transmissible antimicrobial resistance
determinants that have implications for the patient and for the community. Isolates
are also often required to be sent to reference laboratories for epidemiological
tracing in the interests of public health and protecting the food supply.
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Dependent on volumes of samples received for testing in the laboratory the
microscopy component of the diagnostic workup can be onerous. This may be
reflected in repetitive strain injury or more commonly general fatigue from the efforts
required in scanning large numbers of slides by medical technologists. Microscopic
diagnosis of parasitological disease is documented to have inferior sensitivity
compared to molecular based testing. The sensitivity of microscopy for detection of
parasites has been shown to be approximately 60% at best (1). The high specificity
of molecular techniques is also superior for organisms which resemble each other
closely or are indistinguishable by visual examination but have quite different
relevance clinically e.g. Entamoeba histolytica, Entamoeba dispar and Entamoeba
moshkovskii (2).
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The typical viral workup for gastrointestinal disease includes rotavirus and group F
adenovirus. When outbreaks are identified in a community or health care setting,
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testing for other viral agents such as Norovirus, Astrovirus or specific enteroviruses
supplement the virological workup as required.
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It has been reported that in approximately 40% of investigations for symptomatic
diarrhoeal illness no aetiological agent is identified (3). Evidence suggests that
expanding the range of pathogens tested is likely to increase the rate of diagnosis of
an aetiological agent.
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Molecular techniques have proved very useful for expanding the diagnostic
repertoire and have become a routine component of infectious disease diagnostics
since the mid 1990’s. From the late 1990s diagnostic virology underwent a shift from
what was primarily cell culture based diagnostics to molecular virology, most
commonly employing Polymerase Chain Reaction (PCR) as the nucleic acid
amplification technique (NAAT) utilised. As sequence based information is generally
required to develop NAAT based assays, reference laboratories have continued to
maintain cell cultures for emerging viruses. However recent advances using direct
application of nucleic acid detection techniques have been used as a culture
independent methodology for viral discovery to good effect (3-7).
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The clinical microbiology laboratory has seen a gradual shift from the conventional
methods of culture and microscopy towards molecular testing. The pathogens
involved have been those that generally fall within one or more of the following
contexts: difficult or impossible to recover in culture, poorly diagnosed by
conventional means, recovered by conventional means after lengthy incubation, or
those with tedious and/or expensive methodology required for diagnosis. The
transition from conventional methods to molecular testing is most evident in clinical
diagnostic virology, closely followed by diagnosis of Chlamydia trachomatis and
Neisseria gonorrhoeae. As an example C. trachomatis, N. gonorrhoeae and Herpes
simplex virus testing together now account for 51% of total molecular testing
volumes in our laboratory. Organisms that have been difficult to culture including
Mycoplasma pneumoniae, Bordetella pertussis, Bartonella henselae and
Tropheryma whipplei are also now commonly diagnosed by molecular technology.
Slow growing organisms such as Mycobacteria continue to see expansion in the
utilisation of molecular technology. Recently the WHO endorsed the use of a
molecular cartridge based integrated assay called the ‘Xpert MTB/RIF’ developed by
Cepheid as the diagnostic test of choice in developing countries. Rapid detection of
disease states that are only caused by a specific subset of strains e.g. Human
papillomavirus infections of the cervix, has also been possible utilising type specific
nucleic acid based diagnostic techniques. In recent years, the lack of sensitivity of
microscopy techniques has also led to adoption of molecular assays for the routine
diagnosis of Trichomonas vaginalis. Pneumocystis jirovecii diagnosis has also
transitioned away from direct fluorescent antibody testing of samples to the use of
quantitative analysis available by real-time PCR.
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When looking at the remaining conventional activities of a clinical microbiology
laboratory, diagnosis of gastrointestinal disease is potentially the next most ready
component for progressing to routine molecular based testing. The organisms
currently known to cause gastrointestinal disease are relatively well characterised
and this enables the collation of a definitive number of organisms for specific
molecular detection. This number of organisms is not overly large in comparison to
recent advances in the diagnostic density formats of molecular techniques. As
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therapy is often empiric or not recommended, antimicrobial resistance testing is not
as linked to the diagnostic protocol to the extent it is when characterising organisms
from extra-intestinal disease. This allows the application of molecular methods
focused on detection of organisms without the absolute need to elucidate
antimicrobial resistance. The use of bacterial culture for provision of isolates for
public health outbreak tracing however remains an important consideration. Using
molecular technology as an enteropathogen screening technique with a strategy of
follow up ‘reflex’ conventional culture based on molecular detection of certain
bacterial pathogens appears a plausible strategy to preserve this important public
health requirement (8, 9).
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Current Techniques for Diagnosis of Gastrointestinal Infections
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Diagnostic techniques currently utilised in clinical microbiology laboratories for stool
examination can be broadly classified into three components: Microscopy, Culture
and Immunodiagnostic antigen detection assays. Often all three are combined
within the workflow of a laboratory to provide the final results to clinicians. Antibody
testing from serum, plasma or whole blood for certain enteropathogens is also of
use.
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Microscopy
Conventional processing of stool typically involves a macroscopic description being
recorded. Stool is added to normal saline and a drop placed on a microscope slide.
Use of a small amount of Lugols iodine mixed with the stool suspension assists
observation and identification of parasites. A cover slip is applied to produce a wet
preparation for microscopic analysis. This initial microscopic examination provides
diagnostic information on the presence of white cells, red cells, Charcot-Leyden
crystals, fat globules, observable pathogenic and commensal parasites and yeast.
Further processing of stool is then undertaken by combining chemical clarification
and centrifugal concentration to prepare what is known as a ‘conc’ or faecal parasite
concentration microscopy preparation(10). This concentrated deposit is examined
microscopically for the presence of parasites and improves diagnostic yield.
Examination for certain parasites such as Schistosoma requires that all of the
concentrate be examined, which can number several slides. Smears of faecal
concentrate and/or stool supplied in parasite preservation fixatives can also be made
on slides. Smears are fixed and subsequently stained with stains such as the
Wheatly modified Gomori Trichrome, Iron Haematoxylin or the Modified Kinyoun
Acid Fast which allow targeted observations for parasites amongst stool debris(1113). Some staining methods also highlight morphological characteristics important in
the identification of the parasite. Use of microscope eyepieces with calibrated
measuring scales printed within allows accurate measurement of ova, cysts and
parasites which can also be required to differentiate and correctly identify them.
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Culture
Culture on/in bacteriological media is the primary diagnostic method for bacterial
enteropathogens. As one third of the solid matter in stool is comprised of bacteria,
recovering pathogenic bacteria typically involves the combination of selective agars
and the use of selective enrichment broths to reduce normal flora. Plates are
incubated at specific temperatures and appropriate atmospheric conditions. Media
for recovering Campylobacter is incubated in a modified microaerophilic atmosphere
and media for Clostridium difficile is incubated anaerobically. Incubation periods
from two to five days are commonly used before plates are discarded and final
results issued. The typical media applied for routine investigation of bacterial
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enteropathogens generally covers Salmonella, Shigella, Campylobacter,
Aeromonads, Vibrionaceae and Yesinia. Additional non-routine media may be
added if clinical information indicates it may be appropriate e.g. recovery of Listeria
monocytogenes or O157 sorbitol non fermenting shiga toxigenic Escherichia coli.
Some laboratories also routinely diagnose toxigenic Clostridium difficile infection by
recovery of the organism on solid media in anaerobic incubation conditions. Use of
chromogenic agar media (e.g. bioMerieux ChromID Salmonella) which assists in
improving recognition of pathogenic bacteria amongst normal faecal flora has also
become commonplace (9, 14).
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Immunodiagnostic Assays for Enteropathogen Antigens
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In settings of limited resources including at point of care (PoC) and/or where
expediency is required within the laboratory rapid easy to use lateral flow
immunochromatographic assays including those packaged in cartridge form have
been of value. These lateral flow assays are rapid and often provide a result in a
time ranging from 10 to 30 minutes with minimal hands on time. However, the
techniques are generally not amenable to routine processing of large numbers of
samples. Costs range from $5 to $16 AUD per single test dependent on
manufacturer, antigen target and quantity ordered.
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The second formats are the classical serological assays of ELISA (enzyme-linked
immunosorbent assay), CFT (complement fixation test) and IHA (indirect
hemagglutination assay) which are typically utilised in microplate format. These
classical techniques generally involve the use of equipment and reagents within the
laboratory setting and generate results in timeframes between and hour and two
hours. Many of these microplate format assays are also adaptable to high
throughput automated instrumentation where a larger number of samples need to be
tested. There is a large range of commercially produced assays available for
enteropathogen antigen detection. Costs range from $4 to $9.50 AUD per test well,
again dependent on manufacturer, antigen target and quantity ordered.
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Use of “combi” type formats where more than one antigen are detected on/in the
same cassette, strip or microwell reduces the overall costs for the common
applications of these tests. Some of the common products available in the
Australian market are presented below.
Two distinct immunoserological formats are utilised for enteropathogen antigen
detection.
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Parasite Enteropathogen Antigen Detection
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The performance of several antigen detection assays for E. histolytica are presented
in the reviews of Fotedar et al. (2) and Tanyuskel et al.(15) When compared to
microscopy the antigen detection assays were mostly in the range of >90% sensitive
and generally 99-100% specific.
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Rapid Methods
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Cassette or immunochromatographic strip products have mainly been developed
with a focus on the larger market of Cryptosporidium and Giardia, often in a
combined format. Examples of these assays include the TechLab
Giardia/Cryptosporidium QUIK CHEK; R-Biopharm RIDA®QUICK (cassettes or
strips) for Cryptosporidium, Giardia, and in combination; Savyon CoproStrip™ for
Cryptosporidium, Giardia, and in combination; Remel Xpect® for Cryptosporidium,
Giardia, and in combination; and the Meridian Bioscience ImmunoCard
STAT!®Crypto/Giardia.
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The Alere Triage® Micro Parasite Panel and R-Biopharm RIDA®QUICK
Cryptosporidium/Giardia/Entamoeba Combi products provide for detection of Giardia
lamblia, Entamoeba histolytica/dispar, and Cryptosporidium parvum in the one
product.
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ELISA Microplate Methods
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ELISA antigen detection kits have been produced that cover a slightly wider range of
enteric parasites than the rapid methods. Cryptosporidium and Giardia ELISA tests
are available both as single assays and in combination assays. Examples include;
Techlab GIARDIA II, CRYPTOSPORIDIUM II, GIARDIA/CRYPTOSPORIDIUM
CHEK®; R-Biopharm RIDASCREEN®Cryptosporidium, Giardia; Savyon
CoproELISA™ Giardia, CoproELISA™ Cryptosporidium and CoproELISA™ GiardiaCryptosporidium; Remel ProSpecT™ Giardia, ProSpecT™ Giardia/Cryptosporidium,
ProSpecT™ Giardia EZ, ProSpecT™ Cryptosporidium; Cortez Diagnostics
Crypto/Giardia Ag Combo, Cryptosporidium, Giardia; Novagnost Giardia lamblia
Antigen in stool and Cellab Giardia CELISA.
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Comparative or improved sensitivity compared to microscopy and the ability to
automate the testing have seen the adoption of combo microplate ELISA assays for
Cryptosporidium and Giardia utilised as screening methodology within the workflow
of our laboratory.
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Several E. histolytica ELISA assays are available such as the; Techlab E.
HISTOLYTICA II, R-Biopharm RIDASCREEN®Entamoeba; Savyon CoproELISA™
Entamoeba; Remel ProSpecT™ Entamoeba histolytica; Cortez Diagnostics E.
histolytica/dispar ELISA kit; Novagnost Entamoeba histolytica and Cellab
Entamoeba CELISA Path.
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Savyon also produce an ELISA product for Blastocystis (CoproELISA™
Blastocystis), and have a Dientamoeba ELISA (CoproELISA™ Dientamoeba) under
development.
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Indirect immuno-fluorescent antibody assays (IFA):
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Slide based IFA assays are also utilised to detect parasitic antigens from stool.
Bordier Affinity produces an IFA to detect antigens of the microsporidia
Enterocytozoon bieneusi and Encephalitozoon intestinalis from stool.
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Bacterial Enteropathogen Antigen Detection
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Rapid Methods
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A wide range of cassette and immunochromatographic strip tests for bacterial
enteropathogens is also available. The largest product range are assays that detect
Clostridium difficile toxins and/or cellular antigens. Examples include; Techlab C.
DIFF QUIK CHEK®, C. DIFF QUIK CHEK COMPLETE® , TOX A/B QUIK CHEK; RBiopharm RIDA®QUICK Clostridium difficile Toxin A/B, RIDA®QUICK Clostridium
difficile GDH, Remel Xpect® C. difficile Toxin A/B; and Meridian Biosciences
ImmunoCard®Toxins A&B, ImmunoCard®C. difficile GDH.
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Rapid format assays for Campylobacter have been developed including; RBiopharm RIDA®QUICK Campylobacter and Meridian Biosciences ImmunoCard
STAT!®CAMPY;
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Shiga toxins of STEC and O157 cell wall antigen specific rapid tests also exist for
direct testing from stool. These include the R-Biopharm RIDA®QUICK
Verotoxin/O157 Combi and the Meridian Biosciences ImmunoCard STAT!® E. coli
O157 Plus and ImmunoCard STAT!®EHEC.
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SD Bioline (Alere) produce a rapid test for Vibrio cholerae, the SD BIOLINE Cholera
Ag 01/0139 which is specific the Ogawa serotype 01 and Inaba serotype 0139.
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In addition to rapid direct testing of stool, rapid format antigen detection kits are also
available for testing of blood for diagnosis of typhoid fever including the Cortez
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Diagnostics Salmonella Typhi Antigen Rapid Test RapiCard™ Insta Test. The
Biocan TELL ME FAST™ Salmonella typhi/paratyphi A Test Strip is also available
which can be used to test stool, serum or plasma. LumiQuick produce both the S.
typhi/paratyphi Antigen Duo Test and Salmonella typhi Antigen Test for which stool,
serum, plasma or whole blood can be utilised as the input sample. DIALAB
manufacture the Salmonella typhi/paratyphi Ag Cassette for which stool/whole
blood/serum and plasma can all be tested.
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ELISA Microplate and Chemiluminescent Immunoassay (CLIA) Methods
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C, difficile toxin(s) and antigen assays include; Techlab C. difficile Toxin/Antitoxin
Kit, C. DIFFICILE TOX-B TEST, C. DIFFICILE TOX A/B II™ and C. DIFF CHEK™ 60. The R-Biopharm RIDASCREEN®Clostridium difficile GDH and RIDASCREEN®
Clostridium difficile Toxin A/B. The Savyon CoproELISA™ C.difficile GDH and
CoproELISA™ C.difficile Toxin A/B. The Remel ProSpecT™ C. difficile Toxin A/B,
and the Meridian Biosciences Premier® Toxins A&B and Premier® C. difficile GDH.
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Two kits utilising Chemiluminescent Immunoassay methodology are available for C.
difficile on the Diasorin Liaison instrument platform LIAISON® C. difficile Toxins A
and B and LIAISON® C. difficile GDH.
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Campylobacter antigen assays include the R-Biopharm
RIDASCREEN®Campylobacter; Remel ProSpecT™ Campylobacter; Cortez
Diagnostics Campylobacter ELISA kit and the Meridian Biosciences Premier®
CAMPY
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Detection of STEC and/or serotype O157 is available using R-Biopharm
RIDASCREEN® Verotoxin; Remel ProSpecT™ STEC (Shiga Toxin E.coli); Cortez
Diagnostics E.Coli O157 (Fecal) ELISA kit and E.Coli Verotoxin (Fecal) ELISA kit;
Meridian Biosciences Premier® EHEC
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Viral Enteropathogen Antigen Detection
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Rapid Methods
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R-Biopharm RIDA®QUICK Norovirus, RIDA®QUICK Rotavirus, RIDA®QUICK
Rotavirus/Adenovirus Combi; Remel Xpect® Rotavirus; Cortez Diagnostics
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Rotavirus Rapid Test (Cassette) and Adeno / Rota Antigen Rapid Test (Cassette)
RapiCard™ InstaTest; bioMerieux Vikia ROTA-ADENO; Meridian Biosciences
ImmunoCard STAT!®Rotavirus; Alere SD BIOLINE NOROVIRUS Ag Test and SD
BIOLINE ROTA/ADENO Ag Rapid Kit; Famouze Diarlex MB (Rota and Adeno) strip
and Actim NORO Cassette.
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Sensitized latex particle agglutination: Famouze Diarlex Rota-Adeno and Rotalex
(Rota).
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ELISA Methods
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R-Biopharm RIDASCREEN® Rotavirus, RIDASCREEN® Adenovirus,
RIDASCREEN® Astrovirus, RIDASCREEN® Norovirus; OXOID IDEIA Amplified
IDEIA Astrovirus kit , IDEIA Adenovirus kit, IDEIA Norovirus kit and IDEIA Rotavirus
kit; Remel ProSpecT™ Rotavirus, ProSpecT Adenovirus ProSpecT Astrovirus
Microplate Assay; Cortez Diagnostics Adenovirus (Fecal) ELISA kit and Rotavirus
(Fecal) ELISA kit; Meridian Biosciences Premier® Rotaclone®, Premier®
Adenoclone®and Premier® Adenoclone®– Type 40/41.
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Immunodiagnostic Assays for Enteropathogen Antibody Production
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Several antibody detection assays are available for enteropathogens. These assays
have been developed in several formats including rapid tests and each of the
routinely utilised serological techniques (ELISA, CFT, and IHA) in microplate
formats. Slide and tube agglutination assays have also been manufactured.
Detection of host response to infection has generally proven most useful in patients
from areas of low endemicity of the particular enteropathogen that is being tested for
(2). A positive antibody result from a returning traveller with no previous risk of
exposure to the enteropathogen is of high value, whereas positive IgG serology for
enteropathogens that can be encountered locally may represent prior exposure and
not active infection (16). IgM and IgG combination assays are useful in elucidating
the currency of the infection, particularly in areas where the enteropathogen is
endemic (17, 18).
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Examples of available antibody assays and assay formats include the following.
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Rapid lateral flow/immunochromatographic tests
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Rapid format antibody detection kits are available for testing of blood for diagnosis of
typhoid fever including the SD BIOLINE Salmonella typhi IgG/IgM Test. LumiQuick
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produce the Quick Profile™ S. Typhi IgG/IgM Duo Test Card for use whole blood,
serum or plasma.
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Rapid slide or tube agglutination kits
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Famouze manufacture an extraintestinal BLA-Bichrolatex Amibe Slide test for
Entamoeba histolytica. IDL Biotech manufactures the TUBEX TF magnetic and latex
particle agglutination assay in tube format that detects IgM antibodies to Salmonella
Typhi O9 antigen. Both of these assays compare very favourably performance wise
to other immunodiagnostic assays, can be completed in very rapid time (5-10
minutes) and both are useful in front line/PoC settings and within the laboratory(18,
19).
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ELISA format antibody kits
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R-Biopharm RIDASCREEN® Entamoeba histolytica IgG, RIDASCREEN® Taenia
solium IgG; RIDASCREEN® Adenovirus IgA, IgG.
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Cortez Diagnostics Fasciola IgG ELISA kit, Strongyloides IgG ELISA kit,
Cysticercosis IgG (T. Solium) ELISA kit, E. histolytica IgG ELISA kit and
Schistosoma IgG ELISA kit.
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DIASORIN Adenovirus IgA, Adenovirus IgG, Adenovirus IgM, Ascaris lumbricoides
IgG, Entamoeba histolytica IgG, Schistosoma mansoni IgG and Taenia solium IgG.
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Novatec Adenovirus IgA/IgG/IgM, Ascaris lumbricoides IgG, Schistosoma mansoni
IgG, Taenia solium IgG and Entamoeba histolytica IgG.
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Virion-Serion Campylobacter jejuni IgA/IgG/IgM, Yersinia IgA/IgG/IgM and
Campylobacter jejuni IgA/IgG/IgM.
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Bordier Affinity Schistosoma mansoni ELISA, Strongyloides IgG ELISA and
Microsporidial ELISA.
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CFT format kits
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Virion-Serion Adenovirus, Campylobacter jejuni , Listeria monocytogenes, Rotavirus,
Yersinia enterocolitica O3 and Yersinia enterocolitica O9.
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IHA format kits
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Famouze Schistosome IHA, Amoebiasis IHA and Fasciola hepatica IHA; Cellognost
Amoebiasis Combipack and Cellognost Schistosomiasis.
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Immunodiagnostic Assays Specifically for Bacterial Toxins
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An additional diagnostic modality for gastrointestinal disease is direct detection of the
excreted toxins of certain bacterial enteropathogens in the stool sample. In addition
to the commercial assays for the toxins of C. difficile and STEC, immunodiagnostic
antigen tests also exist for other bacterial toxins involved in gastrointestinal
disorders.
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For some bacteria (e.g. Bacillus cereus, Staphylococcus aureus and Clostridium
perfringens), clinical symptoms represent ingestion of large numbers of organisms
and/or their toxins within foodstuffs rather than infection of the intestinal tract by the
organism (20-22). Commercial assays that detect these toxins generally employ
reverse passive latex agglutination (RPLA). Examples include the set of RPLA kits
available from OXOID: E. coli heat-labile enterotoxin and Vibrio cholerae enterotoxin;
shiga toxins of EHEC; Bacillus cereus; Clostridium perfringens enterotoxin and a kit
for Staphylococcal enterotoxins. ELISA based assays for C. perfringens enterotoxin
are also produced by Techlab (Techlab C. perfringens Enterotoxin Test) and RBiopharm (RIDASCREEN®Clostridium perfringens Enterotoxin).
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Comparison of Immunodiagnostic Assays to Molecular Assays
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Rapid and conventional immunoassays for enteropathogens generally show similar
specificities but are often reported to be less sensitive than molecular testing. The
rapid lateral flow kits and agglutination methods offer superior time to result than
molecular techniques, and require only basic technical skill and no specialist
equipment to perform. Direct protein toxin detection is of course unable to be
performed using nucleic acid based molecular testing. Detection of a specific nucleic
acid sequence encoding the toxin is a useful proxy for some organisms such as
toxigenic C. difficile and STEC which both carry specific genetic sequences
responsible for toxin production and which differentiate these strains from nontoxigenic strains.
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Immunoassays for C. difficile toxins A and B and glutamate dehydrogenase (GDH)
antigen are used widely within clinical microbiology laboratories. The GDH assays
compare favourably to molecular methods sensitivity wise but are not quite as
specific as PCR or immunoassay based toxin detection(23, 24). Eastwood et al.
compared nine ELISA toxin assays, a GDH assay and a Toxin B real time PCR
against the gold standard assay of cytotoxin neutralisation on cell lines. Sensitivity
and specificity of the GDH assay was reported as 90.1% and 92.9%, a PCR assay
for TcdB was 92.2% and 94% and the mean of the toxin assays was 82.8% and
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95.4% respectively(24). An international review of the immunoassays for detection
of toxin A or B by Planche et al. found that despite reasonable sensitivities the low
prevalence resulted in unacceptably low positive predictive values for all of the six
common assays examined (Meridian Premier, TechLab Tox A/B II, TechLab Tox A/B
Quik Chek, Remel Xpect, Meridian Immunocard and BioMérieux VIDAS(25). To
address these issues several screening and confirmation algorithms have been
proposed incorporating combinations of the immunoassays and PCR testing of
specific genes in the pathogenicity locus (PaLOC) (23, 26, 27). The suggested
algorithm in Australia is that proposed by the Australasian Society for Infectious
Diseases (ASID) which recommend the use of a lower cost sensitive screening
assay such as GDH, followed by a specific confirmation assay such as Toxin A/B or
PCR against genes in the PaLOC (26).
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Two STEC immunoassays were compared to stx1/2 PCR and chromogenic
CHROMagar STEC cultures by Hirvonen et al.(9). The R-Biopharm RIDA®QUICK
Verotoxin/O157 Combi rapid assay detected 14 of the 16 positives, whereas both the
Premier® EHEC ELISA assay and stx1/2 PCR assays detected all 16 positives.
There are several studies examining the use of ELISA based kits for detection of
STEC (28-31). The Premier® EHEC, and R-Biopharm RIDASCREEN® Verotoxin
assay have been reported to have similar sensitivity when compared to culture, with
sensitivity of 89% (29, 31). The Remel ProSpecT™ STEC, was found to be ten fold
less sensitive than those two assays in the study of Willford et al. (31).
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Rovida et al. performed a comparison of the R-Biopharm RIDASCREEN® and
RIDA®QUICK immunoassays against molecular assays for the four main viral
enteropathogens (32). Results of the immunoassays were variable, specificity was
high for all assays, but sensitivity was generally lower than PCR. The Astrovirus
ELISA assay performed equivalently to PCR testing with a sensitivity and specificity
of 100%. The Norovirus ELISA product had a poor sensitivity of 49.5%, an outcome
that was inconsistent with other investigations that reported sensitivity between 60%
and 90% (32-34). The Adenovirus component of the combi rapid product performed
poorly with sensitivity of 28.6%, however the Rotavirus component had a sensitivity
of 88.8% (32).
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Three papers have been published by an Australian laboratory comparing two rapid
Norovirus immunoassays and an ELISA assay against RT-PCR (35-37). Reported
sensitivity of the immunoassay products was 62%, 83% and 66% for the Bioline SD
Norovirus, RIDA®QUICK Norovirus and the DAKO IDEIA Norwalk like Virus kit
respectively. Specificities of 100%, 98.6% and 85% were also reported respectively
for those tests. These results are consistent with international reports that also
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indicate high specificity but lower sensitivity of Norovirus immunoassays when
compared to RT-PCR methods (38-40).
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All of the R-Biopharm assays for Cryptosporidium, Giardia and E. histolytica,
including the rapid tests and the ELISA kits were evaluated by Goni et al. (41). The
immunoassays were compared to microscopy results and conventional PCR assays.
Each assay provided similar sensitivity and specificity against the two reference
methods. Sensitivity compared to PCR for Cryptosporidium was 65-70% with a
specificity of 92.9-96.4%. Sensitivity compared to PCR for Giardia was 87.5-90.6%
with a specificity of 99.2-96.9%. And sensitivity compared to PCR for E. histolytica
was 62.5-75% with a specificity of 92.8-96.1% (41). A positive predictive value of
just 56.5% for Cryptosporidium with the ELISA method was also reported (41). This
low PPV is consistent with low prevalence of target and use of lower sensitivity
assays. In our laboratory, positive results for Cryptosporidium in ELISA screening
methodologies are confirmed using stained microscopy for this reason.
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An IFA assay by Alfa Cisse et al. using monoclonal antibodies against
Enterocytozoon bieneusi and Encephalitozoon intestinalis was shown to generate
comparable results to conventional PCR with 100% sensitivity and specificity
observed (42). Testing time was reported as three hours with hands on time of one
hour and conventional PCR was listed as an eight-hour test including nucleic acid
extraction.
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Bessede et al. compared one rapid (ImmunoCard STAT!®CAMPY ) and two ELISA
based Campylobacter immunoassays (RIDASCREEN®Campylobacter and
Premier® CAMPY) to two PCR assays and culture (43). The sensitivity of all three
immunoassays was reported as being 5-10% higher than that of either PCR method.
All five alternate assays were significantly more sensitive than culture, which was
only 63% sensitive in the study.
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Results for the immunoassays were consistent with those reported by Granato et al.
who compared two ELISA methods Premier® CAMPY , and ProSpecT™
Campylobacter and the rapid ImmunoCard STAT!®CAMPY to culture utilising PCR
analysis for discrepancy assessment(44). The two ELISA tests had identical
sensitivity of 99.3% and specificity of 98%, and the rapid ImmunoCard
STAT!®CAMPY had a sensitivity of 98.5% and specificity of 98.2% (44).
Interestingly the study of Granato et al. reported a sensitivity of 94.1% for culture,
which was significantly different to that of Bessede et al. at 63% sensitivity.
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Irrespective of comparative analytical performance, in Australia there is an important
consideration that often makes the choice of using immunodiagnostic assays or
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molecular assays a “one or the other” decision. The funding of testing for
enteropathogens by both of these methods is reimbursed from the same Medicare
items 69494-69496. These items can only be claimed just once per patient sample.
The text of the items are; ‘Detection of a virus or microbial antigen or microbial
nucleic acid (not elsewhere specified)’. Consequently combining both methods in
the diagnostic workflow needs careful consideration of costs.
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Current Molecular Approaches to Diagnosis of Gastrointestinal
Infections
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Human gastrointestinal infection is the second leading cause of deaths due to
infectious disease worldwide, responsible for millions of deaths in children under age
five each year, predominantly in developing countries (45-48). In Australia
community and hospital acquired infectious diarrhoea is a significant cause of
morbidity reportedly causing 17.2 million episodes of gastroenteritis a year (49).
OzFoodNet estimated that there are approximately 3.7 million visits to general
practitioners for gastroenteritis and that this resulted in more than 500,000 stool tests
being performed in Australian diagnostic laboratories (49). The 80% discrepancy
between actual doctor visits and estimated cases arises due to the self-limiting
nature of the majority of gastrointestinal diseases. Australian health departments
have estimated that, for every case of salmonellosis notified by a laboratory there
may be up to 7 infections that remain unreported in the community, and that there
are may be up to 8 cases unreported in the community for every notified case of
Campylobacter or Shiga toxin producing Escherichia coli (STEC) (49, 50). Based on
those estimates there is an 86% difference between doctor visits for gastrointestinal
disease and stool samples actually submitted for testing. This may also reflect that
the existing diagnostic tests are not viewed as offering results in a relevant time
frame and that by the time results are available the patient is most likely well. In the
majority of cases of uncomplicated gastroenteritis, only supportive therapies are
recommended by clinicians. In an Australian national gastroenteritis survey it was
found that for most patients symptoms lasted 24-48 hours with an average of five
episodes of diarrhoea per 24 hours (51). The same survey indicated that the age
groups with the highest disease burden were children under five years, and women
between 20-40 years of age. Those aged over 60 often did not report gastroenteritis
but were reported to have the longest duration of symptoms (51). It was also
observed that most samples are submitted when people have symptoms that
continue for three days or more. If symptoms were less than three days only one in
142 cases are submitted for testing. As the duration of symptoms increased to 3-4
days, one in 14 cases were submitted for testing. Patients with symptoms of 5 or
more days had samples submitted for testing in 25% of cases.(51). This appears to
correlate well with conventional detection methodologies generally not being
complete before 3 days and therefore often providing only retrospective information.
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Transmission of enteric disease within the community and the healthcare setting
causes a significant economic drain due to time losses. In Australia it was estimated
at over six million days of lost paid work, nearly half due to a carer needing to look
after the affected individual (51). Any improvement in diagnostics that can assist
with greater coverage and earlier identification of enteric pathogens may help to
reduce these burdens. Sinclair et al. examined pathogens causing community
gastroenteritis in Australia and concluded that, “Many of the pathogens responsible
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for cases of gastroenteritis in the Australian community are likely to go undetected by
current surveillance systems and routine clinical practice” (52). Incorporation of
molecular technologies into clinical practice due to the “unsophistication of
conventional diagnostic methodologies and strategies” has also been promoted by
Bennett & Tarr (53). The overwhelming challenge in diagnosing infectious
diarrhoea is the large number of causative enteropathogens, including viruses,
bacteria and protozoa. Current methodologies are inefficient, as the protocols require
multiple tests and testing modalities. This results in a workflow that is time
consuming and labour intensive. Standard viral enteropathogen diagnostics
available in the routine laboratory are also limited. Routine molecular diagnosis of
an extended panel of enteropathogens appears warranted. Amar et al. published
data on a retrospective analysis of samples from the English case-control Infectious
Intestinal Disease Study (1993-1996) where PCR techniques were utilised to
examine 4627 archived faecal samples for enteropathogens. The percentage of
archived samples from cases in which at least one agent was detected increased
from 53 to 75% after the application of PCR assays (54).
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Addressing several needs not met by conventional methods, the high sensitivity and
rapid time to results provided by molecular diagnostic technologies have seen
increasing application of these techniques to the diagnosis of gastrointestinal
disease. Selected examples of the use of molecular amplification technology for
diagnosis of bacterial, viral and parasitic enteropathogens are presented below.
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Bacterial Pathogens
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The traditional diagnosis of bacterial enteropathogens involves growth of isolates on
solid media after direct inoculation of faeces to the plates, and enrichment/selection
in liquid broths followed by inoculation to solid media. The approach utilises several
types of media and incubation atmospheres to provide appropriate selectivity and
growth parameters for the target organisms and to reduce the recovery of normal
flora. Plates are examined by skilled laboratory staff and suspicious isolates are
confirmed via morphological, serological and biochemical methods, and more
recently by use of proteomic mass spectrometry. The culture methodology performs
well but is dependent on the skill of the individual observing the cultures and the
initial fitness of the organisms in the specimen. Unfortunately, observational
characteristics may not always allow appropriate recognition of pathogens. For
example it is not possible to distinguish all pathovars of E. coli from commensal
varieties based on the phenotype of the organism on culture media. The major
disadvantage of the culture techniques however is that time is necessarily required
for bacteria to grow into colonies that can be adequately observed by the human
eye. Bacterial culture from faeces can take from 2-5 days to complete dependent on
the protocol utilised and what bacterial pathogens are sought. Diagnostic yields
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between 6% and 11% have been reported for the bacterial culture techniques (8,
55). In our laboratory for the period 2007-2011 a yield of 19080 bacterial
enteropathogens recovered from a total of 254940 stool cultures (7.5%) was
observed (56).
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Many PCR assays have been developed for the detection of human bacterial
enteropathogens (8, 57-77), including several multiplex PCR assays targeting more
than one organism in the one assay (54, 78-85). Compared to conventional culture
techniques the use of PCR approaches has reduced detection times from 2-5 days
to a single day turn around (8, 85). Many molecular assays exist for Salmonella and
Campylobacter, as these two genera are responsible for the majority of bacterial
gastrointestinal disease. However there are at least 12 other genera which contain
species that have been reported to cause human enteric infections. Table 1
provides a list of the majority of bacterial enteropathogens known to be responsible
for cases of human gastroenteritis. Diagnostic PCR based assays have been
developed for all of the bacteria listed in Table 1 and the use of real-time PCR is the
most common approach. The use of real-time PCR offers two significant benefits
over conventional amplification techniques. The first benefit is speed due to the
combination of amplification and detection. The second is that the reaction is ‘closed
tube’ and amplicon detection does not require open tube manipulation thereby
reducing the risk of amplicon derived contamination of future assays. The cost of a
‘home-brew’ or ‘in-house’ developed real time PCR assay including extraction is
between $10 and $20 per reaction dependent on the reagents and extraction
methodology utilised. Multiplexing on modern real time PCR instruments generally
allows up to five or six unique fluorescent labels to be reported. In our experience
with the development of four or five target multiplex assays where more than two of
those targets are reasonably expected to amplify concurrently is normally
problematical. In circumstances where more than one target amplification process is
occurring other assay components may not achieve optimal sensitivity. This is
because of competitive inhibition deriving from amplification of the most abundant
target sequences. Dual amplification methodologies employing a nested PCR
approach with limited cycle preliminary target enrichment have been designed to
overcome some of the multiplexing deficiencies that arise due to variation in multiple
target abundance (86, 87).
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Bacterial enteropathogens can be classified into those acquired in the community
from sources such as food, water or fomites; and those acquired as a healthcare
facility associated disease due to C. difficile, generally as sequelae to antibiotic
treatment. Unfortunately the incidence of community acquired C. difficile
gastroenteritis also appears to be rising (88). Discussion of the common molecular
approaches to diagnose specific groups of bacterial enteropathogens follows.
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Enterobacteriaceae (Salmonella spp., Shigella spp., Yersinia enterocolitica,
Plesiomonas shigelloides, Edwardsiella tarda, pathogenic Escherichia coli,
Escherichia albertii)
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Salmonella
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Salmonellosis is caused by Salmonella enterica subsp. enterica which is the second
most common bacterial enteropathogen in Australia after Campylobacter. The
disease is characterised by rapid development of symptoms including abdominal
pain, fever, diarrhoea, muscle pain, nausea and/or vomiting. People can become
infected via faecal-oral transmission, ingesting contaminated food, animal contact
and from environmental exposures (89). There were 11,993 notified cases of
salmonellosis in Australia in 2010; a rate of 53.7 per 100,000 (89). Salmonella
enterica serotype Typhi is only isolated from humans where person-to-person
transmission results in a serious bacteraemia called Typhoid fever. There were 96
notified cases of typhoid during 2010 (rate 0.4 per 100,000). Overseas travel was the
primary risk factor for notified cases in 2010, with 76% of notified cases known to
have been acquired overseas (89). Routine diagnostic procedures require selective
culture methods preceded by enrichment broth, and subsequent biochemical or
proteomic identification, as such it can take 3-4 days before a final result is available
(8). A number of authors have published molecular assays for use on human faecal
samples which have included Salmonella detection in multiplex with other bacterial
enteropathogens (8, 80-82, 84, 85). The genetic targets utilised have primarily been
the invasion gene invA, although the ttrBCA (tetrathionate reductase response
regulator) locus has also been used. Results from multiplex enteropathogen assays
for detection of Salmonella indicate either equivalent performance to culture (85) or
indeed improvement in sensitivity for Salmonella detection of up to 13% (8).
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Shigella
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Shigellosis is characterised by acute abdominal pain and fever, small-volume loose
stools, vomiting and tenesmus(89). Humans and apes are the only known reservoirs
of Shigella and transmission is generally person to person via the faecal-oral route,
but has also been caused by ingestion of contaminated food or water (90). In
Australia in 2010, there were 552 notified cases of shigellosis; a rate of 2.5 per
100,000 (89). Successful molecular detection methods for Shigella targeting the
ipaH invasion plasmid antigen H gene have been published including multiplex
methods with other bacterial enteropathogens (80, 84, 85, 91-93). Two authors have
reported that utilisation of a molecular detection methodology has shown improved
sensitivity for detection of Shigella from human samples as compared to culture
techniques (92, 94).
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Pathogenic Escherichia coli
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E. coli is generally the most abundant commensal in the human intestine. There are
however, certain pathotypes of E. coli which have acquired distinct virulence
determinants and are recognised bacterial enteropathogens. The use of
conventional techniques offers minimal assistance in recognising the majority of
these pathogenic E. coli. Recognition of sorbitol non fermenting shiga toxin
producing E. coli (STEC) of particular serotype O157 is possible on specific
variations of MacConkey media, and on chromogenic agars. There were 81
notifications of STEC in Australia in 2010; a rate of 0.4 per 100,000 population (89).
Due to the lack of utility of conventional culture for detection of STEC or other
enteropathogenic E. coli presenting in community samples, the reported rates of
STEC infection in Australia are influenced by state policy. South Australia routinely
tests all bloody stools by PCR for genes coding for shiga toxins and other virulence
factors, and consequently has the highest notified rates at 2.0 per 100,000 (89). In
our laboratory, any stool sample exhibiting 2+ or more red blood cells in a wet
preparation during faeces microscopy is also screened for STEC. Screening is
performed using an assay published by Paton and Paton, which is a five target PCR
for associated virulence factors including stx1, stx2, eaeA, ehxA and saa (95). The
diagnosis of the entire range of pathovars of E. coli is generally achieved only via
molecular detection of the associated virulence factors that confer the pathogenic
phenotype. Vidal et al. describe a multiplex assay to identify the six categories of
diarrhoeagenic E. coli. The multiplex assay was applied to testing 509 stool samples
from children <9 years of age presenting with acute diarrhoea in Chile and 76
patients were found to have a diarrhoeagenic E. coli present (77). Pathovars and
their typical virulence mechanisms enabling molecular identification are as follows.
Enterotoxigenic E. coli (ETEC), LT and ST toxin genes. Enteropathogenic E. coli
(EPEC), eae encoding intimin (without the presence of stx1/2), the EAF plasmid and
the bfp pilus gene. Enterohaemorrhagic E. coli (STEC or EHEC), stx1, stx2 shiga
toxin genes and eae. Enteroinvasive E. coli (EIEC), ipaH invasion plasmid though
this is also present in Shigella. Enteroaggregative E. coli (EAggEC), AAF-II
adhesion fimbriae. And Diffusely Adherent E. coli (DAEC) have the daa gene
producing F1845 fimbriae.
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A bacterium originally described as Hafnia alvei has also been implicated in
gastrointestinal disease via the same attachment and effacement mechanism
associated with STEC and EPEC (96). Hyma et al. reported that subsequent studies
have identified similar H. alvei-like strains that were positive for an intimin gene (eae)
probe. Based on DNA relatedness these were classified as a distinct Escherichia
species, Escherichia albertii (96). These E. albertii strains were found to be closely
related to strains of Shigella boydii serotype 13(96). Molecular detection of eae
(although not species specific) could assist in recognising gastrointestinal disease
potentially due to E. albertii.
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Yersinia
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Yersinia enterocolitica can cause gastroenteritis via the ingestion of contaminated
food (and in particular raw pig meat) or water (90). Y. enterocolitica is divided into a
number of biogroups some of which are not pathogenic or only weakly pathogenic.
Accordingly the significance of detection of this organism in stool also requires
clinical input. Biogroup 1B is the most pathogenic and possesses a virulence
plasmid as well as chromosomal genes that encode the heat stable toxin YST(90).
The specific gene targets that have been utilised for molecular detection of Y.
enterocolitica from stool include the ail outer membrane attachment, ystA, ystB, virF
transcriptional activator, yadA adhesion and the lysP lysine transporter (67, 80, 82,
84, 85, 97, 98). Yersinia pseudotuberculosis has also been known to cause a self
limiting diarrhoea in young children and adults and other species of Yersinia are of
uncertain significance (90). The Yersinia spp. assay of Cunningham et al., targeting
lysP was found to detect thirteen species of Yersinia excluding Y. kristensenii (85).
The use of such an assay may fulfil recommendations that detection of any Yersinia
species from stool in symptomatic patients with no other diagnosis should be
reported (90).
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Plesiomonas shigelloides
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Plesiomonas shigelloides generally causes a self limiting secretory watery diarrhoea
but can also present as a dysenteric bloody diarrhoea (90). Gastroenteritis due to P.
shigelloides is generally observed in travellers returning from tropical countries and
is associated with ingestion of seafood (99). Molecular detection of the organism
has been performed from human faeces utilising an assay targeting the 23S rRNA
gene, and also from fish portions using the hugA haem receptor protein gene (64,
65).
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Edwardsiella tarda
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Edwardsiella tarda is typically associated with water and animals such as turtles or
fish. The organism is recognised as a rare cause of gastroenteritis and possesses
cell associated haemolysins and cell invasion mediators (90). In our laboratory, all
of the cases of gastroenteritis from which we have recovered E. tarda have been
associated with owners of pet turtles cleaning their tanks. The most recent case we
have observed was in 2011. To date all of the published molecular detection
assays for E. tarda have been directed towards diagnosis in marine animals (100,
101).
Page 24
831
Vibrionaceae (Vibrio cholerae, Vibrio parahaemolyticus, Vibrio fluvialis)
832
833
834
835
836
837
838
839
840
Vibrio species inhabit marine and brackish water systems, all requiring some amount
of Na+ for growth. V. cholerae causes endemic, epidemic and pandemic cholera
disease. There are three distinct sub groups V. cholerae O1, non-O1 and O139. O1
and O139 disease is similar with copious watery diarrhoea where severe dehydration
can lead to death, non-O1 results in a milder disease as cholera toxin is rarely
produced (90). Vibrio parahaemolyticus can cause a watery diarrhoea and is
usually acquired via ingestion of contaminated raw fish or shell fish (102). Vibrio
fluvialis is also recognised as being responsible for sporadic gastroenteritis
outbreaks and has been reported as the cause of severe gastroenteritis (103, 104).
841
842
843
844
845
A number of multiplex PCR assays for identification of several species of Vibrio
including V. cholerae, V. parahaemolyticus and V. fluvialis have been published
using target genes such as dnaJ, rpoB, ftsZ, tdh, trh and toxR (105-109). The
performance of the multiplex assay of Nhung et al. (106) was evaluated specifically
utilising human stool and a sensitivity of 105 to 106 cells/mL of stool was observed.
846
847
Aeromonadaceae (Aeromonas hydrophila complex, caviae complex, veronii
complex)
848
849
850
851
852
853
854
855
856
857
858
859
860
861
862
Gastroenteritis caused by Aeromonas most commonly presents as an acute self
limiting watery diarrhoea (90). Although cholera like presentations have been
reported which have dysenteric presentations similar to that observed with Shigella
(90). Complications can include HUS, and recently homologs to the stx1 and stx2
genes of STEC were identified in strains of Aeromonas (110). The organisms are
inhabitants of aquatic environments and are recovered from food and water sources
associated with human infection. The consensus is that not every isolation of
Aeromonas from stool reflects involvement in intestinal disease, though some most
certainly do. Both conventional and molecular detection requires careful
interpretation in relation to the significance of detecting the organism (90, 111). An
Aeromonas hydrophila complex specific Taqman molecular assay has been
published with primers and probes that target specific sequences of the 16S rRNA
gene as well as the cytolytic enterotoxin gene (aerA) in a duplex assay (74).
Aeromonas hydrophila complex is more associated with severe infections and
septicaemia than the other species groups (90).
Page 25
863
864
Curved and Spiral Shaped Gram Negative Rods (Campylobacter spp.,
Arcobacter butzleri, Brachyspira)
865
866
867
868
869
870
871
872
873
874
875
876
877
878
879
880
881
882
883
884
885
886
887
Campylobacter jejuni and Campylobacter coli are the most common cause of
bacterial gastroenteritis in Australia and globally (59, 89). Sporadic outbreaks are
also caused by two other species C. lari and C. upsaliensis (90). The severity of
campylobacteriosis varies and is characterised by diarrhoea often with blood,
abdominal pain, fever, nausea and or vomiting. In 2010, there were 16,966 notified
cases of campylobacteriosis in Australia, a rate of 112 per 100,000 (89).
Conventional culture requires selective agar incubated in a microaerophilic
atmosphere for up to three days. In attempt to simplify identification and to improve
the speed of diagnosis a number of authors have published assays specifically for C.
jejuni and C. coli detection and/or differentiation targeting genes such as hipO, glyA,
mapA and ceuE (60, 102, 112). Debruyne et al. published an assessment of seven
different assays for detection of C. jejuni and C. coli reporting that two multiplex
assays were highly reliable (113). Other assays have been published for detection
of the four major species infecting humans including an assay targeting the glyA
gene and using hybridisation techniques (114) and an assay using a genus specific
16SrDNA PCR assay followed by a second bi-probe melting curve PCR analysis to
determine species identity (115) . Real time molecular detection assays for
Campylobacter in food products have also been published for C. jejuni and C. coli
(68) as well as an assay that includes specific detection of C. lari (70). The results
reported indicate that molecular techniques when used for detection of
Campylobacter in complex matrices such as stool or food result in significant
improvements of 15-20% or greater in sensitivity of detection compared to culture (8,
59, 70).
888
889
890
891
892
893
894
895
896
897
898
899
900
901
902
903
In our laboratory spiral and curved organisms are sometimes observed in faecal
microscopy techniques but without Campylobacter being recovered in culture, a
phenomenon also observed by Bessede et al. (59). Aside from the previously
mentioned poor sensitivity of culture methods for Campylobacter, some of the
observations of spiral or curved bacteria when followed up in our laboratory have in
fact been Arcobacter butzleri and Brachyspira pilosicoli. A. butzleri can be
recovered on certain Campylobacter culture media if the incubation temperature
utilised is 37°C, and it has been reported as the fourth most common
“Campylobacter like organism” isolated from patients with diarrhoea (116). A recent
study in New Zealand found Arcobacter as the causative agent in 0.9% of diarrhoeal
patients which is consistent with a report from France where A. butzleri was
recovered in 1% of patients (117, 118). Molecular assays for detection of Arcobacter
have targeted several genes including rpoB/C, 16S rDNA and 23S rDNA in both
human stool and food matrices (57, 61, 63, 71, 117, 119, 120). Brachyspira pilosicoli
and Brachyspira aalborgi are spirochaetes which colonize the intestines of a number
of animals, B. pilosicoli causes disease in pigs and both species may be under
Page 26
904
905
906
907
908
909
910
reported as human pathogens though this is not yet proven (90, 121). Large
numbers of spirochaetes attached to the intestinal epithelium may form a ‘false
brush border’, and this condition is referred to as ‘intestinal spirochaetosis’ or IS (90).
Gad et al. suggest that “heavy infestation of the gut surface epithelium by
spirochaetes acts as a barrier for the normal absorptive processes and leads to
diarrhoea” (122). Brachyspira have been detected in human faeces utilising PCR
assays targeting the 16S rDNA gene (123, 124).
911
Clostridia (Clostridium perfringens, Clostridium difficile)
912
913
914
915
916
917
918
Diarrhoea due to Clostridium perfringens type A results from ingestion of 108 or more
vegetative cells from contaminated food (generally meat). Release of an enterotoxin
(CPE) during sporulation causes mild self limiting diarrhoea within 7-30 hours of
digestion of the food. It has also been implicated as a cause of persistent diarrhoea
in the elderly in healthcare facilities (90). CPE is encoded by the cpe gene carried
on a large plasmid of enterotoxin producing strains and has been utilised as a
diagnostic PCR target (125).
919
920
921
922
923
924
925
926
C. difficile strains that possess certain toxin genes can cause a spectrum of enteric
disease from mild self limiting diarrhoea, a bloody-slimy diarrhoea often referred to
as C. difficile associated diarrhoea ‘CDAD’, or the more serious pseudomembranous
colitis (126). The organism causes a significant burden and is prevalent in
healthcare facilities due to a combination of factors including resistance of the spore
form to disinfectants, interference of the protective effect of normal flora via the use
of antibiotics (in particular clindamycin, expanded spectrum cephalosporins and
fluoroquinolones) and patient age (90).
927
928
929
930
931
932
933
934
935
936
937
938
939
940
941
Toxigenic strains of C.difficile that are capable of causing symptomatic intestinal
disease possess a pathogenicity locus termed the ‘PaLoc’(127, 128). Genes within
the PaLoc including tcdA (enterotoxin), tcdB (cytotoxin) and the accessory genes
tcdC, and tcdE have also been utilised as molecular diagnostic targets. The
existence of strains without tcdA or with variant tcdA has seen tcdB become the
most common molecular target even though tcdB itself contains variability (129,
130). In Australia, approximately 1.1% of toxigenic isolates have been reported as
tcdA-/tcdB+ (131). This is an important consideration as one of the more common
commercialised molecular technologies being implemented in Australian diagnostic
laboratories this year has been the Illumigene C. difficile LAMP assay (Meridian
Bioscience) which targets non-variant regions within the tcdA gene with isothermal
loop- mediated amplification. In part to address the question of performance of the
Illumigene assay for detecting tcdA-/tcdB+ isolates the manufacturers have
published data that has shown that the 5’ end of tcdA is not deleted in the common
tcdA-/tcdB+ toxinotypes VI and VII and these strains are still detected by the
Page 27
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943
944
945
technology (132). Of all of the bacterial enteropathogens, toxigenic C. difficile is
rapidly becoming the most widely detected by molecular technologies in diagnostic
laboratories and it has the greatest number of commercialised molecular testing
products available.
946
947
948
949
950
951
Hyper virulent strains of C. difficile (PFGE type NAP1), which are more correctly
known as PCR ribotype 027 strains have been observed in Australia and elsewhere
and have been associated with increased morbidity and mortality (133). Molecular
investigation of deletions within tcdC and/or the presence of the binary toxin genes
cdtA and cdtB have been used as indicators of the possible presence of a hyper
virulent strain requiring further investigation at a reference laboratory (134).
952
953
954
The presence of C. difficile in human faecal flora by PCR requires clinical correlation
due to the possible presence of asymptomatic toxigenic C. difficile at high levels in
children and neonates, and also in approximately 3-5% of healthy adults (90, 135).
955
956
957
958
959
960
961
Arguments have been made for C. difficile to be included in future commercial multi
analyte assays for two reasons. The first is an observation that 30% of requests for
single investigation of C. difficile associated diarrhoea based on clinical suspicion
were negative for the organism but were in fact positive for other enteropathogens
(136). The second is the increased observation of community acquired symptomatic
C. difficile diarrhoea that supports the inclusion of this organism in any multi panel
enteropathogen investigation screening technique.
962
Listeria monocytogenes
963
964
965
966
967
968
969
In the immunologically competent host gastroenteritis due to Listeria monocytogenes
generally occurs 24 hours after ingestion of a food source containing large number
(105-109 CFU/g or mL) of the organism and the illness lasts about 2 days (90).
Gastroenteritis and invasive listeriosis following ingestion may affect the elderly or
immunocompromised. Listeriosis in pregnancy can result in infections of the foetus.
In 2010, there were 71 notified cases of invasive L. monocytogenes infection in
Australia (89).
970
971
972
973
974
975
976
977
978
The majority of PCR assays targeting L. monocytogenes have been developed in the
context of screening the food supply or for applications involving assessment of
waste water treatment efficacy (73, 75, 83, 137). The hly (hemolysin listeriolysin O)
gene target has been utilised most successfully and provides a L. monocytogenes
species specific assay target with a detection limit between 3 and 30 template
molecules per PCR reaction (72). One author incorporated three marker genes for
L. monocytogenes (iap, hly and prfA) as targets for a PCR-Array based diagnostic
assay for human stool samples but none of the 34 clinical samples tested contained
the organism (84). L. monocytogenes has also recently been included in the
Page 28
979
980
bacterial enteropathogen PCR panel developed by local diagnostic company Genetic
Signatures (Easy-Screen Enteric Bacteria Detection Kit) (138).
Page 29
981
Viral Pathogens
982
983
984
985
986
987
988
989
990
991
992
993
994
995
Several viruses are known to cause gastroenteritis and together they are responsible
for significant morbidity and mortality. On a global basis viral enteric disease
accounts for 60% of illness in developing countries and 40% in developed countries
(139). The most common viruses causing gastrointestinal disease are Rotaviruses,
Noroviruses, Astrovirus and certain Adenoviruses. Other less recognised viruses
include Sapovirus, certain Enteroviruses and human Parechovirus. Given many
symptomatic patients are not diagnosed with a known enteropathogen, suspicion
remains that undiscovered enteropathogens and in particular viruses are likely to
exist. In fact recent work using new technologies to enable sequence independent
viral discovery techniques has identified unique viral nucleic acids and variants of
known enteropathogenic viruses in stools from symptomatic patients (3). Though
treatment for viral gastroenteritis is supportive only, rapid diagnosis assists in
isolation, outbreak tracing and cessation of antibiotic courses if they have been
started due to suspicion of bacterial or parasitological disease (102).
996
997
998
999
1000
Most of the enteropathogenic viruses are RNA in nature and require a reverse
transcriptase component to convert RNA into cDNA prior to utilising any DNA based
molecular amplification technique for diagnosis or characterisation. Discussion of
the common molecular approaches to diagnose specific viral enteropathogens
follows.
1001
Rotavirus
1002
1003
1004
1005
1006
1007
1008
1009
1010
1011
1012
1013
1014
1015
1016
1017
1018
1019
Rotavirus is the most common viral cause of gastroenteritis. It has a segmented
dsRNA genome and is divided into seven serogroups with serogroup A being the
predominant cause of infections in humans (140). In Australia, the dominant
genotype changes year to year, in 2010-2011 the genotype G2P[4] was predominant
representing 51% of samples examined under the Australian Rotavirus Surveillance
Program (141). The development of two live oral rotavirus vaccines Rotarix®
(GlaxoSmithKline) and RotaTeq® (Merck) and their use in Australia since 2007 has
shown an early impact with significant declines in hospitalisation and emergency
room visits reported since vaccine introduction (141). Conventional testing
techniques are generally rapid antigen tests either as RPLA, lateral flow
immunoassays, or ELISA detection kits, and usually detecting the VP6 antigens.
Molecular methodologies have not been widely adopted for initial diagnostic testing
despite the fact that they have been shown to have superior analytical and clinical
sensitivity compared to immunoassays (142). The potential benefits of using
multiplex approaches for multi viral detection from stool has resulted in the
development of a number of highly sensitive multiplex assays targeting viral
enteropathogens (139, 140, 143). Aside from reducing the overall costs,
multiplexing for viral enteropathogens is viewed as worthwhile because many are
Page 30
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1021
1022
1023
1024
1025
clinically indistinguishable (102). Molecular diagnosis of Rotavirus has focused
predominantly on the VP7 or VP6 outer capsid protein genes as the diagnostic
targets. It has also been suggested that assay specific cycle threshold cut-offs be
utilised for real-time PCR based rotavirus diagnostics, due to the observation that
higher Ct value detection (e.g. Ct of >28) of Rotavirus was less associated with
gastroenteritis and that 20-40% of rotavirus infections are subclinical (139).
1026
Adenovirus
1027
1028
1029
1030
1031
1032
1033
1034
1035
1036
1037
1038
1039
1040
1041
1042
1043
1044
A subgroup of Adenoviruses known as Group F serotype 40 and 41 are associated
with gastroenteritis (90). Adenoviruses have a dsDNA genome and conventional
diagnosis is normally via the use of rapid immunoassays or ELISA. Variation in the
fiber gene determines amino acid changes conferring serotype in the terminal portion
of the fiber protein (144). Many of the other Adenovirus serotypes are shed in
faeces so it is important that Group F specific molecular assays are utilised for
diagnosis of enteropathogenic Adenovirus (102). At least two Adenovirus real-time
PCR assays have been developed targeting specific regions of the fiber protein gene
to deliver Group F specificity when detecting diarrhoea due to Adenovirus serotype
40 or 41. The fiber gene Group F specific assay of Jothikumar et al. reportedly has a
sensitivity of 3-5 copies per reaction (145, 146). The authors also designed a
second assay targeting a conserved region of the Hexon gene to report all
Adenovirus groups A-F (145). As was found for rotavirus, adoption of RT-PCR
methodology has been reported to increase the recovery of Adenovirus significantly,
Logan et al. reported a 175% increase compared latex agglutination testing when
using a Group F specific molecular assay (146). Two of the three multiplex/panel
viral enteropathogen assays previously mentioned also include primers and probes
specific for Adenovirus Group F (140, 143).
1045
Norovirus and Sapovirus
1046
1047
1048
1049
1050
1051
1052
1053
1054
1055
1056
1057
Norovirus and Sapovirus are both members of the Caliciviridae possessing ssRNA
genomes which exhibit a high level of genetic diversity and are divided into five
genogroups GI to GV, GI and GII predominantly cause acute viral gastroenteritis,
and GIV occasionally (140). Each genogroup can be further subdivided into multiple
genotypes with GII/genotype 4 strains predominant globally (147). The burden of
disease from these viruses is high, the CDC estimated that each year in
industrialised countries these viral infections cause 64,000 episodes of diarrhoea
that require hospitalisation as well as 900,000 clinic visits by children and up to
200,000 deaths of children under five years old in developing countries (147). As
there is no effective method for culturing these viruses, molecular technology has
been the dominant diagnostic modality and is already considered the gold standard.
Consequently there are a significant number of commercialised molecular test kits
Page 31
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1059
1060
1061
1062
1063
1064
1065
1066
1067
1068
available, as well as a substantial number of published molecular assays. The most
successful molecular target utilised for detection of Norovirus is a region at the
ORF1-ORF2 capsid-polymerase junction and derives from the work of Kageyama et
al. (148). Singleplex or multiplex Taqman based adaptations containing specific
component assays for GI and GII genogroups designed in this region have become
widely used with clinical sensitivity reported as 91-97% (102, 149-151). Sapovirus
detection assays have evolved identically to Norovirus assays and the most common
approach is also real-time Taqman assays (152-155). Diagnostic targets utilised
are the capsid-polymerase junction or conserved regions of the polymerase. Two of
the three multiplex/panel viral enteropathogen assay publications include assays for
Norovirus GI and GII as well as Sapovirus (139, 140).
1069
Astrovirus
1070
1071
1072
1073
1074
1075
Astroviruses are positive sense, ssRNA viruses and cause acute gastroenteritis in
children and sporadic outbreaks in adults (156, 157). A number of real time PCR
assays have been published for the detection of Astrovirus and the ORF1a, 3’NCR
and capsid sequence regions have been used (143, 158, 159). Logan et al.
compared RT-PCR to electron microscopy and observed a tenfold increase in the
rate of detection of Astrovirus (154).
1076
Human Parechovirus (HPeV)/Enteroviruses
1077
1078
1079
1080
1081
1082
1083
1084
1085
1086
1087
1088
1089
1090
1091
Human Parechoviruses (HPeVs) are associated with gastroenteritis however the
aetiology is not clear (139). Although the majority of HPeV infections occur in
children without specific symptoms, symptoms have been described ranging from
gastroenteritis and respiratory infections to neurological disease especially in the
very young (160). Braun et al. recently published a study following viral
gastrointestinal disease in a childcare centre and observed HPeV was present in
21% of symptomatic children, but also in 27% of samples from asymptomatic
children (161). Molecular detection techniques have been published and generally
target HPeV specific sequences in the 5’UTR giving specificity compared to other
Picornaviridae (139, 162, 163). Amongst the Enteroviruses a number have been
associated with diarrhoea, in particular certain Echovirus types (164, 165).
Molecular detection targets are also primarily designed at conserved specific regions
of the 5’UTR, however a recent publication by Harvala et al. discusses specificity
problems with 5’UTR assays for diagnosis of enteroviruses in symptomatic patients
presenting to hospital due to apparent gastrointestinal rhinoviruses (166).
Page 32
1092
Parasitic Pathogens
1093
1094
1095
1096
1097
1098
1099
1100
An Australian study by Bowman et al. reported a detection rate of 2.0% (10/505)
from submitted stools for all parasites by combination of routine and concentration
microscopy (167). For the period 2007-2011 in our laboratory a diagnostic rate of
12.3% (31299/255018) for all parasites was observed from wet preparation, faecal
parasite concentration and fixed stain microscopy techniques on 255018 stool
samples (56). Ova, cysts and parasite examination by faecal concentration
microscopy and/or wet prep, has been reported as having a diagnostic yield of 2.9%
for Entamoeba complex in an Australian study by Fotedar et al. (168).
1101
1102
1103
1104
1105
1106
1107
1108
1109
1110
Dependent on volumes of samples received for testing, the microscopy component
of the diagnostic workup poses a large burden to the workflow of a lab and indeed on
the general health of the laboratory staff examining the slides. Repetitive strain
injuries are becoming more common as volumes of samples requiring extended field
examination by microscopy increase. The sensitivity of microscopy for detection of
parasites has been shown to be approximately 60% at best (1). The high specificity
of molecular techniques is also superior for organisms which resemble each other
closely or are indistinguishable by visual examination but have quite different
relevance clinically e.g. Entamoeba histolytica, Entamoeba dispar and Entamoeba
moshkovskii (2).
1111
1112
1113
1114
There have been a significant number of publications for molecular parasitology in
recent times. Amoebae, Flagellates, Coccidia, Blastocystis hominis, and
Microsporidial assays predominate, but there are also an increasing number of
publications of molecular assays for Helminths.
1115
1116
1117
1118
1119
Specific conventional diagnostic techniques for soil transmitted helminths require
several days of observation for results and can pose an infection risk to laboratory
staff e.g. the Harada-Mori larval stage nematode culture (169) and the Strongyloides
faecal culture (170). Both of these conventional techniques appear highly suited to
replacement by molecular technology.
1120
1121
1122
1123
1124
1125
1126
1127
1128
1129
1130
The utility of real-time multiplex PCR for concurrent testing of multiple parasites is
viewed favourably due to decreased cost/target, an increased range of parasites
examined and improved speed of analysis (171). Articles using at least triplex
multiplex testing include the following combinations: E. histolytica/G. intestinalis/C.
parvum (172, 173); E. histolytica/G. intestinalis/Cryptosporidium sp./Dientamoeba
fragilis (1); Ancylostoma/Necator americanus/Ascaris lumbricoides/Strongyloides
stercoralis (174); E. histolytica, E. dispar and E. moshkovskii (175); a Luminex
implementation for E. histolytica/G. intestinalis/Cryptosporidium sp./ Ancylostoma
duodenale/ Necator americanus/Ascaris lumbricoides/Strongyloides stercoralis
(176); Ancylostoma duodenale/Necator americanus/Oesophagostomum bifurcum
(177); and Cyclospora/Cystoisospora/Microsporidia (178).
Page 33
1131
Amoebae
1132
1133
1134
1135
1136
1137
1138
Amoebae present in the human intestine can be divided into three groups. The first
is the pathogen Entamoeba histolytica, which is responsible for amoebic dysentery
and invasive disease. The second is Entamoeba moshkovskii and Entamoeba
dispar which are generally considered non pathogens, but both have various case
reports postulating their involvement in intestinal disease and discomfort (2). And
the third is a range of non-pathogens which include Entamoeba coli, Entamoeba
hartmanni, Entamoeba polecki, Endolimax nana and Iodamoeba buetschlii.
1139
1140
1141
1142
1143
1144
1145
1146
1147
1148
1149
1150
1151
Molecular techniques have been particularly useful for detection and differentiation of
E. histolytica from E. dispar and E. moshkovskii as these organisms are
morphologically identical by conventional microscopy techniques and clearly have
very different clinical relevance. The target utilised is generally 18S rDNA which
encodes the small subunit rRNA as it offers appropriate specificity for distinguishing
between the three species and is also multi copy which enhances assay sensitivity.
Many other molecular targets have been utilised and the review of Fotedar et al.
provides a comprehensive overview (2). The multiplex assay of Verweij et al. for E.
histolytica/G. intestinalis/C.parvum was shown to exhibit 100% sensitivity and
specificity (173). A multiplex real time PCR assay for detection and differentiation of
E. histolytica, E. dispar and E. moshkovskii has also been published which targets
18S rDNA and utilises hybridisation probes and melting temperatures for species
specific identification (175).
1152
1153
1154
1155
1156
1157
1158
1159
1160
1161
1162
1163
1164
1165
1166
Traditionally the presence of the non pathogens E. coli, E. hartmanni, E. polecki, E.
nana, and I. buetschlii has been made via observing cysts and/or trophozoites in
faecal specimens via concentrated wet mounts and/or permanent stained smears.
One of the disadvantages of the current multiplexed molecular assays for parasites
is that the non-pathogens are no longer identified. Arguments exist both for and
against the need to incorporate detection of non-pathogenic protozoa. What is clear
is that acquisition of the non-pathogenic protozoa is normally via the same faecaloral route as pathogenic protozoa and the detection of non-pathogens indicates
possible exposure to faecal material warranting further examination in symptomatic
patients (179). Santos et al. also promote the value of reporting non pathogenic
amoebae when observed and have published an 18S rDNA based PCR-DNA
sequencing approach for identifying the Entamoeba species present in samples
(180). A sequencing based approach is not realistic for routine diagnostic detection
of the non-pathogenic Entamoeba and the development of real-time PCR assays for
non-pathogenic protozoa would be a better approach.
1167
Flagellates
Page 34
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1169
1170
Flagellates in the human intestine include two pathogens; Giardia intestinalis and
Dientamoeba fragilis, and three non-pathogens; Chilomastix mesnili, Retortamonas
intestinalis and Trichomonas hominis.
1171
1172
1173
1174
1175
1176
1177
1178
1179
1180
1181
1182
1183
1184
1185
1186
1187
1188
Giardia intestinalis is the most common non viral enteropathogen causing
gastroenteritis in developed countries (173). Prevalence of 2-5% in developed
countries and 20-30% in developing countries has been reported (181). G.
intestinalis accounted for 18% (5752/31299) of the total reported parasites from our
laboratory between 2007 and 2011 as shown in Table 3. Diagnosis can be via
microscopy and observation of cysts and trophozoites. However often the initial
screening is performed using ELISA or other immunoassay methods which generally
also detect Cryptosporidia. Molecular assays for G. intestinalis also primarily target
specific regions of the multi copy ribosomal genes including the 18S rDNA and
intergenic spacers (182, 183). Other targets utilised in real-time PCR assays include
the beta-giardin gene (184). As G. intestinalis is the second most reported parasite
after Blastocystis hominis it features prominently in all of the multiplex parasite
methods published excluding those designed for helminths alone (1, 172, 173, 185187). The performance of molecular testing for G. intestinalis has proven to be very
good, several publications show PCR to have excellent sensitivity and specificity
when compared to ELISA methodology and PCR exhibited at least a 10%-13%
improvement in sensitivity compared to microscopy (182, 183). Bruijnesteijn et al.
reported PCR for G. intestinalis to be 34% more sensitive than microscopy (185).
1189
1190
1191
1192
1193
1194
1195
1196
1197
1198
1199
1200
Dientamoeba fragilis genetically is characterised as a flagellate, but in fact is
‘aflagellate’ and morphologically resembles amoebae (102). D. fragilis is particularly
difficult to diagnose even when using preserved stools and fixed stain microscopy
based techniques due to the fact the trophozoites degenerate rapidly and lose
nuclear definition (188). The Australian prevalence of D. fragilis has been reported
as being from 0.9 to 16.8% in patients symptomatic with gastroenteritis (189). In our
laboratory despite the difficulties in diagnosing the organism, it comprised 2.5%
(791/31299) of the total parasites that were reported between 2007 and 2011, Table
3. Molecular detection of D. fragilis has been shown to offer considerably better
performance and has been reported to be 42%-65% more sensitive than microscopy
(185, 188). Targets utilised for molecular detection of D. fragilis have been the 5.8S
rRNA gene (185, 190) and the SSU 18S rDNA (191).
1201
1202
1203
1204
The relevance of detecting the non-pathogenic flagellates is similar to the detection
of the non-pathogenic amoebae as previously discussed. The non-pathogenic
flagellates have also not had molecular methods published other than the
sequencing approach also previously described for amoebae.
1205
Coccidia
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1208
Coccidial enteropathogens include Cryptosporidium parvum, Cryptosporidium
hominis, Cyclospora cayetanensis and Cystoisospora belli (previously Isospora
belli). All are obligate intracellular pathogens.
1209
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1212
1213
1214
1215
1216
1217
1218
1219
1220
1221
1222
1223
1224
1225
Cryptosporidiosis is a parasitic infection of the lower intestine; symptoms generally
include abdominal cramping and usually large-volume watery diarrhoea. The
Ingestion of contaminated water is the major risk factor for infection (89). In 2010,
there were 1,480 notified cases of cryptosporidiosis reported in Australia (89). The
major burden of disease is generally in children <5 years of age (172). In our
laboratory Cryptosporidium spp. comprised 7.4% (2313/31299) of the total parasites
we reported between 2007 and 2011, Table 3. Diagnosis is generally via modified
acid-fast staining techniques and observation of the oocysts using microscopy.
Often the initial screening is performed using ELISA or other immunoassay methods
which generally also detect G. intestinalis. Consensus primers/probes for molecular
detection of Cryptosporidium spp. are recommended, as there are a number of
Cryptosporidium species known to infect humans for example; C. parvum, C.
hominis, C. felis and C. meleagridis (1). Molecular assays have targeted a variety
of genes including the SSU 18S rDNA (1, 192), a C. parvum specific ’segment A’
sequence (172, 173, 185, 193), the COWP gene (194), actin gene (195), and the
hsp70 gene (196). PCR has been shown to be specific and was reported as being
16% more sensitive than microscopy techniques (197).
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1227
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1230
1231
1232
1233
1234
1235
1236
Cyclospora cayetanensis is considered an emerging pathogen, generally associated
with ingestion of contaminated food in travellers returning from SE Asia or South
America (198). Symptoms reported are prolonged diarrhoea including nausea and
abdominal cramps (199). Conventional diagnosis is similar to other coccidian
parasites and relies on modified acid-fast staining techniques such as the modified
Kinyoun acid-fast stain and observation of the oocysts via microscopy. In our
laboratory 12 cases of Cyclospora cayetanensis were diagnosed between the years
2007 and 2011 from 255018 stool samples submitted for enteropathogen testing
(Table 3). The molecular target utilised in published methods is the SSU 18S rDNA
(178, 198, 199). Sensitivity has been reported as 1 oocyct per 5uL PCR reaction
volume (199).
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1242
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1245
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1247
Isospora belli is primarily associated with immunologically compromised patients in
which it can cause chronic and severe diarrhoea. However, I. belli has also been
reported as causing infections in children and travellers to tropical regions (200,
201). For the other coccidian parasites conventional diagnosis again relies on
modified acid-fast staining techniques such as the Kinyoun stain and observation via
microscopy. At our laboratory, we did not detect I. belli in the four years between
2007 and 2011. Ten Hove et al. have developed a real time PCR assay for the
detection of I. belli with very good performance characteristics, reporting 100%
sensitivity and 100% specificity (201). The molecular targets utilised for I. belli PCR
assays include the internal transcribed spacer 2 (ITS-2) region of the ribosomal
genes and the SSU 18S rDNA (201, 202).
Page 36
1248
Ciliates
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Balantidium coli is an opportunistic parasitic pathogen with pigs as the primary host,
humans become infected via direct or indirect contact with pigs and develop
balantidiasis. The infection may be subclinical (203), or can develop as a fulminant
infection with bloody and mucus containing diarrhoea and possible perforation of the
colon (203). To date no PCR detection assays have been published but ITS
sequencing (204) and the SSU 18S rDNA (205, 206) have been utilised in
taxonomical studies with other Balantidium species and would likely be suitable
targets for the development of specific real-time PCR assays.
1257
Blastocystis hominis
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Blastocystis hominis is classified in its own Kingdom (Stramenopila) (90). The
organism is by far the most common parasite observed in human stool samples in all
countries (207). In our laboratory, Blastocystis hominis comprised 64.4%
(20148/31299) of the total parasites we reported between 2007 and 2011 (Table 3).
Some patients from which B. hominis is recovered report abdominal pain, diarrhoea,
bloating, and flatulence. However detection of B. hominis is of uncertain clinical
relevance, recent studies indicate that the parasite is actually a species complex with
perhaps up to nine different types found in humans, all with variation in virulence
(208). Subtype 3 is the most common in prevalence studies, followed by subtype 1
(208). Roberts et al. have published a very well designed assay targeting the SSU
18S rDNA with primer sets carefully designed to avoid the variation inherent in the
wide range of genotypes (209). Sensitivity was reported as 94% (92/98) which was
51% better than the sensitivity of microscopy from a modified iron-haematoxylin
permanent stain (209). An incidence of 19% in symptomatic Sydney population was
much higher than that observed by our laboratory in Brisbane which was 6.3%
(3576/56876) for 2011, Table 3.
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Microsporidia
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Microsporidia are small spore-forming obligate intracellular organisms and were
originally considered protozoa but have since been recognised as being fungi (102).
Enterocytozoon bieneusi, and Encephalitozoon intestinalis are the predominant
microsporidia infecting humans and for the most part only those patients with
immunosuppression are infected. The small size, intracellular nature and poor
staining with histological stains results in under-reporting of microsporidial infections
(200). The conventional diagnostic technique utilised is modified trichrome staining
and examination under the microscope for the typical morphology. Several
molecular assays have been published, and potentially the most useful is the
TaqMan multiplex of Verweij et al. which detects both E. bieneusi, and
Page 37
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Encephalitozoon intestinalis via the ITS of the ribosomal genes (210). Another
molecular approach uses a multiplex Luminex bead array assay including E.
bieneusi, and E. intestinalis and this method yielded 87–100% sensitivity and 88–
100% specificity (178).
1289
Helminths
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1301
1302
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Soil-transmitted helminths (Strongyloides stercoralis, Ascaris lumbricoides,
Ancylostoma duodenale, Necator americanus and Trichuris trichiura) infect an
estimated one-sixth of the global population (211). The rate of infection by helminths
is reportedly the highest for children living in sub-Saharan Africa, and this is followed
by Asia, Latin America and the Caribbean (211). Molecular PCR amplification as a
diagnostic modality has been compared against both the Harada-Mori larval stage
nematode culture (169) and the Strongyloides faecal culture (170) . Gotuzzo
reported a sensitivity of 95% for Strongyloides diagnosis by PCR, equivalent to the
performance of both the Harada-Mori which had a sensitivity between 70 and 100%
(dependent on the study) and also Strongyloides Agar Plate Culture which had a
sensitivity of 85-97% (also dependent on the study) (212). Several multiplex real
time molecular assays have been designed combining some of the soil transmitted
helminths and are listed as follows; Ancylostoma/Necator americanus/Ascaris
lumbricoides/Strongyloides stercoralis (174); a Luminex implementation for
Entamoeba histolytica/Giardia intestinalis/Cryptosporidium sp./ Ancylostoma
duodenale/ Necator americanus/Ascaris lumbricoides/Strongyloides stercoralis (176)
and an assay for Ancylostoma duodenale/Necator americanus/Oesophagostomum
bifurcum (177).
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Molecular targets in the Luminex bead array assay reported by Taniuchi et al.for
Ascaris lumbricoides and the hookworms were the ITS1 and ITS2 regions
respectively. The SSU 18S rDNA target was utilised for Strongyloides stercoralis.
Reported sensitivity of the multiplex compared to parent singleplex assays was
between 83% and 100% (187). Basuni et al. utilised the same target genes for a
pentaplex multiplex for Ancylostoma/Necator americanus/Ascaris lumbricoides and
Strongyloides stercoralis, which also included a phocine herpesvirus internal control
reaction (174). Basuni et al. compared the results of the multiplex to those of the
conventional methods and reported that 48/77 samples were positive with the
molecular test and just 6/77 were positive with the microscopy.
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A limited number of real time PCR based molecular assays have been published for
other helminths. Design and validation of real-time assays for many of the helminths
listed in Table 1 would be required to complete any comprehensive molecular
approach.
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An assay was published for detecting the trematode Clonorchis sinensis in stool
samples (213) . The internal transcribed spacer 2 (ITS2) region was utilised to
Page 38
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design a specific TaqMan real time PCR assay. The authors reported a diagnostic
sensitivity of 95.9% and 100% sensitivity at 100 eggs/g, though egg counts as low as
1egg/g were PCR positive.
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A multiplex real-time PCR assay for detecting Schistosoma mansoni and S.
haematobium in stool samples was published by ten Hove et al. (214). This assay
utilised primers and probes targeting the cytochrome c oxidase gene. A detection
rate of 84.1% compared favourably to microscopy at 79.5%. The authors reported
100% sensitivity for the S. mansoni assay at 100 eggs/g (EPG).
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Alasaad et al. published a real-time PCR assay for the identification of the trematode
Fasciola spp (215). The ITS-2 region was utilised to design genus specific primers
for Fasciola and to design species specific internal TaqMan probes to identify F.
hepatica and F. gigantica. The authors reported a sensitivity of 1pg/µL of Fasciola
DNA. Other diagnostic targets have also been utilised and Le et al. have published
an assay targeting the mitochondrial DNA genes cox1-trnT-rrnL (216).
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Intapan et al. designed a real-time FRET based PCR assay for detection of the
trematode Opisthorchis viverrini in human faecal samples (217). The primers and
probes were designed to bind to a specific region of the pOV-A6 specific DNA
sequence. Diagnostic sensitivity was reported as 97.5% and specificity as 100%.
There was 100% sensitivity at 100 EPG, 94.4% at 1-99 EPG and the lowest EPG
detected by the PCR was 17 EPG.
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Conventional PCR has been utilised with a wide range of genetic targets for the
following helminths. ITS2 for the trematodes Paragonimus westermani, Fasciolopsis
buski and Fasciola gigantica (218). The non-coding HDP2 sequence and the cox1
sequence has been used for the tapeworms Taenia solium and Taenia saginata
(219, 220). The 28S D1 ribosomal DNA (rDNA) and mitochondrial cytochrome c
oxidase subunit I (mtCOI) fragments contain appropriate species specific regions for
designing a real time PCR assay for the small trematode Metagonimus yokogawai
(221). The repetitive element DL1 has been utilised for detection of the cestode
tapeworm Diphyllobothrium latum (222) as has the cytochrome c oxidase subunit 1
gene of mitochondrial DNA (223). ITS1 was utilised to detect the nematode round
worm Trichostrongylus spp. (224). SSU 18S rDNA was utilised to detect the
whipworm Trichuris trichiura (225). And the mitochondrial Cytochrome C Oxidase
Subunit 1 (cox 1) has been used for species specific detection of the cestodes
Hymenolepis nana and H. diminuta (226).
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Current Commercially Produced Multi Analyte Molecular Products
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1371
1372
A summary of commercial molecular assays and the enteropathogens detected is
presented in Table 2. The enteropathogen coverage of the currently available multi
analyte molecular assays is quite variable. Before adopting these products, the
scope of enteric pathogens usually observed by the laboratory and the desired
workflow would need to be considered. In general, bacterial pathogens are
reasonably well represented. Coverage of enteric viral disease ranges from none
(Savyon) to a reasonably comprehensive set of five viruses (Genetic Signatures and
Film Array). The commonest parasites detected in developed countries are
represented in the majority of assays although Seegene currently has no parasite
detection component. The maximum number of different parasites detected by the
commercial assays is six (Genetic Signatures). When compared to faecal parasite
concentration microscopy the available molecular assays provide limited parasite
coverage.
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In our laboratory a review of the enteric pathogens reported from 255018 faeces
samples submitted for testing between 2007 and 2011 indicated that 17 different
parasites were reported and 11 different bacterial species, refer Tables 3 and 4 (56).
Viral agents reported reflected the existing scope of testing which included
Rotavirus, Adenovirus and Norovirus. Comparison to conventional methodology
shows that the most comprehensive multi analyte option for parasites the Easy
Screen Enteric Parasite Detection Kit (Genetic Signatures) which provides 6
parasitological assays would have covered 95.9% (30010/31299) of the total
parasites reported by our laboratory. The NanoChip Gastrointestinal Panel (Savyon)
and combinations of Easy-Plex kits (AusDiagnostics) offer four parasites (differing
from Easy Screen by exclusion of Blastocystis hominis and Entamoeba complex)
and would have resulted in coverage of 28.7% (8997/31299) of the parasites
reported. The Luminex xTAG GPP parasite assay component (Cryptosporidium,
Giardia intestinalis and Entamoeba histolytica) would have resulted in coverage of
26.2% (8206/31299) of the parasites reported. Based on types of parasites
detected, using the molecular assay with the widest coverage would have resulted in
1299 patients between 2007 and 2011 (324/year) not having a parasite reported that
would otherwise have been when using faecal parasite concentration microscopy.
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Overall detection of bacterial enteropathogens at our laboratory from 2007-2011
was 19080 reported positives from 254940 cultures with a total of 10 different
bacterial enteropathogens reported, refer Table 4 (56). In addition to the 10 bacterial
species reported, toxigenic C. difficile has also been reported by PCR based testing
alone in our laboratory since 1997. The coverage of gastrointestinal disease due to
bacterial enteropathogens by the available molecular kits is also variable. The
Luminex XTag GPP bacterial target range would have resulted in 79.4% coverage
(15143/19080) of bacteria reported by our conventional culture. The ‘Diarrhea ACE’
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product (Seegene) compares very well to the range of organisms we reported using
conventional culture based methods and would have resulted in 99.8% coverage
(19078/19080) of the bacteria reported. Thirty seven Arcobacter, one Plesiomonas
and one Edwardsiella isolate would not have been reported by the Seegene
molecular assay. The Savyon NanoChip Gastrointestinal Panel and the
AusDiagnostics Easy-Plex Panel bacterial target range are identical and would have
resulted in 77.3% coverage (14748/19080) of the bacteria reported by our
conventional culture. The Genetic Signatures EasyScreen Enteric Bacteria
Detection kit target range would have resulted in 79.1% coverage (15093/19080) of
bacteria reported by our conventional culture methods.
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For laboratories receiving moderate to high volumes of samples per day use of the
available commercial molecular options might also present processing challenges.
In our laboratory more than 240 samples/day are received six days per week for
testing. The highest single instrument commercial kit ‘throughput/single test batch’ is
provided by the NanoChip (Savyon), at up to 95 patient samples per batch in 4
hours, however this solution does not offer any diagnostic coverage of enteric
viruses. The other methods are just 24, 32, 7 and 15 samples per batch (Luminex,
Seegene, AusDiagnostics and Genetic Signatures respectively) in similar time
frames on single instrumentation. One solution to throughput constraints is use of
multiple instruments, but this adds cost and the need for additional laboratory space.
Available laboratory space, particularly in controlled purpose built molecular
laboratories, is an important consideration in modern pathology laboratory practice.
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1425
A number of evaluation studies examining analytical performance have now been
completed for some of these commercial multi analyte enteropathogen detection kits,
in particular the Luminex xTAG GPP test which was the first to carry approval as an
IVD assay. Reported performance characteristics from various sources for the
commercial products are as follows.
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Luminex xTAG GPP
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The xTAG GPP assay is based on the Luminex bead array hybridisation platform
and offers up to 15 bacterial viral and parasitic pathogens in a single test as
summarised in Table 2. The assay is based on a single one-step RT-PCR, a target
specific primer extension, subsequent hybridisation to specific xTAG beads and then
laser based detection on the Luminex Analyzer.
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Chong et al. examined the performance of the GPP assay on 313 stools collected in
a high prevalence paediatric population from Botswana for the comparative detection
of viral targets including: Norovirus (GI/GII), Rotavirus A, and Adenovirus (serotypes
40 and 41) (227). The xTAG GPP assay results were compared to the authors inhouse multiplex real-time PCR viral assay that detected Adenovirus, Norovirus GI
and GII and Rotavirus A. Discordant results were subsequently tested with
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singleplex PCR assays for the individual viruses (227). Six samples were shown to
be inhibitory by failure of the MS2 internal control assay, leaving 307 valid results in
the study. Overall 280/307 (91.2%) of the stools tested with the GPP assay were
positive and 108 (35.2%) of these were positive for more than one pathogen (227).
Positivity rates for various targets were as follows: Norovirus GI/GII 43/307 (14.0%),
Rotavirus A 176/307 (57.3%), Adenovirus 40/41 34/307 (11.1%), C. difficile 13/307
(4.2%), Campylobacter 49/307 (16.0%), ETEC LT/ST 28/307 (9.1%), Shigella 35/307
(11.4%), E. coli O157 4/307 (1.3%), Salmonella 15/307 (4.9%), STEC stx1/stx2
4/307 (1.3%), Yersinia enterocolitica 1/307 (0.3%), Cryptosporidium 19/307 (6.2%)
and Giardia 10/307 (3.3%) (227). When comparing the xTAG GPP with the authors’
in house viral multiplex real-time PCR assay, the Luminex platform showed a
sensitivity of 98.3% (176/179) for Rotavirus A, 97.7% (42/43) for Norovirus and
100% (34/34) for Adenovirus 40/41. Three rotavirus samples were negative by GPP
and positive by multiplex and singleplex rotavirus assays, subsequent examination of
the crossing points for the in-house assays were greater than 33 cycles, indicating
low viral loads in these samples (227).
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Vocale et al. compared the xTAG GPP kit on 385 stool samples from hospitalized
symptomatic patients against culture for bacterial detection, EIA assays for virus
detection and microscopy for parasite identification (228). xTAG GPP produced
positive results in 27% of the samples tested (104/385) compared to a 13%
combined detection rate of the conventional methodologies. 10% of the positive
patients had two enteropathogens detected by the xTAG GPP system, with one
patient having three enteropathogens detected (228).
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Marimón et al. assessed the performance of the xTAG GPP against a panel of 387
stool samples including 233 retrospective frozen samples and 154 prospective
samples (229). Conventional techniques were positive for 225 of the stools (173/233
retrospective and 52/154 prospective), and there was an 84% agreement with the
xTAG GPP. The xTAG GPP detected most of the Campylobacters (61/63),
Rotaviruses (63/65) and Noroviruses (25/26)(229). The performance for detecting
Salmonella was less reliable with 7/42 Salmonella not detected. Use of another
commercial molecular assay for discrepant analysis of these seven Salmonella
results also resulted in a not detected result (229).
1471
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Ciardo et al. assessed the performance of both the xTAG GPP and the Seegene
‘Diarrhea ACE’ kits on 126 stool samples from symptomatic patients (230, 231).
Results were compared to culture on selective media, EIA testing, and Norovirus RTPCR. Pathogens were found in 42 of the stool samples. In six samples only one of
three methods was positive, in a further 6 samples 2 of three methods were positive.
The two multiplex assays gave identical results in 115/126 samples. No pathogen
was found in 64 specimens(231).
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Mansuy et al. utilized the xTAG GPP assay on 365 diarrhoeal stools samples
collected from children and adults in the hospital context and compared results to
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bacterial cultures, detection of E. coli shiga toxin like genes and two immunoassays,
the Exacto Combo Adeno Rota All Diag and the ImmunoCard STAT Norovirus (232).
Utilising all testing modes there were 156 enteropathogens detected. This included
98 viruses, 42 bacterial targets, and 16 parasites. There were also 29 co-infections
which all included a viral pathogen (232). Contrasting markedly with the findings of
Marimón et al. the xTAG GPP assay was found to be more sensitive than
Salmonella culture. Results also indicated the GPP assay was more sensitive than
the alternate assays used for C. difficile Toxin and the three viral enteropathogens
(232). The xTAG GPP produced similar results to conventional methodologies for
Campylobacter detection (232). The xTAG GPP assay was reported as being less
sensitive than PCR detection for STEC (232).
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Wessels et al. repeated a previous analysis of 300 samples using a more recent
version of the Luminex xTAG GPP assay. The newer assay included improved
Campylobacter species specific targets replacing a 16S rDNA target that was used
in a preliminary version of the xTAG GPP kit in the prior study (233). 62 out of 200
prospectively collected samples were found positive in the GPP assay, and 80
pathogens were detected in total. In 55 cases Campylobacter was detected in the
preliminary version of the kit. Other targets were C. difficile (n=7), Giardia (n=4),
Norovirus GII (n=4), Shigella (n=4) and the ETEC ST toxin (n=4)(233). 74 of the
100 selected samples were positive by GPP and 101 pathogens were detected.
Campylobacter was the most prevalent (n=39), followed by Giardia and Norovirus
(n=10), rotavirus (n=8), C. difficile (n=7), Cryptosporidium (n=6). In total there were
94 Campylobacter GPP positive results from the preliminary version of the kit,
compared to only 10 recovered isolates. When the improved IVD kit was utilised,
just the 16 specific C. jejuni, C. lari and C. coli positives were detected, which
matched what was previously confirmed by real-time PCR (233). The high rate of
detection of Campylobacter by the preliminary 16S target was attributed by the
authors as representing ‘apathogenic Campylobacter’(233). It must be noted that
using bacterial 16S rDNA as a diagnostic molecular target has resulted in
unexpected specificity issues in other assays, and therefore this should also be
considered as an explanation for the findings (234). Nucleic acid testing on a
microbial biosphere as diverse as the human faecal flora necessitates careful
evaluation of primers and/or probes in respect of the expected specificity.
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Seegene ‘Seeplex Diarrhea ACE’ Detection Kits
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The Seeplex Diarrhea ACE assays involve the use of three PCR amplification
reactions containing dual priming oligonucleotides. Amplicons are then detected and
specifically identified by size using auto-capillary electrophoresis (ACE) (235). When
used together the kits cover four viral enteropathogens, nine bacterial pathogens and
the toxin gene of toxigenic C. difficile. Seegene does not currently have a parasite
kit available.
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Higgins et al. utilised the Seegene Diarrhea-V ACE viral component to perform a
retrospective analysis of 200 clinical specimens. From these samples 177 had also
been previously examined for viral enteropahogens by electron microscopy and/or
real-time RT-PCR (236). Discordant analysis was performed with other commercial
kits or in-house assays. The diagnostic sensitivities of the kit were 100% for
Adenovirus, Rotavirus, and Norovirus GI and 97% for Norovirus GII with specificities
of 100% for each of the first three viruses and 99.4% for Norovirus GII (236). The
limits of detection were reported as 31, 10, 2, and 1 genome equivalent per reaction
for Adenovirus, Rotavirus, and Norovirus GI and GII respectively (236). Viral coinfections were observed for 12 (6.8%) stool specimens (236).
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Bessède et al. examined the performance of the Seegene Seeplex Diarrhea-B1
bacterial component for detection of Campylobacter on 242 samples (59).
Conventional culture, three immunoassay methods (ProSpecT Campylobacter,
RidaScreen Campylobacter and ImmunocardStat!Campy) plus an in-house PCR
were also performed on the samples. In total there were 23 positive samples , which
included 16 positive by culture. Seven other samples were culture negative but
positive for Campylobacter using both the immunoassays and PCR (59). A total of
37 specimens were positive by at least one method (59). Of the 16 positive culture
specimens, three were not detected by the Seegene PCR and four were not
detected by the in-house PCR. Two positive cultures were not detected by the
ImmunoCard , and one was not detected by the ELISA methods (59). For 6 of 7
samples that fulfilled the positivity criteria when the culture was negative, all of the
non culture methods were positive (59). Of 14 cases where only one technique was
positive, 9 were positive using immunoassay methods only, and 5 were positive
using molecular methods only (59).
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Results for Salmonella enterica detection were also presented. The Seeplex
Diarrhea-B1 multiplex kit detects Salmonella spp. (Salmonella enterica and
Salmonella bongori), Shigella spp. (Shigella flexneri, Shigella boydii, Shigella sonnei,
and Shigella dysenteriae), Vibrio spp. (Vibrio cholerae, Vibrio parahaemolyticus, and
Vibrio vulnificus), and C. difficile toxin B (235). The assay detected 14 samples of S.
enterica that were also positive by culture. There were another eight samples which
were positive by the multiplex PCR only, and five samples that were culture positive
and PCR negative (59).
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Ciardo et al. also assessed the performance of both the xTAG GPP and the
Seegene ‘Diarrhea ACE’ kits on 126 stool samples from symptomatic patients and
found the two multiplex assays gave identical results in 115/126 samples (230, 231).
Results were compared to conventional bacterial detection techniques of culture on
selective media, EIA testing, and Norovirus RT-PCR and pathogens were found in
42 of the stool samples. In six samples only one of three methods was positive, in a
further 6 samples 2 of three methods were positive.
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Savyon NanoCHIP Gastrointestinal Panel
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The NanoCHIP Gastrointestinal Panel from Savyon Diagnostics is a microarray
panel that can detect four bacterial and four parasitic enteropathogens. Detection of
the array occurs on the NC400 NanoChip molecular electronic microarray system.
The bacterial detected include Salmonella, Shigella, Campylobacter, and C. difficile.
The parasites detected include Entamoeba histolytica, Giardia intestinalis,
Dientamoeba fragilis and Cryptosporidium spp.. No viral enteropathogens are
detected by the assay.
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Savyon have recently presented data on the NanoCHIP microarray based
Gastrointestinal Panel (237), however no third party evaluation has yet been
published. The NC400 NanoCHIP system itself has been successfully applied to
detection of Influenza A and B, Respiratory Syncytial and Parainfluenza viruses
(238). According to Greenberg et al. from Savyon Diagnostics the performance of
the NanoCHIP Gastrointestinal Panel was compared against culture, microscopy,
EIA and RT-PCR for 142 known positive samples and the NanoCHIP detected all as
positive except for two of the 39 samples positive for Giardia and two of the 38
samples positive for Dientamoeba fragilis (237).
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AusDiagnostics EasyPlex FaecalProfile-10
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The analysis uses the principle of Multiplexed Tandem PCR employing two
sequential PCR steps (239). Firstly, a short (15 cycles) multiplexed pre-amplification
reaction using primers homologous to all targets in the panel and including a
reverse-transcriptase component is performed. This is then followed by amplicon
dilution into individual wells for real time PCR reactions using primers ‘nested inside’
those used in the initial short amplification step (86). This process is automated by
the Easy-Plex liquid handling robotic system. The secondary PCR reaction is
performed in the Rotor-Gene instrument and positives are determined by observing
increasing fluorescence of the intercalating dye Eva-Green associated with the
accumulation of amplification products. Specificity of the amplicons generated for
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the diagnostic targets and result calling is determined by post amplification melting
curve analysis. The ten targets and controls are amplified together in the initial
amplification and then real-time PCR is performed in individual wells of a 72 position
Easy-Plex ring (86). The specific targets included in the kit are Salmonella spp.
(tetrathionate reductase structural gene), Shigella spp. (invasion plasmid antigen H
gene), Campylobacter (16s rRNA gene), C. difficile (toxin B gene), Adenovirus
subgenus F (hexon gene), Norovirus GII/1 and II/4 (capsid protein gene), Norovirus
GII/2+3 (capsid protein gene), Giardia spp. (small subunit Ribosomal RNA),
Cryptosporidium spp. (18S Ribosomal RNA). There is also an artificial sequence
detected for the assay control which is termed ‘SPIKE’ (86).
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Data has been published for sub component assays of the AusDiagnostics Faecal
Profile-10 Assay, but none yet for the complete assay. As the initial amplification is a
large combined outer primer multiplex assay, the sensitivities quoted for the
subcomponent assays may or may not necessarily reflect the sensitivities observed
in the Faecal Profile-10 assay. High level multiplexing requires appropriately
designed compatible primer sets, preferably with little or no cross homology at the 3’
ends of any two primers in the multiplex mix. Any 3’ homology will be conducive to
selective amplification of primer dimers formed when two primers anneal to each
other via their 3’ ends and result in short length amplification products. Dependent
on the stringency of the reaction, cross homology in as little as three consecutive
bases from the 3’ end is sufficient for primer dimer formation due to the excess of
primer compared to target in PCR reactions. The selective amplification of short
length amplicons drains PCR reaction resources sufficiently to reduce optimal
sensitivity of the intended PCR reactions. The problem may not be as acute with
MT-PCR due to the ‘pre-amplification’ function of the initial multiplex, prior to the
second stage PCR testing, and of course would be eliminated if the manufacturer
has designed the 10 outer primer pairs to be free of any cross homology leading to
dimerisation.
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Stark et al. published an evaluation of the MT-PCR ‘Gastrointestinal Parasites – 5’ kit
comparing the MT-PCR to conventional microscopy techniques for 472 faecal
samples (1). The authors compared the assay to iron haematoxylin microscopy and
an existing set of real-time PCR assays (1). Enteropathogenic parasites detected by
the MT-PCR were as follows: 28 G. intestinalis, 26 D. fragilis, 11 E. histolytica, and 9
Cryptosporidium spp. (1). The results indicated 100% correlation to the RT-PCR
assays. In comparison the microscopy results were reported as having sensitivities
and specificities of: 56% and 100% for Cryptosporidium spp., 38% and 99% for D.
fragilis, 47% and 97% for E. histolytica, and 50% and 100% for G. intestinalis (1).
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Thomas et al. presented performance data on the MT-PCR ‘Faecal Profile – 6’ kit
comparing the MT-PCR to conventional bacterial culture techniques and
immunoassay (TECHLAB C diff Quick Chek Complete antigen/Toxins A and B) for
147 selected stool samples (240). The samples contained the following
enteropathogens: 41 Salmonella, 4 Shigella, 73 Campylobacter spp., 18 C . difficile
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toxinB positive stools and 11 negative stools (240). Of the 73 Campylobacter
culture‐positive stools tested; 72 were MT‐PCR positive (98.6% sensitivity 100%
specificity)(240). Interestingly the authors state that the only negative sample was a
mixed infection of C . difficle (toxinB) and Campylobacter which raises questions
regarding competitive inhibition for samples containing multiple enteropathogens.
No data is provided for the relative abundance of these two targets in that sample.
Two positive Shigella culture results were identified and both were MT-PCR positive
(100% sensitivity) (240). There were 41 Salmonella culture‐positive stools of which
40 were MT‐PCR positive (97.8% sensitivity)(240). There were also 9 Salmonella
culture‐negative but MT‐PCR positive stools, of which one stool was positive on reculture, two more were confirmed as positive by alternative methods, five were
determined as negative by other methods, and one that was determined to be an
incorrect melt temperature (240). The resultant specificity for Salmonella was
therefore 96%. All 18 of the TECHLAB C. difficile toxinB positive stools were
identified correctly by the MT-PCR (240).
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Genetic Signatures ‘EasyScreen Enteric’ Kits (Parasite, Bacteria, Viral)
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The most recent commercial product in Australia is the Genetic Signatures set of
EasyScreen kits.
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The three kits utilise target nucleic acids that have undergone a bisulfite conversion
of unmethylated cytosine bases into uracil bases under the trade name of ‘3base’
testing (241). This conversion reduces sequence complexity from four possible
bases to three possible bases (uracil and thymine bases being able to hydrogen
bond to adenine). Two distinct advantages are promoted for utilising the ‘3base’
technology (241). Firstly it allows unique design opportunities for assays without
requiring degenerate designs in regions of known variation, and subsequently offers
some ‘future proofing’ of assay designs potentially reducing the impact of unforseen
future sequence variation at the location of primes and/or probes. Secondly, as the
target sequence is effectively converted to a synthetic nucleic acid, patents
applicable to the originating target regions no longer apply (241).
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The kits ship complete with a choice of specific extraction protocols both manual and
automated which can be adapted to most common automated nucleic acid extraction
instruments. The extraction of the stool is a rapid process with swab of stool,
placement of the swab in a buffer, heating for 15 minutes and then purification by
standard Boom (242) based extraction methodology on silica columns or magnetic
silica beads (243). As the nucleic acids are bisulfite converted in the extraction
process they may not be suitable for other downstream molecular assays that have
not been designed to utilise the converted sequences.
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The EasyScreen Bacterial Detection Kit is a real-time PCR kit for the detection of
Salmonella, Campylobacter, Shigella, Listeria, Yersinia and C. difficile (243). The
EasyScreen Viral Detection Kit is a real-time PCR kit for the detection of Norovirus
I/II, Astrovirus, Adenovirus, Sapovirus and Rotavirus (243). The EasyScreen
Parasite Detection Kit is a real-time PCR kit for the detection of E. histolytica, D.
fragilis, Cryptosporidium, Giardia and B. hominis (243). Microsporidial detection is
listed as ‘coming soon’. All of the three kits are each multiplexed over two reaction
wells per patient per kit and include an Internal Positive Control and an Extraction
Control.
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Melki et al. from Genetic Signatures presented assay performance data indicating
that all of the component assays in the kits were linear from 101 to 106 target copies
and that there was no specificity issues when the assay was validated against a
collection of bacteria and fungi (138). Data was presented for more than 400 clinical
samples that had been compared to culture, EIA and microscopy and results
indicated equivalent performance for bacteria and viruses, and improved recovery of
parasites (138). Stark et al. have a manuscript currently accepted in press that
presents an evaluation of the parasite component of the Easy Screen kits (244).
Results indicated a 92%-100% sensitivity and 100% specificity. The authors also
comment that this panel should not routinely replace microscopy as it does not cover
C. cayetanensis or C. belli (244).
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Idaho Technology/BioFire – FilmArray GI Panel
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This product is yet to be made available to the Australian market, but has been
utilised in selected laboratories. The manufacturer has very recently presented
performance data on the use of the film array nanofluidic pouches containing the
Gastrointestinal Panel at several conferences (136, 245-247). The data is derived
from comparative testing at the Primary Childrens Medical Center Laboratory, a
laboratory located in the manufacturers’ local area in Salt Lake City, Utah. The
nanofluidic pouch technology employed in the FilmArray Gastrointestinal Panel is
currently FDA and CE-IVD cleared for a 20 target respiratory panel for respiratory
viruses and bacteria, and has been shown to perform very well compared to
conventional viral and bacterial respiratory diagnostics and also a commercial xTAG
Luminex respiratory panel (248). The pouch itself contains compartments for cell
lysis, DNA/RNA purification, an initial large multiplex reaction chamber, and then a
102 chamber reaction module also referred to as a ‘chemical circuit board’ in which
individual singleplex second-stage PCR reactions occur, result calling is performed
by end point melting curve analysis (87). The two stage nested PCR amplification
and melt curve detection strategy is very similar to that employed in the MT-PCR
utilised in the AusDiagnostics kits, but in a totally closed system. The construction of
the nanofluidic pouch is illustrated in Figure 1.
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The FilmArray GI Panel allows rapid simultaneous identification of 26
enteropathogens as summarised in Table 2. Testing requires minimal preprocessing of specimens. The stool sample is diluted 1:10 in Cary Blair media,
subsequently filtered by gravity-flow through a coarse strainer, and loaded into the
FilmArray GI pouch using a novel filter-injection vial (245). An automated report
listing all of the pathogens detected is ready approximately one hour later.
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Vaughan et al. presented data from clinical testing results of 620 sequential patient
stool samples submitted to the Primary Children’s Medical Center laboratory (247).
From the 620 stool investigations by conventional methodologies 13.7% of patients
were diagnosed with a causative pathogen and 86.3% remained undiagnosed (247).
The FilmArray GI panel was then utilised on 118 samples out of the 620 submitted.
The 118 samples comprised 43 known positive samples and 75 samples negative
by the conventional methods in use (247). FilmArray GI panel testing detected 95
organisms compared to 45 detected by standard clinical methods and as ordered by
the patients clinician (247). The authors attributed the increased rate of positives to
two factors: improved detection sensitivity of the FilmArray GI Panel for
enteropathogens, and the increased range of enteropathogens in the panel some of
which were not requested or offered in the routine workup at that laboratory. Overall
concordance of the FilmArray system and conventional methods was reported as
97% (247). In 30% of cases multiple pathogens were detected, and 20 of the 75
previously undiagnosed specimens were found positive with at least one pathogen
using the FilmArray GI Panel, leading to a potential 26% increase in pathogen
detection (247).
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Harrel et al. presented data specific to the performance of the FlimArray GI panel for
viral enteric disease using a set of previously characterised samples and also
prospective clinical samples (245). Assessment of the FilmArray GI Panel was
made in comparison to the conventional methods used in the clinical laboratory.
Specimens previously determined to be positive for viruses were re-tested using the
Film Array GI Panel. 66/69 (95.7%) RT-PCR confirmed Norovirus samples (36
Norovirus GI and 33 Norovirus GII) were identified as Norovirus by the FilmArray GI
Panel (245). 19/22 (86.4%) Rotavirus EIA positive faecal specimens were identified
as Rotavirus by the FilmArray GI Panel (245). The authors suggested that sample
degradation during storage could be responsible for the false negatives observed.
The second component of the evaluation utilised randomly selected clinical samples
obtained from children with undiagnosed diarrhoea. All samples were tested with the
available conventional methodology in the laboratory but these did not cover all of
the viruses present in the FilmArray GI Panel (245). All five viruses were detected,
Rotavirus 4 positives/134 tests, Adenovirus 6/150, Norovirus 26/210, Astrovirus 2/97
and Sapovirus 5/88 from the clinical samples (245). Compared to the routine testing
performed in the laboratory the data suggested that the Film Array GI Panel would
potentially improve viral enteropathogen detection by an additional ~25% in children
who remained undiagnosed by existing common conventional methods (245). The
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multi-target testing available in the FilmArray GI panel also indicated that multiple
infections were common and these were again found in about 30% of clinical
specimens (245).
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In a separate study, Harrel et al. reported the performance of the FilmArray GI Panel
on samples submitted for assessment of C. difficile alone, or in combination with
requests for other pathogens. In the 406 samples submitted for C. difficile testing
alone, 74 were determined as positive by the Illumigene C. difficile LAMP assay
(Meridian Diagnostics). 83 of the 406 specimens, comprising 35 positive and 48
negative specimens by the Illumigene method were retested with the FilmArray GI
panel and showed 100% concordance. In this sample set of 83, the authors also
reported the detection of 16 other enteropathogens in the 48 samples that were
negative for C. difficile by using the Film Array GI panel. The pathogens detected in
the C. difficile negative samples included Aeromonas , Campylobacter, EPEC (eae),
Giardia intestinalis, Astrovirus, Norovirus, Rotavirus, Sapovirus and ETEC (lt/st).
Interestingly in 15 of the 35 C. difficile positive samples co-infections were also
detected by the FilmArray GI Panel. Additional enteropathogens detected in the C.
difficile positive subset included Adenovirus F 40/41, Campylobacter, EAggEC (pAA
Plasmid), EPEC (eae +/- bfpA), Norovirus, Plesiomonas, Salmonella and Sapovirus.
The authors argued that the inclusion of C. difficile into the FilmArray GI panel was
clearly warranted given that another enteropathogen was detected in 19.4% of
samples which were originally only requested for C. difficile testing (136).
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Asymptomatic carriage rates of toxigenic C. difficile are contentious but have been
reported from 4-20% in adults dependent on the population and technology utilised
to determine the data (249). Interestingly in this data set, 18% (15/83) of the samples
tested showed co-infection of toxigenic C. difficile with other recognised
enteropathogens. It is worth noting that the laboratory performing the evaluations is
the ‘Primary Childrens Medical Center Laboratory’. As 49.7% of the samples tested
were reported to be from patients <6 years of age (136), a bias to samples collected
from children may reflect the origin of the findings. Asymptomatic carriage of C.
difficile has been reported at levels from 75% to 23.5% in infants and children at 1
year of age and 5 years of age respectively (135).
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Whilst the FilmArray GI Panel product offers a very comprehensive suite of targets
and a very simple workflow incorporating the nucleic acid extraction component,
throughput is limited at just one pouch per instrument per hour. Due to this, it is
unlikely that the product could be utilised in larger diagnostic laboratories as a
primary screening methodology without acquiring significant numbers of instruments.
The product would be very useful as a comprehensive ‘stat’ method for urgent
samples, especially within larger laboratories using batched workflows when the
sample might not be able to be tested immediately. Currently our laboratory utilises
a variety of lateral flow immunoassay devices to provide urgent results for toxigenic
C. difficile prior to subsequent PCR follow up, and also for Rotavirus and Adenovirus
testing as required in-lieu of ELISA testing in normal batch process. At present the
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FilmArray products are not yet available in Australia. Cost per test for the FilmArray
system and test kits is expected to be $150 AUD per test and $50,000 AUD for the
instrument platform. Pricing may be revised now that BioFire has been acquired by
bioMerieux.
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Potentially Adaptable ‘High’ Multi Analyte Molecular Technologies
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Existing commercial multi analyte assays for gastrointestinal pathogens range from
eight to 26 targets, and cover up to 95% of the pathogens usually reported. There
are a much greater possible number of known enteropathogens than 26. This
effectively means that using the existing commercial test kit with the highest
coverage would still leave a number of patients for whom conventional methodology
potentially would have reported an enteropathogen being issued a result of no
pathogens detected by the molecular kit. Depending on the multi analyte kit utilised
this ‘missed’ enteropathogen may be bacterial, viral or parasitic.
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Faecal parasite concentration microscopy is labour intensive and has comparatively
poor sensitivity to molecular amplification technologies. Microscopy can however
detect a vast range of parasites by operator skill and visual observation alone. The
highly sensitive molecular assays are limited to reporting only those organisms that
have specific primers/probes included in the assay. At the moment it can be argued
that existing multi analyte nucleic acid amplification kits do not offer sufficient
parasite coverage to supplant parasite microscopy Arguments also exist for the
need to incorporate detection of non pathogenic protozoa as well. Detection of nonpathogenic intestinal protozoa in microscopy and the reporting of these is common.
Many laboratories attach report comments highlighting that the organisms are nonpathogenic or of unknown significance. Opinion is divided in regards reporting nonpathogenic protozoa. Some argue that these should be reported because the
acquisition of the protozoa is generally via the same faecal-oral route as pathogens
and detection in symptomatic patients indicates possible exposure to faecal material
warranting further examination for known enteropathogens, and unknown viral
enteropathogens (3, 179). Routine reporting of non-pathogens also ensures
laboratory staff maintain expertise in differentiating pathogenic from non pathogenic
protozoa (250). Others contend that reporting of non pathogenic protozoa actually
complicates patient management and doing so incurs significant health costs in
unnecessary patient follow up, be that clinician or patient driven (250, 251).
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Molecular assays offer improved sensitivity, simplified workflow, and reduced time to
results. However, in order to offer appropriately comprehensive enteropathogen
diagnostics using molecular assays the use of alternate molecular technologies will
be required. Many of these technologies have been developed for use in human
genetics research but are adaptable to microbial diagnostics. Until recently, use of
these products in pathology diagnostics was prohibitively expensive. This has
recently changed and as summarised in Table 5, if a laboratory has sufficient scale
the cost per patient for testing as many as 80 individual enteropathogen targets
using nucleic acid amplification technology on these systems has reduced to <$25
AUD. The capital expenditure for the equipment required has also dropped
commensurately.
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A considerable amount of literature exists utilising microarray technology in clinical
microbiology and the many and varied uses are summarised in the review of Miller
and Tang (252), and the papers by Booth et al. (253) and Versalovic (254). The
liquid array of the Luminex platform and its commercial application as the xTAG
Gastrointestinal Pathogen Panel has already been discussed. A number of authors
have published work on the detection of bacterial enteropathogens using universal
primer sets in the bacterial 16S and 23S ribosomal RNA genes and intervening
spacer sequences with post amplification hybridisation detection on solid
microarrays tiled with organism specific probes (82, 255, 256). One author has
utilised virulence associated gene targets with microarray detection to good effect
(84). Although care is required as there is evidence that virulence determinants are
not necessarily specific to defined enteropathogenic bacteria. Examples relating to
virulence determinants associated with enteropathogenic strains of E. coli being
detected in other enteric flora include the detection of the eae gene in Escherichia
albertii and Citrobacter rodentium; the stx2 gene in Enterobacter cloacae and
Citrobacter freundii, stx1 and stx2 in Aeromonads, and O157 antigen expression in
Citrobacter freundii, Citrobacter sedlakii, and Escherichia hermanii (96, 110, 257260). Earlier publications with solid phase microarray approaches showed a
detection sensitivity in the order of 103 to 105 CFU/mL and a resultant correlation of
only 80% with culture techniques (255). A more recent publication testing 1700 stool
samples has shown a similar detection sensitivity of 103 CFU/mL but in this study all
440 culture positive samples were also detected by the microarray method, as well
as an additional 51 positive results for which culture was negative (256). Of interest
29 of these 51 were identified by the microarray as Shigella spp., and it is known that
the differences between E. coli and Shigella in particular in the ribosomal gene
sequences are minimal. Solid phase microarray platforms in current form are viewed
as unlikely to be suitable for high volume routine use in a diagnostic laboratory as
detection platforms for amplified nucleic acids. This is due to a number of potential
problems. The current high cost of the microarrays themselves, the limits on depth
of multiplexing possible in the amplification component whilst preserving appropriate
compatibility of the resultant amplicons to stringency parameters for the hybridisation
processes, and finally the ongoing contamination risk due to requirement for open
tube manipulation of amplicons for hybridisation onto the arrays.
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A large amount of work has been undertaken towards profiling the microbial
biosphere and the human microbiome including the human intestinal tract using a
variety of techniques (261-266). Several interesting clinical applications have
emerged from this work, in particular culture independent approaches to
identification of organisms (3, 6, 267). Metagenomic approaches based on
sequence independent PCR amplification and sequencing of 384 clones per sample
have been successful in identifying existing, highly divergent and also novel viruses
from faecal samples of 12 symptomatic patients (3). The review article by Barnard et
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al. summarises the applications evolving for detection of novel viruses directly from
clinical materials (4). A commercial system the Plex-ID (Abbott-IBIS) exists for
profiling samples for unknown organisms. The PLEX-ID Analyzer is a high
resolution electrospray ionization mass spectrometry (ESI-MS) instrument which
uses multi-locus nucleic acid base composition analysis based on combined mass
assessment of amplicons for microbial identification (5). At present the current cost
excludes the use of this platform for routine enteropathogen diagnostics.
Underpinning much of the future technology in profiling microbial content in clinical
samples is improvements in nucleic acid sequencing using next generation high
throughput sequencing technologies. Leveraging the depth of sequencing available
in next generation sequencing technologies allows determination of the bacterial
population either directly or via selective enrichment of pathogens by specific preamplification (268-270). However, the direct application of these technologies to
routine enteropathogen diagnostics is at present too costly, has insufficient
throughput and requires significant computational analysis and associated
information technology resources. Delivering several hundred patient results per day
in clinically relevant time frames is not currently realistic, though it is worth noting
that the technology is rapidly advancing.
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Amongst the currently available adaptable new technology platforms that can offer
appropriate numbers of target enteropathogens at an appropriate price point and
which are suited to current diagnostic laboratories, three genetic technologies
appear to meet the requirements. The three systems and relevant performance
parameters for this application are summarised in Table 5. As test scale increases
so does risk and the resultant cost of assay failure. Table 5 summarises the
potential lost testing as a percentage of the daily workload should an initial batch of
testing fail for some reason for each of the three systems on which it is currently
viable to perform 96 samples for 80 targets in a realistic processing time.
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Life Technologies Open Array Plate and QuantStudio 12K Flex
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OpenArray technology utilizes a microscope slide-sized plate with 3,072 through
holes as shown in Figure 2. Each plate contains 48 sub arrays with 64 through holes
and each through-hole is 300μm in diameter and 300μm in depth. Through holes are
coated with hydrophilic and hydrophobic coatings. Plates with user defined Taqman
assays are manufactured by Life Technologies and dried down into the appropriate
through holes. Liquid reagents, which are the mastermix base and the patient
nucleic acids are then added to complete the PCR reaction and are retained in the
through holes via surface tension. QuantStudio 12K Flex OpenArray plates come
encased with an alloy base to assist with handling of the array without touching the
through-holes prior to sealing and filling the case with immersion fluid and loading
onto the QuantStudio 12K Flex instrument for amplification and detection. Four
individual OpenArray plates can be run in a single run on the QuantStudio 12K Flex
system, which equates to 32 traditional 384-well qPCR plates (12,000 data points).
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Assay time on the QuantStudio 12K Flex system is 2.5 hours. The use of a
specialised liquid handler called the AccuFill System is required to dispense master
mix and patient sample nucleic acid onto the appropriate number of target wells for
the assays required for that sample. The final reaction volume is 33nL and the
manufacturer claims this has been shown to enable 7 logs of dynamic detection
range and a sensitivity to 1 copy (271). The nanolitre scale reaction volume enables
low cost for multiple reaction targets whilst still using the reliable performance of
Taqman probe based nucleic acid amplification and detection. The ‘128’ format
array plate is utilised to obtain up to 128 singleplex Taqman q-PCR enteropathogen
targets for 24 patients per slide, and with 4 slides able to be loaded on to the
QuantStudio 12K Flex at the same time. This equates to a throughput of 96 patients
tested for up to 128 targets by Taqman PCR chemistry every 2.5hours. Costing of
format 128 OpenArray plates and reagents for detecting 80 targets based on
volumes of 250 patient samples a day, 6 days a week have been quoted as 20cents
per data point which equates to <$22 per patient. An example of 80 possible
enteropathogen targets is provided in Table 1. Being able to expand the parasite
coverage economically is a key feature of this technology and suggests that it is
possible to realistically replace conventional microscopy of faeces concentrates or
fixed smears.
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The QuantStudio 12K Flex system also has the capability of interchangeable blocks
allowing the use of 384-well TaqMan Array Cards, and TaqMan assays in 96 and
384 well plates. Life Technologies have recently presented data at the ECCMID
conference on the use of a TaqMan array card developed by their company for the
detection of 16 enteropathogens. Van Hannen et al. described the use of the
microfluidic TaqMan Array molecular device to detect these 16 enteropathogens
(272) . The authors assessed the real-time PCR assays in the card against several
hundred clinical stool samples and compared the results to bacterial culture and
microscopy (272). The assay was also compared directly to a set of in-house PCR
assays for 103 of the samples. The enteropathogen targets contained in the array
were 3 viruses (Adenovirus, Norovirus and Rotavirus), 8 bacteria (STEC, Shigella,
Salmonella, C. coli, C. lari, C. jejuni, Y. enterocolitica, C. difficile) and 5 parasites
(Giardia intestinalis, Entamoeba histolytica, Cryptosporidium parvum, Dientamoeba
fragilis and Blastocystis hominis) (272). Assays were designed with the assistance
of Life Technologies who used specific assay design software (272). Internal
controls for inhibition and/or inefficient nucleic acid extraction were included. Three
co-extracted seeded internal control materials were used, the cyanobacterium
Synechococcus, RNA Phocine Distemper Virus and the DNA Phocine Herpesvirus
(272). The authors first tested the assay designs in 96 well plate formats for
sensitivity and specificity against clinical isolates after which the assay designs with
acceptable performance were spotted on 384 well, microfluidic cards (272). Results
showed that the TaqMan Array Card was more sensitive than culture and
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microscopy and correlated well to the in-house PCR assays (272). Life
Technologies is currently working with our institution on a 32 target OpenArray plate
format enteropathogen panel to be used as proof of principle for a subsequent 80
target enteric OpenArray 128 format solution.
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Fluidigm Genetic Analysis System with 96.96 Dynamic Array IFC and
BiomarkHD
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A miniaturised microfluidic circuit in the form of a dynamic array suitable for nanolitre
scale qPCR testing is available from Fluidigm. The Integrated Fluidic Circuits (IFC)
are available in a variety of formats including a 96.96 IFC which allows combining up
to 96 patient samples or controls with up to 96 individual 5’ nuclease assays in
singleplex or up to triplex formulation within the space of the array. Dynamic Array
IFCs have an on-chip network of microfluidic channels, chambers and valves that
automatically assemble individual PCR reactions (273). Figure 3 shows the
construction of the IFC 96.96 product. The IFC requires filling in a specialised
loading instrument called the IFC-HX IFC Controller which takes 80 minutes (273).
Once the IFC is loaded the amplification and fluorescence based detection take
place in a second instrument called the BioMark HD System in real-time or in end
point modes which takes a further 60 minutes (273).
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Reaction volume is lower again in this system at 6nL which offers 5.5logs of dynamic
range (273). The drop of precision correlates with decreasing reaction volumes due
to stochastic limitations as defined by the Poisson distribution, i.e. by the application
of small volumes of patient nucleic acid these samples may simply by chance not
contain the target of interest if it is also present at limited concentration in the nucleic
acid extract (274). The principle just described underpins the use of ‘Digital PCR’ on
these platforms as a quantitative methodology which does not require comparison to
internal or external standards (275, 276).
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The disadvantage of this system is the requirement to pre-prepare up to 96 master
mixes and then load these to one side of the IFC chip, and then to load up to 96
patient samples to the other side of the IFC chip. Whilst the IFC-HX controller
conducts the filling and mixing process this still equates to a significant amount of
pipetting and construction of PCR mastermixes for use on the IFC plate. The
additional pipetting required extends the time required from assay setup to result to
around 5 hours. The costs provided by the supplier indicate a price point between
$15 and $18 AUD per patient for up to 96 targets. Capital costs are in the order of
$480,000 AUD per system including the IFC HX Controller and Biomark HD
instrumentation.
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Seqenom MassARRAY Genetic Analysis System in 384 well PCR Plate format
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Sequenom have a high-resolution mass spectrometry solution for characterisation of
nucleic acids called the MassARRAY System. Combined with software called iSEQ,
an analysis of sets of highly multiplexed PCR amplicons is possible following
extension of a target specific probe and measurement of the specific probe mass in
the MALDI-TOF instrument. In genetics the application effectively reports SNP
information at the 3’ end of the probe location. In this application a given PCR
amplicon derived from the large homogenous multiplex reactions and to which a
target specific probe needs to hybridize onto for single base extension will either be
present (positive for the enteropathogen) or not present (negative for the
enteropathogen). The compatibility of the amplification primers as well as the
designs of the probes for the specific target amplicons is managed in the design
phase by specific assay design software. This software is capable of creating
compatible single reaction multiplex assay designs of up to 29plex. Even at such
high multiplexing, the use of appropriate assay design preserves the ability of the
MALDI-TOF system to distinguish each extension probe specifically by its possible
full-length masses.
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In order to achieve the specific aims of 80 enteric targets, three multiplex reactions at
29plex would provide up to 87 enteric PCR targets per patient. Using 384 scale
microwell plates at three reaction wells per patient allows up to 128 patient samples
or controls per 384 well microplate for 87 targets. Conventional thermocyclers with
384 compatible blocks are utilised. The amplification reaction volumes are 5µL and
as there are no fluorescent dyes required by this technology the costs of the
amplification component are comparatively low. The manufacturer has quoted a
price of between $10 and $12 AUD per patient for up to 87 enteric microbial targets
based on the volume of patient testing previously described.
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To adapt the MassARRAY workflow to direct detection of microorganisms there are
important additional considerations relating to the design of the amplification and
probe extension components. Optimal microbial targets for detection are those
which are specific to the intended target only and where possible those that are also
multicopy to enhance sensitivity. The use of multicopy genetic targets can be
problematic, as often these targets comprise part of mobile genetic elements such as
insertion sequences or plasmids. An unintended consequence of using mobile
genetic elements is that they may at some point in time transfer to other species,
could already be present in other species that have not yet been recognised, and
indeed could also potentially even be lost altogether from certain strains of the target
organism. Examples of these events have been observed on a number of occasions
where molecular techniques have been applied to microbial diagnostics. Examples
include loss of the cryptic plasmid of Neisseria gonorrhoeae containing the cppB
target resulting in false negative results (277). False positives being reported due to
the use of the cytosine DNA Methyl transferase cmt gene target in N. gonorrhoeae
that was found to also have homology to other Neisseria species that were transient
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colonisers of urogenital sites (234). False positives reported by using the IS481
multicopy insertion sequence for detecting Bordetella pertussis that was later found
to be present in B. holmesii and certain strains of B. bronchiseptica (278, 279).
Structural defects that emerge can also result in false negative results where
sequence might be deleted in certain strains of the target organism. Such a deletion
was observed in the Chlamydia trachomatis cryptic plasmid resulting in false
negative results for many commercial assays (280, 281).
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The specificity of the nucleic acid amplification technologies is underpinned by the
base content of primers and probes and the ‘pair bonding” of these bases specifically
to the intended target sequence. In general, the combination of 20 or more base
pairs from each of two primers and a probe interrogates upwards of 60 bases of
information contributing to the pair-bonding responsible for amplification and
detection to successfully occur. Assay stringency controlled via the temperature of
annealing, cation concentrations and buffering work to ensure that in a well
optimised PCR reaction this will reflect in the outcome of amplification of the
intended target only. The reality is however that detection assays can generally
tolerate minor single base mismatches of sequence information under the primers
and/or under longer probes. This generally results in delayed cycle threshold values
for real time PCR. Mismatches to target template at the 3’ ends of primers are not
tolerated well and generally will prevent amplification.
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In respect of the MassARRAY assay design, the amplification reaction and the probe
could be expected to confer similar specificity and tolerance of mismatches to other
nucleic acid amplification technologies, however the MassARRAY result
interpretation relies heavily on the single base extension reaction. Since the
identification of this single base is what determines the final mass of the analyte in
the flight tube, it is in effect the arbiter of whether a result is scored as positive or
negative for the microorganism. Temporal variation in the local and global
epidemiology of the organism is also an important consideration when validating
molecular assays. A reliance on a single extension base to determine a specific
identification requires that additional assay validation steps be performed so that
there is appropriate confidence in the reliable conservation of that base at that
genetic location. The examination of sequence data from genomic databases should
be performed as should sequencing of organisms sourced from divergent locales
and from different chronological periods. Closely related organisms should also be
thoroughly examined for cross homology at the target region for the assay.
Performing these investigations in the design phase is important so that there is
confidence that the sequence targeted in general and the location of the single base
extension more specifically are both sufficiently robust as to provide reliable results.
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Despite the extra rigour required in the design phase, the major problem with the
MassARRAY approach is that the process also takes much longer than the other
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technologies. The process is described in Figure 4 and it requires up to 570 minutes
(9.5 hours) from PCR setup to the first batch of 128 patient results despite the use of
liquid handling robotics (MassARRAY Liquid Handler) in the time consuming iterative
primer extension process. Nanodispensing to the MALDI-TOF SpectroCHIP (Figure
5) is performed by the MassARRAY RS1000 Nanodispenser. The MassARRAY
RS1000 Nanodispenser can hold up to two 384-well microplates, and can dispense
all of 384 samples to the SpectroCHIP in less than 10 minutes. The actual MALDITOF mass spectrometry takes only 45 minutes. Whilst the total time to the first
batch of results is quite long, overlapping setup using staggered amplifications in
multiple thermalcyclers and the speed of the Nanodispenser and mass spectrometry
allow the full workload to be achieved in a ten hour time frame. This is similar to the
overall time taken for the daily workload on the other two platforms. There is
however a considerable risk that if the first batch fails for some reason then
troubleshooting and repeating is both costly and almost time prohibitive at 9.5 hours.
When using overlapping batch processing, if the same problem is evident in other
batches in progress, then the entire days workload might be lost by the time the
results of the first batch indicate a problem. The possibility of backlog might require
that two entire systems running from entirely separate reagent lots would be needed
for redundancy.
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Healthcare Cost Implications in the Australian Context
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Funding of pathology testing is under increasing pressure, cost containment
measures including fee schedule freezes, tendering of service provision and caps on
testing or growth rate have all been implemented in response to pressures on
healthcare budgets around the world (282). The great proportion of diagnosis of
gastrointestinal disease in Australia occurs in the private sector based on community
disease burden. Reimbursement of private pathology testing in the Australian
context primarily occurs via recovery of payments for service from the federal
government under a taxation funded national health insurance scheme called
Medicare. The Medicare benefits paid per service from January 2000 to June 2008
grew by 6%, in the same time period average weekly earnings grew by 48% and the
Consumer Price Index by 32% (283). In Australia the lack of reimbursement growth
in real terms has contributed to the merger of many individual pathology companies
into large market listed private entities where economies of scale can be achieved.
Economies of scale include the heavy use of automation and access to supplier
volume discounting that are justifiable only by access to sufficient test volumes.
Public sector pathology services have also seen an increase in ‘centralisation’ of
services in order to achieve appropriate efficiencies associated with combining test
volume but are primarily funded via State Governments for public patient services.
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Diagnostic technology that delivers more with the same (or less) funding will indeed
become of further importance due to projections of significant retirement in the
workforce and an increasingly ageing population placing increasing demands on the
health care sector (284).
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The funding of gastrointestinal disease diagnostics in Australia is facilitated within
Medicare by using a number of benefit ‘items’. The 85% rate is the rate normally
recovered by private pathology laboratories for testing performed and reimbursed
under these items. Four items in particular are of relevance in regards conventional
diagnostic techniques performed on stool for the purposes of detecting
enteropathogens.
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ConventionalTechnology Items:
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Item 69300
Microscopy of wet film material other than blood, from 1 or more sites, obtained directly
from a patient (not cultures) including:
(a) differential cell count (if performed); or
(b) examination for dermatophytes; or
(c) dark ground illumination; or
(d) stained preparation or preparations using any relevant stain or stains;
1 or more tests
Fee: $12.60 Benefit: 75% = $9.45 85% = $10.75
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Item 69336
Microscopy of faeces for ova, cysts and parasites that must include a concentration
technique, and the use of fixed stains or antigen detection for cryptosporidia and giardia including (if performed) a service mentioned in item 69300 - 1 of this item in any 7 day
period
Fee: $33.65 Benefit: 75% = $25.25 85% = $28.65
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Item 69339
Microscopy of faeces for ova, cysts and parasites using concentration techniques examined
subsequent to item 69336 on a separately collected and identified specimen collected within
7 days of the examination described in 69336 - 1 examination in any 7 day period
Fee: $19.25 Benefit: 75% = $14.45 85% = $16.40
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Item 69345
Culture and (if performed) microscopy without concentration techniques of faeces for faecal
pathogens, using at least 2 selective or enrichment media and culture in at least 2 different
atmospheres including (if performed):
(a) pathogen identification and antibiotic susceptibility testing; and
(b) the detection of clostridial toxins; and
(c) a service described in item 69300;
- 1 examination in any 7 day period
Fee: $53.25 Benefit: 75% = $39.95 85% = $45.30
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The reimbursement available for the standard workup of a stool submitted for
analysis for Micro/Culture/Sensitivity and Ova/Cysts/Parasites amounts to $73.95
(Item 69336 + Item 69345).
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Three items in particular are of relevance in regards utilising Molecular diagnostic
techniques performed on stool for the purposes of detecting enteropathogens.
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Molecular Technology Items
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Item 69494
Detection of a virus or microbial antigen or microbial nucleic acid (not elsewhere specified)
1 test
Fee: $28.85 Benefit: 75% = $21.65 85% = $24.55
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Item 69496
3 or more tests described in 69494
Item 69495
2 tests described in 69494
Fee: $36.10 Benefit: 75% = $27.10 85% = $30.70
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Fee: $43.35 Benefit: 75% = $32.55 85% = $36.85
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The reimbursement available for three or more targets in a multi analyte molecular
diagnostic assay for enteropathogens is defined by the item 69496 and is $36.85.
This calculates as a difference of $37.10 in funding between the conventional
techniques for enteropathogen detection of culture and parasite concentration, and
the available funding for a multi analyte molecular technique.
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At the moment diagnostic molecular testing in Australian laboratories as applied to
gastrointestinal pathogens is primarily supplemental and provided by a limited set of
in-house assays at a cost base of about $10-16 for a single reaction (singleplex or in
multiplex up to a maximum of 4 or 5 targets). Reimbursement for the molecular
testing is then obtained from items 69494 – 69496 dependent on the number of
targets to a maximum of three. The use of in-house PCR testing even in multiplex
format is therefore cost-prohibitive if attempting to examine a large number of
enteropathogens. When more than three to four reaction tubes per patient are
utilised the cost will exceed the reimbursement available under the MBS microbial
nucleic acid detection items.
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As described in Table 2, current commercially available multi analyte molecular
based enteropathogen diagnostics range in cost from $25 to $51 with variable
enteropathogen coverage. From the currently available kits arguably the EasyScreen product at $51 per test matches most closely the scope of pathogens
regularly detected by the conventional methodologies but at $51 the cost per test
exceeds the remuneration available.
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On that basis the use of multiplex PCR assays on existing common molecular
equipment either has too restricted a scope of pathogens and/or is too costly
compared to conventional techniques of microscopy/culture and antigen detection to
be viewed as a suitable replacement technology.
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There is also a reticence by patients to pay any premium ‘co-payment’ for pathology
testing, and bulk billing to Medicare item fees without patient contribution occurs for
90% of pathology tests performed (283). Though there is precedent for patient
contributions where referrers and patients can clearly perceive value. The take up of
liquid based cytology (Thinprep, SurePath) for cervical screening which does not
have a Medicare reimbursement item and is an additional cost compared to
conventional Papanicolaou screening has proved that if a particular testing modality
is seen to provide improved diagnostic value, referrers will recommend such testing.
And importantly more than 25% of patients will accept additional out of pocket costs
of up to $40 for this testing (285). To improve the value proposition for referrers
and patients in enteropathogen diagnostics two improvements are required.
Matching and extending the range of enteropathogen targets beyond what is
currently routinely reported. And achieving significant improvement in the time to
results. If those two challenges can be met it might be possible that such an assay
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would become preferred by referring practitioners and be recommended to patients
as offering improved value perhaps worthy of an additional cost. Recent advances
in molecular technology look promising for delivering this ideal solution of an
improved range of enteropathogens detected in a significantly reduced time frame
and potentially within the available molecular funding available under Medicare Item
69496 ($36.85).
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The price per patient sample based on 80 PCR targets performed in acceptable time
frames using the previously described adaptable high throughput, ‘high’ multi analyte
molecular technologies has been calculated based on volumes of 250 patient
samples per day, six days of the week. The cost/patient test using these
technologies is $25 or less, and under the $36.85 maximal funding available for
multiple microbial nucleic acid detection. In the context of the growing fiscal
pressures on healthcare budgets, delivering routine detection of both existing and
additional enteropathogens with higher sensitivity, in shorter time frames and at
lower cost appears to meet many aims.
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Aside from standalone use of the molecular technology, a proposed model of parallel
multi analyte molecular testing with concurrent minimised bacterial culture is
proposed in a later section of this paper. The primary rationale for maintaining a low
cost parallelised bacterial culture system is recovery of isolates for epidemiological
use and sensitivity testing. In such a dual scenario the minimum criteria required of
the bacterial culture item 68345 - culture in two different atmospheres and wet prep
analysis could also be achieved, but with the primary diagnostic screen being a ‘high’
multi analyte molecular test. Funding for such an algorithm could also then be
recovered from the MBS Item 68435 at $45.30. The additional remuneration in this
model would offset the costs of labour and additional processing required for
performance of simple wet preparation and reflex culturing bacterial enteropathogen
PCR positive samples.
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Government funding models eventually adapt to the emergence and use of new
technology, but usually take time. Governments would need to determine by costbenefit analysis if the additional costs of bacterial isolate recovery alongside
molecular screening justified this additional expenditure.
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Nucleic Acid Extraction from Stool
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Use of any nucleic acid based technology necessarily requires the availability of the
target nucleic acids from sample material. Direct application of nucleic acid based
reporting probes to cells for in-situ hybridisation techniques such as oligonucleotide
or peptide nucleic acid FISH requires only physical preparation of cells on slides.
Free nucleic acid may be present in any sample, but optimal diagnostic utility when
using nucleic acid amplification technologies requires specifically recovering intra
organism DNA and/or RNA. The nucleic acids must also be recovered in a
sufficiently intact state for later enzymatic amplification processes. They should also
be recovered free from inhibitors of amplification, and in a format that preserves
them from degradation. The extraction may also serve as a process that
concentrates the nucleic acids relative to the abundance in the original sample
processed.
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Composition of the Stool Matrix and Inhibition
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Recovery of nucleic acids from stool poses some particularly unique challenges.
The stool matrix itself is comprised of 75% water and 25% solid matter in normal
stool. The solid matter is comprised of 30% as bacteria, 30% as food matter (1020% as fats), 10-20% inorganics, 2-3% protein, and the remainder derives from the
patient (cells, bile, secretions, leucocytes, bilirubin) (286). PCR can be inhibited by
known inhibitors in stool specimens. Bile acids, bilirubins, haem, pectins, phenolic
compounds, glycogen and complex carbohydrates have been reported as potential
inhibitors (286, 287). Clearly many inhibitors of PCR amplification are prevalent in
stool. Macro sized particulates that remain in solutions prepared from stool can also
block liquid handling pipettes and columns that are utilised in the nucleic acid
extraction processes. Blocked pipettor tips and columns can result in failure of
extractions or reduced yields of nucleic acids. Diluting the nucleic acid extracts prior
to testing has also been described as a method for removing inhibition(286). The
dilutions performed reduce the concentration of inhibitory substances in the extract
to a level where they are no longer inhibitory to the PCR process. However diluting
out inhibitors also carries the risk of diluting out the intended diagnostic target nucleic
acid below the limit of detection of the assay resulting in a false negative result.
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Processing of Stool
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Processing of faeces in a pathology lab is normally performed within the
Microbiology laboratory. Molecular testing for toxigenic C. difficile has contributed to
processing of stool in molecular laboratories also becoming common in recent years.
Stool extraction protocols that simplify and/or limit the hands-on component offer
improved workflow in the molecular laboratory. One of the techniques that assists
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handling is the use of ‘flocked’ nylon swabs as a tool for sub sampling from faeces
collection jars. This provides a sample format (i.e. a swab) that molecular
technicians are already very comfortable processing. Diatherix Laboratories who
offer a ‘Fed-Ex’ in sample service in Huntsville AL utilise the provision of E-Swab
(Copan, Spain) collection in transport media either from rectal swab or from a swab
collected from a stool sample, subsequently testing 11 enteric pathogens via a TEM
(Target Enriched Multiplex) PCR (288). Genetic Signatures provides a flocked swab
for sampling from the stool collection into proprietary extraction buffer utilised to
produce nucleic acid for 3base molecular testing of stool samples (138). Despite
increasing use of stool swabs, most of the commonly available stool specific
extraction methods utilise an input sample of 200mg of stool or 200uL liquid stool
into a buffer.
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It is important to recognise that stool does not necessarily represent the actual
infection site in the intestine but is an excretion vehicle that delivers a convenient
sample for labs to test. As such a stool sample may not carry consistent numbers of
causative enteropathogens dependent on the diarrhoea frequency and volume.
Additionally the enteric pathogens within the stool matrix from which we wish to
extract nucleic acids are diverse in structure and form. The nucleic acid extraction
process utilised must first be able to disrupt several different varieties of ‘packaging’
i.e. cell and cyst walls and capsids in order to release the nucleic acids. Parasites
can be free living trophozoites, cysts/oocysts or helminth worms and eggs; viruses
can be RNA or DNA and intracellular and/or packaged in capsid protein coats,
bacteria can possess gram negative or gram positive bacterial cell walls with
different amounts of glycopeptide.
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Nucleic Acid Extraction Chemistry
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The protocols for the extraction of nucleic acids from stool can be divided into two
distinct modalities. The first category are those relying on variants of the protocol
first described by Boom et.al. (242). Such protocols utilise buffered chaotropic
agents such as guanidium thiocyanate to facilitate sample lysis, nuclease
deactivation and nucleic acid binding to silica. This is then followed by several
washing steps with ethanol to remove non nucleic acid components from the
silica/nucleic acid and drying to remove residual ethanol. Release (or elution) of the
nucleic acids from the silica is then performed by washing with a low salt buffer or
water. In the available extraction kits, the silica component is either in a membrane
column format in tubes or in microwells, or as liquid paramagnetic silica beads.
Dependent on the format of the silica, one of either centrifugation, application of
vacuum or magnetic attraction are subsequently utilised to achieve manipulation of
the liquid solutions and silica to extract the nucleic acids. The particulate nature of
some stools can make it difficult to utilise columns/microwell membranes and
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2333
2334
pipettes for aspiration of the sample itself. Pre-clarification of a suspension of stool
using centrifugation after the lysis has been performed has been used as a method
for limiting the particulates carried forward to the binding component of the extraction
(172, 227, 289).
2335
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2337
2338
2339
2340
2341
2342
The second category of extraction from stool is a relatively new methodology reliant
on using direct preparation of a stool sample in a single vessel. This form of
extraction is utilised by the Focus Diagnostics Clostridium difficile assay. The stool
is prepared via swab into a buffer which is centrifuged and a heating step applied.
Supernate is then directly added to the mastermix in the test well. The formulation of
the PCR reaction mix is proprietary but clearly it is lytic, preserves nucleic acids and
appears to be effective at preventing the action of PCR inhibitors present in stool
samples.
2343
2344
2345
Commercially Available Kits and Modifications
2346
2347
2348
2349
2350
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2352
2353
2354
2355
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2357
A non exhaustive list of currently available methods includes the following. Manual
methods such as the QIAamp DNA Stool mini kit, Qiagen, Hilden. Semi automated
methods such as the Qiagen Stool mini kit on the QIACube instrument. And
automated applications of specific kits on magnetic bead instruments such as the
Nuclisens Easymag (bioMérieux), Magnapure32 , Magnapure96( Roche),
KingfisherFlex (Thermo), QIASymphony (Qiagen), EZ-1 (Qiagen), Maxwell
(Promega), kPCR (Siemens) and EpMotion 5075 (Eppendorf). Column/membrane
based kits are used on vacuum manifold instruments such as the Xtractor Gene
(Qiagen) and EpMotion 5075 (Eppendorf). Highest throughput extraction is available
using the Magnapure96 or Kingfisher Flex instruments both of which can purify 96
samples by magnetic bead technology in less than 1 hour from the post-lysis silica
binding step.
2358
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2364
A number of modifications to commercial kits have been utilised that improve
extraction of nucleic acids from stool. The Qiagen DNA Stool mini kit utilises
InhibitEX a proprietary substance to remove impurities prior to the silica binding
component of the extraction(289). Use of chelex resins or polyvinyl pyrrolidone
(PVP) has also been shown to assist in removal of inhibitors of amplification
technologies without loss of target DNA (140, 173, 187, 286).
2365
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2367
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2369
Due to greater difficulty in releasing nucleic acids from enteropathogens with grampositive bacterial cell walls, oocysts and ova some authors have also utilised a
variety of physical disruption methods including freeze/thaw and/or mechanical
disruptions prior to extraction. Zirconia, glass, ceramic or stainless steel particles
and beads utilised in homogenising equipment such as the TissueLyser
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(Qiagen,Hilden), MagNAlyser (Roche, Rotkreuz), FastPrep (MP Biomedicals,
Sydney), Mini Bead Beater-8 (Bio-Spec, Bartlesville, Oklahoma) have been used
(139, 187, 290-293). Similar processing has been applied to disruption of fungal
hyphae for downstream nucleic acid testing (294). By selecting an appropriate
specific gravity of particle and an appropriate oscillation frequency, the impact
between the particles and the organism produces effective disruption without
excessive shearing of nucleic acids.
2377
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2380
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2383
2384
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2386
A dual approach has been suggested by Amar (286) whereby a faecal sample is
processed via mechanical disruption with zirconia beads in a MagNAlyser and
concurrently via a parallel traditional lysis followed by recombination of the two
methods after silica bound nucleic acids have been obtained, with washing and
elution performed on the combined silica. This combined method reportedly caters
for the different robustness of viruses and gram negative bacteria (traditional lysis)
versus that of gram positive bacteria and protozoal oocysts (mechanical disruption)
(286). Successfully utilising a mechanical disruption extraction protocol for viral
enteropathogen detection without loss of target has also been reported in the
literature (139).
2387
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2389
Recovery of RNA from Stool Samples
2390
2391
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2397
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2399
2400
2401
2402
2403
2404
2405
2406
2407
2408
2409
2410
Several important viral enteric infections are caused by RNA viruses e.g. Rotavirus,
Norovirus, Coronavirus, Enterovirus, Astrovirus, Sapovirus and Human
Parechovirus. Successful extraction of RNA from faecal samples requires both an
appreciation for the functionality of the extraction methodology utilised as well as
attention to laboratory equipment and practices. In general, RNA is recovered in
sufficient quantity via co-purifying with DNA to enable appropriate detection of viral
enteric illness. Either DNA or ’Total’ Nucleic acid extraction kits have been utilised
successfully for enteric RNA virus diagnostics (139, 140, 295). Nonetheless,
endogenous RNases are released from cells in clinical samples, and inactivation of
these should occur as soon as possible to prevent RNA degradation. The chaotropic
guanidinium isothiocyante found in the majority of extraction kits is also an effective
inactivator of RNAses, and specific nuclease inhibitor solutions are also available.
After elution of RNA or total nucleic acids, RNA degradation may still occur if
attention to laboratory equipment and processes do not meet an appropriate level of
rigour. It is essential that any item that could contact the purified RNA is RNasefree. Laboratory surfaces and molecular equipment such as pipettors should be
decontaminated with a solution such as RNase Away (Molecular BioProducts).
RNase-free certified pipette tips and other laboratory consumables should be utilised
and staff should be encouraged to change gloves frequently. Conversion of RNA to
cDNA by randomly primed reverse transcriptase protocols as soon as possible after
extraction has also been suggested (286).
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2411
Control of Molecular Gastrointestinal Infection Testing
2412
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2415
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2418
2419
2420
2421
As described in the previous section, stool is a complex matrix containing inhibitory
substances that may co-extract with nucleic acids. Modern extraction chemistries
and instruments have however proven to be very reliable at removing inhibitors
within our laboratory. A rate of <0.4% inhibition has been observed from stool
extracted for Clostridium difficile testing in our laboratory. In total, there were 132
PCR inhibitory stools from 35210 total stools extracted for the period 2007-2011.
These 132 stools remained inhibitory even after nucleic extraction was repeated
indicating that in some patient samples the particular matrix might still rarely result in
PCR inhibition and a failure of nucleic acid testing methods. Appropriate controls to
detect inhibition and assay performance in general are required.
2422
2423
2424
2425
2426
The practice of diagnostic laboratory nucleic acid amplification technology in
Australia is controlled by application of standards from the National Pathology
Accreditation Advisory Council (NPAAC) (296). Several distinct layers of controls
are mandated or recommended and these are consistent with best practice
internationally.
2427
2428
Positive Controls
2429
2430
2431
2432
2433
2434
2435
2436
Positive control material controls for acceptable performance of the testing reagents.
When examined quantitatively via crossing points (which correspond to target
abundance), positive control material may also control for acceptable sensitivity of
the amplification reaction. The use of a positive control near the limit of detection of
the assay is required by the standards applicable in Australia. Failure to amplify a
positive control with a target abundance set near to the limit of detection would
indicate that the current test batch is exhibiting lower than optimal sensitivity and
should be repeated.
2437
2438
2439
2440
2441
2442
2443
2444
2445
2446
2447
2448
2449
Positive controls can be obtained or prepared from a number of potential sources.
Previously positive patient material can be utilised, purified nucleic acid from
cultivated organisms produced within the laboratory or from commercial culture
collections, synthetic nucleic acids such as plasmids, target matching oligonucleotide
sequences and synthetic viral particles can also be produced or obtained
commercially. When selecting or preparing positive control material it is prudent to
incorporate a definable difference that will enable a positive reaction due to the
positive control to be distinguished from a positive reaction from the target analyte.
This is commonly achieved by the positive control material generating a longer
amplicon than the true analyte. Our laboratory routinely includes several Uracil
bases replacing thymine bases in the positive control sequence. Including uracil
bases throughout the positive control material provides the option if the positive
control is suspected to be contaminating other processes that it can be specifically
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removed/destroyed from test wells pre-reaction with the enzyme Uracil-NGlycosylase.
2452
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2455
In event of a positive control testing negative in a test batch, the status of any
positive patient test is not in doubt, but all negative test results need repeat testing
and an investigation of the cause for failure of the positive control is required.
2456
2457
Negative Controls
2458
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2466
Negative control materials are required to be extracted along side the test samples.
These negative controls are utilised as a control measure that confirms that there is
no unexpected positivity in the test batch. Unexpected positivity can have a number
of causes which are all related to contamination events in any of the analytic
process. Contamination between a positive sample and negative samples could
occur in primary sample handling, contamination during sample loading into
extraction methods, contamination during the extraction process, contamination
whilst handling extracts, contamination during PCR assay setup, and contamination
between reaction wells during amplification.
2467
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2469
In addition, contamination of the testing process deriving from positive control
material (as opposed to positive patients in the test batch) is also a consideration if
the negative control is unexpectedly positive.
2470
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2472
It is also possible for the Negative control material itself to become contaminated and
be positive before it is introduced to the current test batch and this needs to be
considered in investigations of unexpected positivity.
2473
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2475
In event a Negative control returns a positive reaction in a test batch, the reliability of
negative results on the test batch are not in question, but all positive results are in
doubt and require investigation of potential causes and repeat testing.
2476
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2479
Negative control material should ideally be of the same matrix as the samples
themselves such that they are handled identically through the full test process. In
most cases the negative material is derived from a pool of patient samples known to
be negative for the analytic target.
2480
2481
No Template Controls
2482
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2485
A ‘No Template Control’ (NTC) is a specific type of negative control that serves
primarily as a sentinel for contamination introduced upon preparation of the PCR
mastermix. The NTC also detects inadvertent cross-contamination during loading of
sample materials during the PCR setup, and also contamination occurring during the
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PCR run, NTCs in most laboratories represent a test well containing PCR
mastermix with molecular grade water substituting for the usual patient sample.
2488
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If all samples are positive on a test run including the NTC this almost certainly
represents contamination of the PCR mastermix or contamination of the entire batch
during amplification.
2491
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2493
If all patient samples and the negative control are positive on a test batch, but the
NTC remains negative this indicates severe contamination of the pre-PCR processes
including sample preparation and extraction.
2494
2495
NTC reaction wells are normally placed between patient test samples and positive
control wells to assist with detection of contamination during PCR loading.
2496
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Co-Extracted Seeded Internal Controls
2499
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2501
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2503
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2505
To prove unequivocally for each patient that a target was truly “Not detected” by an
amplification assay another specific type of control is required. This control is
defined s a Co-Extracted Internal Control, and optimal utility is obtained if it is also
calibrated and seeded at a specific target copy number. The specific target copy
number will produce expected cycle threshold results in the amplification component
that provide valuable information regarding extraction and amplification assay
performance.
2506
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2512
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2522
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2524
The use of the control involves a calibrated seeded internal control target being
placed into the patients sample before the extraction process begins. A specific
amount of ‘seeded co-extracted’ internal control in which the quantity of seeded
material reflects the minimal amount that can routinely be extracted and recovered
as a detectable result with an amplification assay. Co-recovering this nucleic acid
and detecting it in the same well as the patient sample provides the only reliable
confirmation of a patients ‘not detected’ result for a diagnostic target. Using an
internal control in duplex reactions (or greater plex) provides ‘in well’ control over
several performance characteristics of a molecular assay. The control can detect
the presence of total or partial PCR inhibition by either failure to be detected or being
detected with delayed cycle threshold respectively. The co-extracted internal control
can also detect if there has been unacceptably low yield or failed nucleic acid
extraction performance for each patient sample. Total failure of extraction would be
observed as a negative result, and reduced performance of the extraction
methodology would reflect as a delay to the controls cycle threshold. It is not
possible to immediately determine if total or partial failure of the internal control is
due to PCR inhibition or problems with the extraction (or both). Importantly the coextracted internal control is the only control that monitors and controls for events in
the amplification environment in the individual reaction vessel for each patient.
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An important design consideration is that the seeding level of the control and the
robustness of the molecular assay designed to detect the internal control both
require careful development. The assay that detects the internal control should be
designed and optimised such that if the diagnostic target is also present then the
sensitivity of the diagnostic assay(s) should not be compromised by competitive
inhibition from the internal control reaction. In properly designed internal
control/assay combinations, this commonly results in what is a perfectly acceptable
outcome of the Internal Control assay testing as negative due to competitive
inhibition from the diagnostic target assay. In this scenario, a detectable result is
validly reported for the specific diagnostic target even though the internal control has
apparently “failed” albeit by design.
2536
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2538
Control of Reverse Transcription for RNA Targets
2539
2540
2541
2542
2543
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2546
2547
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Multi analyte testing of faeces including viral enteropathogens requires both RNA
and DNA to be recovered and tested as some of the common enteric viruses have
RNA genomes. Consequently, a seeded co-extracted control for both RNA and DNA
should be utilised. It is important to control for recovery of sufficient amplifiable viral
RNA from stool samples. It is also important to control for the acceptable
performance of the enzymes responsible for conversion of RNA to cDNA in the PCR
mastermix prior to DNA amplification. Controls such as MS2 phage(297), phocine or
equine herpes virus(187, 272), phocine distemper virus(272), synthetic armoured
RNA (Asuragen) and bacterial ‘BioBall’ (BTF) have been utilised. All of these
products can be quantified appropriately to establish correct seeding levels.
Importantly all contain nucleic acids that require functional extraction from within
capsids or cell walls which makes them superior to naked DNA or RNA controls such
as constructed plasmids, synthetic gene segments or amplified nucleic acids. .
2552
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2554
Comparative Control to Traditional Methodology
2555
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When incorporating molecular technologies as replacements or adjuncts to the
current processes, comparison with the level of control currently practiced with
traditional methods in microbiology is worth discussing.
2558
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Culture media has pre-release QC performed on sub samples, but individual plates
themselves cannot be confirmed as functional before use. When in use, there are
no controls on each specific plate undergoing the daily incubation/removal for plate
reading/ and return to indicate that during this process it was and indeed remained
possible to cultivate a given organism on that plate.
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Culture reading is reliant on the observational skill of the scientist, and this can be
compromised when other flora (normal or pathogenic) is present due to visibility
difficulties. Active inhibitory effector molecules released from other organisms can
also delay or prevent the growth of pathogens. The sample itself may also contain
bacteriostatic or bactericidal substances delaying or impeding possible growth.
Importantly unlike other disciplines very little if any secondary assessment of an
individuals result interpretation (in this case of what has grown) is performed.
2570
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2573
Results of microscopy are also not checked by a second person and are dependent
on operator skill and fatigue levels. Seeding of a control organism into every faecal
parasite preparation or fixed stain is not performed to indicate the success of each
individual preparation.
2574
2575
Attempts at repeat testing of the sample are likely to provide results that differ due to
cellular degradation, overgrowth, death of labile organisms and so forth.
2576
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2580
ELISA testing for antigenic epitopes of pathogens does not have in built controls of
negative results in each well that prove equivocally the sample was applied correctly
and/or did not contain substances that interfered with the performance of the assay.
Control lines in lateral flow immunoassays do offer some control for correct sample
application.
2581
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2588
Conventional microbiology is highly reliant on viability/growth and morphology
techniques which are applied diagnostically at an ecological interface between
humans and microorganisms. This contrasts significantly to the comparatively well
characterised human cells, extracellular proteins and other biochemical metabolites
which are the relatively static analytes in other pathology disciplines. It is possible to
argue that microscopy and culture for microorganisms are perhaps the least
controlled diagnostic pathology techniques and these techniques are applied to
perhaps the most dynamic analytes i.e. microorganisms.
2589
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2593
Molecular technology though also reliant on the individual training, competency and
performance of staff for optimal outcomes benefits from the many layers of process
controls previously discussed. Due to the controls utilised it is likely the molecular
techniques offer improved confidence in the reliability of results for enteropathogen
testing.
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Potential Workflow Replacing Conventional Testing
2595
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2611
An example of a potential workflow using the three ‘high’ multi analyte molecular
systems is described in Figure 6. A similar approach has been proposed by
Schuurman et al. in relation to pre-culture screening of bacterial enteropathogens by
PCR and reflex culture of positive PCR samples (8). The proposed protocol
described in Figure 6 is likely to be more effective at preserving bacterial
enteropathogens than that proposed by Schuurman et al. The suggested protocol
incorporates molecular screening, a ‘mini’ culture bacterial enteropathogen
maintenance/enrichment system, plus wet preparation faecal microscopy on patient
samples. Viral and parasite PCR results (positive or negative) are issued directly to
the referrer, as are the negative bacterial culture results. Positive PCR testing for a
bacterial enteropathogen initiates a reflex culture from the mini culture tubes onto
conventional agar plates to attempt recovery of the bacterial enteropathogens. The
strategy suggested reduces labour costs, significantly reduces time to result for
negative patient samples, and reduces time to result for positive viral and
parasitological enteropathogens. If reporting of ‘presumptively’ positive bacterial
PCR results is also included prior to culture results being available this would
complete rapid result turnaround for all three pathogen types.
2612
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2620
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2622
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The potential multi analyte molecular enteropathogen detection components in this
suggested workflow remove the need for routine faecal parasite concentration
microscopy. Performing simple wet preparation is primarily for meeting the
requirements of the culture funding item. Wet preparations require only minimal field
assessment and reporting of the presence of leucocytes, erythrocytes, observable
parasites, and miscellaneous other findings such as Charcot-Leyden crystals and fat
globules. The ‘mini’ culture is based upon culture media and enrichment broths
being prepared in smaller format tubes that are amenable to automated “cherry
picking” of samples which are PCR positive for bacterial enteropathogens.
Identification of PCR positives requiring subculture can be directed via laboratory
information systems to facilitate efficient workflow and minimisation of human error in
selecting the appropriate tubes. The format of the media is ideally in SBS format
racks, and the use of 1.4mL tubes in a 96-cluster arrangement is suggested. The
three selected media types are Cary Blair (298), Selenite Broth (299) and
Campylobacter Broth (300). Using these three media types enables preservation
and enrichment of all of the bacterial enteropathogens in the time period between
sample receipt and the completion of the molecular testing. Cary Blair media has
also been successfully utilised as the input sample for molecular testing (85) and
therefore serves as a useful source of material if any repeat molecular testing is
required. A semi-solid broth supporting the growth of Campylobacter has been
added to the protocol because decreased sensitivity of culture for Campylobacter
was observed where stool culture was delayed waiting for bacterial enteropathogen
screening by PCR testing in the protocol of Schuurman et al. (8). By miniaturising
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the volume of media the cost is also significantly reduced, as are the requirements
for incubator space. The hands on component of initial stool processing in the
microbiology laboratory would change to using flocked swabs as the inoculation
device for three media tubes, creation of a wet preparation and inoculation of a tube
containing PVP Buffer and micro particles for sample disruption and subsequent
nucleic acid extraction.
2641
2642
2643
2644
2645
2646
2647
As mentioned previously the benefits of recovering isolates reside with epidemiology
and antimicrobial sensitivity testing and surveillance. Additionally a part of ongoing
molecular assay validation requires that there is periodic evaluation of the suitability
of the primer/probe sequences for detecting organisms which might have genomic
drift over time (296). The ability to re-validate performance of the molecular assay by
occasionally culturing all samples including those PCR negative for bacterial
enteropathogens is also possible with the suggested workflow.
2648
2649
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2651
Cost-benefit analysis is required as there are implications in regards what Medicare
item(s) can potentially be billed, with the suggested approach potentially reimbursed
under one or both of Item 69345 $45.30 for the culture and wet preparation and Item
69496 $36.85 for the molecular testing.
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Discussion
2654
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2670
Available molecular technology continues to progress in scale, speed and
affordability each year. It is difficult to predict the form and scope to which the
technology will be applied in routine diagnostic Microbiology or indeed just when
various conventional techniques might be replaced, if at all. There is no doubt
however, that molecular based technology will continue to increasingly supplement
the clinical microbiology diagnostic repertoire in Australian laboratories. The
evolution of high multi target analytical instruments coupled with reducing cost per
data point has recently bridged a gap between the number of potential organism
targets required for comprehensive molecular screening of certain clinical samples
and what has been economically possible with existing multiplex real-time PCR
testing. The utility of screening faecal samples with these systems has been
discussed in this report and proof of principle is in progress in our laboratory.
Arguably traditional mycology especially of dermatophytes may be another
component of the routine laboratory functionality that could potentially be adapted to
such systems. Extension of the current multiplexed molecular testing panels for
respiratory virus diagnostics appears to also be a logical use of the systems
described.
2671
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2674
There are compelling benefits for using high multi analyte molecular technologies for
screening for enteropathogens. Healthcare costs can be reduced from $73.95 per
patient test using conventional methods to $36.85 whilst also delivering extended
scope of testing, enhanced sensitivity and improved time to result.
2675
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2686
For bacterial enteropathogens when compared to culture the completion time is
reduced from 2-5 days to a single day. Equivalent or improved detection has been
observed, particularly for Campylobacter with 15-20% increases in sensitivity. E. coli
pathovars and E. albertii can be detected molecularly via virulence genes where
otherwise they are not obvious. As is currently the case for culture, interpretation of
the significance of certain molecular results also requires clinical correlation. The
detection of Aeromonas or Brachyspira may not always represent the causative
agent, some species of Yersinia are of unknown significance but reporting is still
suggested, and finding toxigenic C. difficile can represent asymptomatic carriage
particularly in children but also in a percentage of adults. Due to emergence of
increased rates of community C. difficile disease a case has also been made for
inclusion of toxigenic C. difficile in any routine panel of bacterial enteropathogens.
2687
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2689
2690
2691
2692
For viral enteropathogens, adopting molecular techniques has always resulted in an
immediate increase in the detection rate. This phenomenon arises for two reasons
(a) increased detection of viruses previously tested for because of increased
sensitivity, and (b) use of multiplex testing and inclusion of additional virus types.
Molecular panels of enteric virus detection assays have also raised issues that
require clinical input including high rates of co-infections, and a recognition that
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asymptomatic and subclinical infections exist for a number of viruses including
rotavirus and human parechovirus.
2695
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2700
2701
2702
Utilisation of molecular technology for parasitic enteric disease has been shown to
offer substantial benefits compared to microscopy preparation techniques and the
requirements on laboratory staff to screen slides. Sensitivity of the microscopy
techniques has been shown to be particularly poor for some organisms such as D.
fragilis, and using molecular techniques results in sensitivity increases of at least
10% (and up to 60% for D. fragilis). Replacement of larval stage nematode culture
methods by multiplex PCR assays has also exhibited at least equivalent sensitivity
for those helminths and reduced time to result from several days to a single day.
2703
2704
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2706
2707
2708
2709
2710
2711
2712
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2714
2715
2716
2717
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2719
Several commercial multi analyte enteropathogen systems/kits are either available or
about to be released. These solutions were reviewed in comparison to the range of
pathogens reported in our laboratory across 4 years of data. When considering
using one of these commercial products as a comprehensive ‘replacement’ solution
to the conventional methodologies there were some gaps in coverage amongst the
kits that would have resulted in more than 300 patients a year failing to have an
enteropathogen reported. The best coverage for bacterial enteropathogens was
99.8% (Seegene), and the worst 77.3% (AusDiagnostics). The best coverage for
parasites was 95.9% (Genetic Signatures) and the worst 26.2% (Luminex) though
Seegene was 0% having no parasite component. Issues with the ability to process a
daily workload of 250 samples were identified and the throughput per
batch/instrument in a 4-hour run time ranged from 95 to 7 patients. The Idaho
Technology FilmArray was comprehensive and rapid with a one hour time to result
but only one patient per run per instrument is possible. Diagnostic yields using
these commercial multi analyte enteropathogen kits were in the order of 30% higher
than conventional methods and co-infections were increasingly observed ranging
from 6.8% to 35% dependent on kit and study.
2720
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2722
2723
2724
2725
The time to obtain negative results for all enteropathogens, positive results for viral
and parasitological infection and also positive presumptive bacterial infection are all
reduced significantly when using molecular assays compared to the existing
conventional techniques found in Australian laboratories. Provision of results in a
timely fashion before an illness has resolved is of use for limiting community
transmission and supports endeavours for appropriate use of antimicrobials.
2726
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2729
2730
2731
2732
Using widely available standard real time PCR instrumentation to perform a large
number of real-time multiplexed home brew PCR assays is cost prohibitive when
four or more of these assays are required. As the existing commercial products
currently also do not provide an appropriate range of organisms to be able to
realistically replace existing methods, three systems have been described for which
a high number of PCR targets can be tested. Each system is capable of turning over
250 samples with 80 PCR targets/sample within 10 hours. Based on a test volume
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of 250 samples/day for six days a week the cost per patient is ~$25 or less
dependent on the system.
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2741
2742
2743
2744
2745
2746
In an environment of increasing health costs and fiscal pressures on health budgets
for pathology in Australia the use of these high multi analyte systems potentially
drives the cost down from $73.95 (Item 69336 + Item 69345) for conventional M/C/S
and O/C/P to $36.85 (Item 69496) for microbial nucleic acid determination. The
potential has been shown for these systems to deliver improved testing for lower
cost when utilised as stand alone methods. If the utility and value of the results
increase due to improved enteropathogen coverage and decreased time to reporting,
potentially more stool samples would be submitted for testing than the current rate of
one stool from every seventh patient presenting to a clinician with gastroenteritis.
This would actually increase testing costs in the health budget in a direct sense but
may help reduce community enteric disease burden and the economic costs of lost
time by patients and carers
2747
2748
2749
2750
2751
2752
2753
2754
2755
2756
2757
2758
2759
2760
2761
2762
2763
2764
2765
Conceptually these systems meet the requirements but there are of course a number
of challenges to overcome. Stool is a complex matrix containing many inhibitors of
PCR and the organism nucleic acids themselves are ‘packaged’ in a variety of ways
from cell walls, cyst walls, capsids, teguments and cuticles. Rapid, effective
automated nucleic acid extraction technology and appropriate control material to
seed into extractions is required. Even so, a percentage of samples will remain
inhibitory and not be suitable for PCR testing, which will require reverting to
traditional diagnosis, or sample recollection for repeat molecular testing. Several of
the helminth targets are yet to have a published real-time PCR developed and
require target evaluation and assay design. Validation of the large number of target
assays many of which detect organisms that have very low prevalence will be very
challenging. Adjusting to the inevitable increases in pathogens identified, the
increased observation of co-infection, and delivering appropriate interpretation to
clinicians and patients for organisms of variable or unknown significance and/or
asymptomatic carriage will all pose a challenge. There will also be an invariable
deskilling of the scientific staff in the techniques of faecal parasite identification by
microscopy, and bacterial enteropathogen recognition. Skills in bacterial culture
reading would be maintained if the proposed workflow of concurrent reflex culture
was adopted.
2766
2767
2768
2769
2770
2771
2772
2773
2774
There appears to be two divergent projections of the functionality of future diagnostic
microbiology laboratories. The traditional global commercial microbiology
companies (bioMerieux and Becton Dickinson) are promoting 1-5 million dollar total
lab automation systems which bring manufacturing style automation and digital
imaging to the traditional phenotypic modalities of agar plates and microscope slides.
The products such as Previ-Isola/Smart Incubator (bioMerieux) or Kiestra (BD) utilise
automated agar plate inoculation robotics, attached track based smart incubation
modules, automated plate handling with high-resolution image capture of plate
growth, analysis of growth remotely via image observation, and downstream colony
Page 77
2775
2776
2777
2778
2779
2780
2781
2782
2783
2784
manipulations for identification techniques and sensitivity test setup initiated by
software based colony picking. There is also rapid progression of molecular
technology which is acting as a disruptive technology to the traditional methods and
replacing ‘agar plate amplification’ with ‘nucleic acid amplification’, the latter being
considerably faster than the former, with equivalent or much improved specificity and
sensitivity, and decreasing cost. The challenges of antimicrobial sensitivity testing
will likely see molecular technology restricted to components of the workflow that are
reliant primarily on detection of the organism in the medium term. However, it
appears likely that enteropathogen detection within Australian laboratories is likely to
transition to routine molecular methods over the next few years.
Page 78
2785
Figures and Tables
Table 1: Example List of 80 Potential Enteropathogen Targets for High Multi-Analyte
Systems. The expanded target list for parasites represents a level of coverage
where consideration might be given to not performing conventional microscopy
based parasite testing on faeces. Many additional bacterial targets are present and
cover numerous pathogens not examined for in routine bacterial culture. The
expanded viral coverage also adds several viruses not routinely examined for by
existing common tests in diagnostic pathology laboratories.
Bacteria
Viruses
Parasites
Salmonella enterica
Salmonella spp.
Campylobacter spp.
Campylobacter jejuni
Rotavirus A/B/C
Adenovirus F 40/41
Norovirus GI/GII/GIV
Norovirus GI
Entamoeba histolytica
Entamoeba dispar
Entamoeba moshkovskii
Entamoeba coli
Campylobacter coli
Arcobacter spp.
Brachyspira spp.
Shigella spp.
Shigella dysenteriae
Shigella flexneri
Shigella sonnei
Norovirus GII
Sapovirus
Mamastrovirus/Astrovirus
Human Parechovirus
Enterovirus
Picobirnavirus
Aichivirus
Entamoeba polecki
Iodamoeba buetschlii
Endolimax nana
Entamoeba hartmanni
Giardia intestinalis
Dientamoeba fragilis
Chilomastix mesnili
Yersinia enterocolitica
Coronavirus
Retortamonas spp.
Yersinia spp.
Cryptosporidium spp.
Listeria monocytogenes
Balantidium coli
Vibrio cholera
Blastocystis hominis
Vibrio parahaemolyticus
Cyclospora cayetanensis
Vibrio fluvialis
Isospora belli
Escherichia coli EHEC
Enterobius vermicularis
Escherichia coli EPEC
Ascaris lumbricoides
Escherichia coli ETEC
Ancylostoma duodenale
Escherichia coli EIEC
Necator americanus
Escherichia coli EAggEC
Trichostronglyus spp.
Aeromonas spp.
Strongyloides stercoralis
Plesiomonas shigeloides
Trichuris trichiura
Edwardsiella tarda
Diphyllobothrium latum
Clostridium difficile
Taenia solium
Taenia saginata
Hymenolepis nana
Hymnenolepis diminuta
Dipylidium caninum
Fasciolopsis buski
Echinostoma ilocanum
Heterophyes heterophyes
Metagonimus yokogawai
Page 79
Clonorchis sinensis
Fasciola hepatica
Opisthorchis viverrini
Paragonimus
westermani
Paragonimus mexicanus
Schistosoma spp.
Schistosoma mansoni
Schistosoma japonicum
Table 2: Comparative Summary of Commercial Enteropathogen PCR Testing Kits.
Manufacturer
Luminex
Seegene
Product Name
xTAGGPP
Technology
Multiplex
RT-PCR,
BeadHyb
Tag
Unknown
24
<5 hours
Diarrhea
ACE
Detection
3 kits
Multiplex
RT-PCR,
CE
Detection
$39
Up to 32
<5 hours
Cost/patient AUD
Throughput/batch
Extraction to
Result Time for
maximum batch
TARGETS:
Salmonella
Campylobacter
Shigella
Yersinia
Vibrio spp.
Vibrio cholerae
EHEC
Other Pathogenic
E.coli
Aeromonas
Listeria
Clostridium difficile
Clostridium
perfringens
Plesiomonas
shigelloides
x
x
x
x
x
X
ETEC
x
x
x
x
x
x
x
x
x
x
Idaho
Technology
FilmArray GI
Panel
Multiplex RTqPCR in Pouch
Nanofluidics
Not available
1
1 hour
Savyon
AusDiagnostics
NanoCHIP
Gastrointestinal
Panel
PCR plus
Microarray
Easy-Plex Faecal
Profile 10
Multiplexed
Tandem RTqPCR
Genetic
Signatures
EasyScreen
Enteric
Detection
3 Kits
3base
Multiplex
RT-qPCR
$40
Up to 95
4 hours
$24.70
Up to 7
<4 hours
$51
Up to 15
3 hours
x
x
x + dysenteriae
x
x
x
x + O157
ETEC,EPEC,EIEC,
EAEC
x
x
x
x
x
x
x
x + NAP1
x
x
x
x
x
x
x
x
x
Rotavirus
Adenovirus
Norovirus
Astrovirus
Sapovirus
x
x
x
x
x
x
x
x
x
x
x
x
Cryptosporidium
parvum
Giardia intestinalis
Entamoeba
histolytica
Dientamoeba
fragilis
Entamoeba
complex
Blastocystis
hominis
Cyclospora
cayetanensis
x
x
x
x
x
x
x
x
x
x
x
x
Other kit
x
x
x
Other kit
x
x
x
x
x
x
x
x
x
x
x
Page 80
Table 3: Analysis of Reported Parasites 2007-2011 Sullivan Nicolaides
Pathology(56). The results show that whilst the majority of recovered parasites are
Blastocystis, Giardia, Cryptosporidium, Dientamoeba and Entamoeba histolytica, in
total up to 13 distinct parasites of clinical significance and 4 types of commensal
parasites have been reported from patients over the time period examined.
Blastocystis
Giardia
Cryptosporidium
Dientamoeba
fragilis
Endolimax nana
Entamoeba coli
Entamoeba
histolytica
Iodamoeba
butschlii
Enterobius
vermicularis
Hymenolepis
nana
Hookworm ova
Chilomastix
Cyclospora
Taenia sp ova
Strongyloides
larvae
Ascaris spp
Trichuris ova
Total Parasites
Total Faeces
2007
4057
1118
321
301
2008
4177
1047
430
122
2009
4636
1179
997
78
2010
3702
1122
214
118
2011
3576
1286
351
172
Total
20148
5752
2313
791
146
182
20
213
201
22
242
198
34
206
152
31
163
132
34
970
865
141
23
24
24
22
18
111
16
13
15
8
18
70
5
6
2
5
0
18
9
19
2
2
9
1
11
1
1
7
9
7
5
0
7
3
8
3
0
1
3
3
1
3
1
25
48
12
6
25
0
1
6230
47781
0
0
6276
48308
2
0
7435
52927
1
0
5596
49126
1
0
5762
56876
4
1
31299
255018
2786
Page 81
Table 4: Analysis of Reported Bacterial Enteropathogens 2007-2011 Sullivan
Nicolaides Pathology (56). The results show that whilst the majority of recovered
bacterial enteropathogens are Campylobacter, Salmonella, Aeromonas, Clostridium
difficile, Yersinia and Shigella, in total up to 13 distinct pathogens have been
reported from patients over the time period examined.
2007
Campylobacter 1927
Salmonella
844
Aeromonas
856
Yersinia
52
Shigella
44
Vibrios
6
Arcobacter
18
Edwardsiella
0
EHEC
0
Plesiomonas
0
2008
2106
703
863
113
45
7
12
0
0
0
Bacterial Culture
2009
1985
827
868
87
36
14
1
0
0
1
C. difficile PCR
stx1/2 PCR
eaeA PCR
328
5
4
Positive by PCR Detection
453
493
5
7
6
15
309
5
8
2787
Page 82
2010
1857
1025
679
82
39
9
6
0
0
0
2011
2205
1077
632
11
28
13
0
1
1
0
962
3
21
Total
10080
4476
3898
345
192
49
37
1
1
1
19080
Table 5: Comparison of Systems Capable of Cost-Effective ‘Comprehensive’ Multi
Analyte Testing and High Throughput – Based on volumes of 250 patients/day and 6
testing days per week
Fluidigm 96.96 IFC Chip Sequenom MassARRAY
Biomark HD
Life Technologies
Open-Array Plate
QuantStudio 12K Flex
Open Array Plate
Format (up to) 112
Assays by 24 samples
per Plate by 4 plates
per run
Format and Density
96x96 IFC Chip
96 assays by
96 samples per run
3 x 27-29plex multiplex
in 384 well plate
format and secondary
primer extension per
sample
Analytical Volume
Dynamic Range
Multiplex PCR
Optimisation
Considerations
6nL
5.5logs
Multiplex capable but
meets criteria as
singleplex
5µL
7logs
Yes – requires
Multiplexing to meet
criteria
33nL
7logs
No - singleplex only
PCR Assay Setup
Considerations
PCR assays to be
individually
constructed and
loaded to IFC chip
ports
<5 hours per 96 batch
dependent on liquid
handling for filling IFC
chip
PCR assays to be
individually
constructed and
loaded into PCR
amplification plates
9.5 hours to first 128,
45 minutes MALDI-TOF
end point analysis
allows overlapping
batch processing
Single System
10.25 hours with
overlapping batches in
process simultaneously
Double System
9.5 hours
Specific PCR Assays
pre-filled into defined
wells at manufacture,
sample loaded with
mastermix base only
3 hours per 96 batch
Requires Accufill liquid
handler to apply
samples and
mastermix base
Single QuantStudio
9 hours (3 runs)
100%
33%
Assay to Result Time
Completion Time 250
samples each for >80
specific PCR targets
Possible daily workload
loss in event of first
batch fails
Capital Cost (AUD)
Contamination
Considerations
Indicative Cost/Patient
including reagent
volume discounting
(AUD)
Excludes Capital Costs
Single Biomark HD
10-12 hours (3 runs)
Double Biomark HD
8-10 hours
33%
$480,000 per system
Closed Amplification
$450,000 per system
Open amplified
reaction tubes for
secondary primer
extension reaction
setup
$15-$18
$10-$12
Page 83
Double QuantStudio
7 hours
$160,000 per system
Closed Amplification
$22
Figure 1: Construction of the FilmArray Nanofluidic pouch, adapted from Poritz et al.
(301). The pouch contains fluidic components manipulated by the Film Array cycler
to achieve integrated extraction of nucleic acids, construction of PCR mastermixes
and PCR in a set of wells in an array format.
Figure 2: Open-Array plate for Life Technologies QuantStudio 12K Flex qPCR
System(302). Each Open-Array plate has 3072 through holes arranged as 48
squares of 8x8 through holes. User defined assays are added to sets of defined
through holes on manufacture of the Open Array. Custom array formats of 32, 64,
128 and 192 through holes per patient test can be defined and primers and probes
are deposited in the through holes assigned. Patient nucleic acid and the remainder
of the reagent required to complete the PCR mastermix are loaded to the
appropriate through hole set by the Accufill liquid handler. A maximum of 4 OpenArray plates can be run at the same time on the QuantStudio 12K Flex real time
PCR cycler.
Page 84
2788
Figure 3: High Precision 96.96 Genotyping IFC Fluidigm Chip for Biomark HD qPCR
System.(303). The 96.96 Fluidigm IFC Chip is loaded with 96 PCR assays on one
side and 96 patient extracts on the other. Each patient sample is combined with
each reaction mix within the IFC in a specific individual chamber utilising integrated
thermal and pneumatic processes.
2789
Page 85
Figure 4: Seqenom MassARRAY Workflow (304).. The amplification and iterative
primer extension steps account for 7 hours of processing before MALDI-TOF
analysis is performed. Failure of a batch of testing would not be apparent until
approximately 8 hours after the first batch had been processed. If the cause of the
failure affected other batches currently part way through the pre-MALDI-TOF
process, this may render an entire days testing unsuccessful. This would delay
patient results and incur high costs for the laboratory.
Figure 5: Seqenom MassARRAY SpectroCHIP for MALDI-TOF Mass
Spectrometer(305). Extension oligo products are dispensed by robotic
nanodispenser onto the SpectroCHIP array which is loaded into the MALDI-TOF
MassARRAY Analyzer. Up to 384 reactions containing extended oligo products can
be loaded to one SpectroCHIP for analysis.
Page 86
Figure 6: Example workflow for incorporation of a high multi analyte PCR and ‘mini’
preservation culture within the laboratory. Stool is processed via three concurrent
modalities ; nucleic acid extraction with high multi analyte PCR testing, inoculation of
Cary Blair, Selenite and Campylobacter Broth with appropriate incubation, and
performing wet preparation microscopy. Positive PCR results for viruses and
parasites are reported directly. Positive bacterial PCR results initiate a presumptive
report and reflex culture from the mini cultures onto full agar plates. Reflex culturing
allows recovery of the bacterial pathogen for antimicrobial sensitivity testing and
epidemiological use where indicated.
Page 87
2790
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