Performance of 16S rRNA Gene
Sequencing on Primary Specimens
Linda Hoang, MSc, MD, FRCPC, DTM&H
Program Head
Bacteriology & Mycology Laboratory
BC Public Health Microbiology & Reference Laboratory
BCCDC Public Health Microbiology & Reference Laboratory
BCCDC Public Health Microbiology & Reference Laboratory
LAB
BCCDC Public Health Microbiology & Reference Laboratory
Introduction
• WHY BETTER FASTER?
– correct treatment agent, duration of therapy, & patient prognosis
• However, in some specimens where infection is
suspected,
•
– Bacterial and fungal cultures may be negative
– Conventional, targeted PCR assays
In these settings, possible options include:
– Conventional, targeted PCR assays
– Serological tests
– Broad based PCR assays
– Next generation sequencing
BC Public Health Microbiology &
Reference Laboratory
• Bacterial identification
– 16S rRNA gene sequencing
– Conventional biochemical analyses
• Fungal identification
– ITS gene sequencing
– Conventional phenotypic
and biochemical analyses
BCCDC Public Health Microbiology & Reference Laboratory
16S ribosomal RNA
• Part of the 30S small subunit of
prokaryotic ribosomes
• Conserved gene target universal in bacteria
• Contains multiple hypervariable regions
• Sequence of 16S gene allows for molecular
discrimination of bacterial strains
• Dependent on a database with gene sequences
(Genbank, RDP, etc)
16S PCR
and
Sequencing
Targets
Advantages of using 16S and ITS targets
are:
– Amplification of a wide range of bacterial and
fungal pathogens
– Available reference database with accurate
identification through sequencing of gene
products
– Identification of culture negative organisms
– Amplification of dead organisms
– Discovery of novel pathogens
Direct 16S???
OK, but
• Consult with MedMicro
• Only on sterile sites
• Lots of polys +/- org seen
• No if mixed organisms seen
BCCDC Public Health Microbiology & Reference Laboratory
16S PCR
and
Sequencing
Challenges with 16S
• There are still challenges with using broad
range amplification targets:
– Typically ineffective for polymicrobial infections
– Cannot give antimicrobial susceptibilities
– Risk of specimen and laboratory contamination
– Little information for the performance of 16S and ITS
assays, for all specimen types, reported in literature
• Don’t know sensitivity of test
Study Objectives
• Our goals were to:
– Evaluate the performance of 16S amplification
and sequencing from direct clinical specimens
– Evaluate factors predicting amplification and
identification success
• Microscopy findings
• Specimen type (Fresh versus FFPE)
Methods
• Retrospective review of 16S over a 4 year period
(January 2009-June 2013)
– Review specimen types submitted
• Fresh vs formalin fixed
– Review sequencing results against microscopy
(e.g. gram stain, histopath, etc) results
– Review types of organisms identified by sequencing
– Statistical analysis performed using Fisher's exact test
(GraphPad), with a p-value of 0.05
Results
Results
Results
Results
Results
Results
Results
Results
Results
Discussion
• 16S had an overall positivity rate of 23%,
– Comparable to published studies
• A positive microscopy result (Gram smear or
histology) was significantly associated with
success (up to 100%)
• There was no significant difference in positivity
rates for 16S subgroups (eg CSF, bone)
Discussion
• The largest group of organisms was
Streptococci
– May reflect high susceptibility to antibiotics, or their
overall prevalence in the microbiology lab
• Next largest groups were anaerobes and
atypicals
– Could be reflective of the difficulty in culturing these
pathogens
• A similar distribution of organisms was noted in
a large study in 2014
Conclusions
• Direct specimen 16S have important roles, particularly in
patients with:
– previous antibiotic treatment
– microscopy positive/culture negative samples
– suspicion of atypical/fastidious pathogens
• Tests labour intensive and costly, need for careful
•
selection of appropriate samples
Based on our findings, 16S cannot be used as tests to
rule out infection, but are helpful if positive.
Acknowledgement
• BCPHMRL Bacteriology Lab Technologists
• Dr. Michael Payne
BCCDC Public Health Microbiology & Reference Laboratory