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Psittacosis : molecular tools for detection and typing of Chlamydophila psittaci
Heddema, E.R.
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Heddema, E. R. (2007). Psittacosis : molecular tools for detection and typing of Chlamydophila psittaci Mercis
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Download date: 15 Jun 2017
Psittacosis
Molecular tools for detection and
typing of Chlamydophila psittaci
Edou Redbad Heddema
Cover design © Mercis publishing, used with permission
ISBN 10: 9073838970
ISBN 13: 97873838970
Printed by F&N Eigen Beheer, Amsterdam
This thesis was financially supported by Bayer Healthcare, Oxoid, Pfizer,
Roche Diagnostics, and the University of Amsterdam
Psittacosis
Molecular tools for detection and typing of
Chlamydophila psittaci
Academisch Proefschrift
ter verkrijging van de graad van doctor
aan de Universiteit van Amsterdam
op gezag van de Rector Magnificus
prof. dr. J. W. Zwemmer
ten overstaan van een door het college voor promoties ingestelde
commissie, in het openbaar te verdedigen in de Aula der Universiteit
op donderdag 22 maart 2007, te 14.00 uur
door
Edou Redbad Heddema
geboren te Weststellingwerf
Promotiecommissie
Promotor:
Prof. dr. C. M. J. E. Vandenbroucke-Grauls
Co-promotores:
Dr. Y. Pannekoek
Dr. C. E. Visser
Overige leden:
Dr. R. E. Jonkers
Prof. dr. P. A. Kager
Dr. J. T. Lumeij
Prof. dr. P. Speelman
Dr. J. van Steenbergen
Prof. dr. D. Vanrompay
Faculteit der Geneeskunde
Table of contents
Contents
Chapter 1. Introduction: Chlamydophila psittaci infections
with the emphasis on zoonotic infections
7
Chapter 2. A woman with a lobar infiltrate due to psittacosis
detected by polymerase chain reaction
31
Chapter 3. Development of an internally controlled real-time PCR
assay for detection of Chlamydophila psittaci in the LightCycler 2.0
system
39
Chapter 4. An outbreak of psittacosis due to Chlamydophila psittaci
genotype A in a veterinary teaching hospital
55
Chapter 5. Prevalence of Chlamydophila psittaci in fecal droppings
from feral pigeons in Amsterdam, The Netherlands
69
Chapter 6. Genotyping of Chlamydophila psittaci strains in human
clinical samples by ompA sequence analysis
79
Chapter 7. Summarizing discussion: molecular tools for the detection
and typing of Chlamydophila psittaci strains causing human and
avian infections
85
Chapter 8. Nederlandse samenvatting, Dankwoord, Curriculum
vitae, Publicaties
95
Chapter 1
Introduction: Chlamydophila psittaci
infections with the emphasis on zoonotic
infections
Edou R. Heddema1, Yvonne Pannekoek1, Caroline E. Visser1, Christina
M.J.E. Vandenbroucke-Grauls1,2
1
) Department of Medical Microbiology, Academic Medical Center,
University of Amsterdam, Amsterdam, the Netherlands.
2
) Department of Medical Microbiology & Infection Control, VU
University Medical Center, Amsterdam, the Netherlands.
7
Chapter 1
History
In 1881, Jacob Ritter wrote an article in which he described seven cases
of psittacosis in one family related to parrots and finches caged in the
study of his brother's house in Uster, Switzerland. Three people died,
including one of his brothers (29,54). Ritter accurately identified the
study as the site of the source of infection, considered the birds as
vectors, and determined both the incubation period and the
nontransmissibility of the disease from human to human. The main
pathologic finding was pneumonia. Besides the respiratory symptoms,
the disease presented with headache and a slow pulse rate compared with
the body temperature (relative bradycardia), that was often seen in
“typhus” (typhoid fever), an endemic disease in Europe at that time.
Therefore, he named the disease “pneumothyphus”. Ritter's article is a
precise description of the clinical presentation, epidemiology, pathologic
findings, and natural history of psittacosis.
Several outbreaks were reported since Ritter’s description. One of the
largest outbreaks in the 19th century was in Paris in 1896 which involved
more than 70 people. In 1893 a Frenchman, named Nocard, isolated a
Gram-negative bacterium belonging to the Salmonella group from an ill
bird. The bacterium was named Nocard’s bacillus and seen as the
causative agent of psittacosis. Only seldom this easy to culture bacterium
could be isolated from birds involved in outbreaks of psittacosis.
In 1929 and 1930 a sharp increase in psittacosis cases was observed
worldwide. This is the so-called psittacosis pandemic. Two reasons for
this increase were established. Firstly, an outbreak of psittacosis occurred
among bird flocks in Argentina that were meant for international bird
trade. Thus, worldwide distribution of infected birds occurred. Secondly,
keeping birds for hobby flourished in the late 1920’s because of good
economic times (63). It took until 1930 before the true psittacosis agent
was identified and cultured (3,4). At first, the psittacosis agent could only
be propagated in birds, but later peritoneal inoculation in mice became
the preferred culture technique (26). The agent was classified as a virus
belonging to the psittacosis-lymphogranuloma venereum group. The
“virus” was stained with Giemsa, Macchiavello or Castaneda technique.
Some properties of the agent revealed a size of 0.22-0.33 µm, a unique
developmental-cycle and it was considered a large virus with bacterial
affinities (38).
8
Chapter 1
For diagnostic purposes, culture was a quite cumbersome procedure.
Laboratory associated infections occurred and the poor growth in culture
of some strains were major problems. Therefore serologic tests were
developed quite soon after the discovery of the causative agent. In 1935
Bedson and co-workers described the complement fixation reaction (CF)
(2). With this test it was possible to diagnose psittacosis when the “virus”
could not be grown. But for many years this method was not very
standardized concerning the antigen used and the amount of complement
added (16). Despite these shortcomings , it became the preferred method
for detection of psittacosis cases. In the 1950’s it became clear that
psittacosis could be effectively treated with tetracycline. In 1966 all
“viruses” in the psittacosis-lymphogranuloma venereum group were
finally assigned to the bacterial genus Chlamydia. (48).
Already in 1928 psittacosis became a notifiable disease in the
Netherlands. The disease was included in the “wet op de besmettelijke
ziekten” (law for the prevention of infectious diseases). In the
Netherlands, one of the first reports on psittacosis was by Herderschêe in
1930 (31). Six people were described who had close contact with
recently acquired parrots. Two of them died and again the main
pathologic finding was pneumonia. In 1937, laboratories in Amsterdam
succeeded in isolating the “psittacosis virus” from a patient with
“atypical pneumonia” by use of mouse inoculation. They stained the
“virus” with the modified Castaneda technique according to Bedson. By
use of the CF test they were able to find serologic evidence for
presumed psittacosis patients in the Netherlands, but they were unable to
isolate the “virus” (72). In 1949 Dekking pointed at racing pigeons as a
potential source for infection (17). Quite soon thereafter, several authors
described cases of psittacosis related to parakeets and pigeons (5,7,34).
Most of these cases were diagnosed by serological tests (CF).
9
Chapter 1
Microbiology
The bacterium
The causative agent of psittacosis is Chlamydophila psittaci. This
pathogen is an obligate intracellular Gram-negative bacterium.
Chlamydophila psittaci (formerly Chlamydia psittaci) has, like all other
members of the order Chlamydiales, a unique developmental-cycle which
was already recognized by Bland and Canti in 1935. Two distinct forms
of this pathogen are recognized: the extracellular infectious, spore-like
elementary bodies (EB) and the intracellular non-infectious fragile,
metabolically active reticulate bodies (RB). The EB is approximately 0.3
µm in size and derives its rigidity from intensive disulfide bridges
between the cysteine rich residues in the so-called cysteine rich outer
membrane envelope proteins (envA and envB). The RB is about 1 µm in
size and is the replicative form. The developmental cycle starts with
attachment and entry of the EB. Invasion is probably by receptor
mediated endocytosis. To date the precise structure of the receptor is still
unknown. Once inside the cell the EB’s are surrounded by a membrane
bound vacuole termed an inclusion that avoids fusion with lysosomes. In
the case of C. trachomatis infection, after entry, EB inclusions start to
fuse into larger vacuoles. This fusion is not observed in C. caviae and C.
psittaci strains. After approximately 8 hours, EB’s start to convert into
the metabolically active RB’s. These RB’s, which divide by binary
fission, are surrounded by an expanding inclusion membrane to
accommodate the growing microcolony. After a period of growth RB’s
convert back into EB’s and the inclusion contents is released from the
cell. The cell lysis and the new released EB’s can infect other cells. This
developmental-cycle takes approximately 48-72 hours depending on the
particular species or genotype studied (42,71).
10
Chapter 1
Figure 1. Chlamydial developmental-cycle
(source www.chlamydiae.com).
11
Chapter 1
Taxonomy
In 1966 all viruses in the lymphogranuloma-venereum group were
assigned to the bacterial genus Chlamydia. Since then, it became clear
that many animal species could be infected by Chlamydia spp.
“Chlamydia psittaci” was isolated from several animal species like
sheep, goats, horses, cats, birds and cattle. It was proven that the ovine,
feline and avian strains of “Chlamydia psittaci” could infect human
beings. In 1986 a new C. psittaci strain TWAR was identified and later
renamed Chlamydia pneumoniae. It was isolated from a student with
upper respiratory tract infection and appeared to be primarily a human
pathogen (27). A zoonotic reservoir was not identified. In the 1990’s
several authors showed that RFLP analysis of the ompA gene of the
members of the Chlamydiales could reliably cluster these strains in
several distinct groups most often closely related to clinical condition and
preferred host (32,36,44). However, until 1999, the taxonomy of the
Chlamydiales was mainly based on phenotypic characteristics of the
bacteria. In 1999, Everett presented sequence data of the intergenic
ribosomal spacer region of the bacteria in the Chlamydia genus. These
data served as the basis for a revision of the Chlamydiales taxonomy
(20). In this proposed taxonomic reclassification the family of the
Chlamydiaceae was divided in two genera, Chlamydia and
Chlamydophila. Chlamydia psittaci became member of the
Chlamydophila genus and was subdivided in 4 Chlamydophila spp. :
C. psittaci, C. abortus, C. felis and C. caviae (Figure 2.) This
classification showed the genetic relatedness of the Chlamydophila spp.
and its typical hosts (birds, sheep, goats, cats, and guinea pigs). Mainly
because this reclassification is based on limited sequence data, several
Chlamydiologists have objected to this proposed reclassification (62).
From the point of view of the animal pathogens, in particular the
zoonotic chlamydiosis agents, this reclassification has in my opinion
reduced confusion. Therefore, in this thesis the new taxonomic
classification is used.
12
Chapter 1
Fig 2. Taxonomic overview (source www.chlamydiae.com). Together
with the genus Chlamydophila, three new families were added in the new
classification. The avian strains of Chlamydia psittaci were reclassified
as Chlamydophila psittaci. Three new Chlamydophila spp. were
recognized with their own specific animal host. In the Chlamydia genus
two new species were added: Chlamydia suis and Chlamydia muridarum.
13
Chapter 1
Host range
Besides new sequence data, the new taxonomic classification also takes
into account that the six Chlamydophila spp. have a different biological
and ecological niche (20). Some species are only found in a single host
(for example C. caviae in Guinea pigs) while others can infect more than
one host. This is called “host range”. All Chlamydophila spp. have a
more or less narrow host range (Table 1a) (8). This host range is
probably the result of long-term adaptation between the Chlamydophila
spp. and their preferred animal hosts. The typical hosts of C. psittaci are
birds. Almost every bird can be infected with C. psittaci. The different
serotypes of C. psittaci have a preference for specific bird groups from
which they are predominantly isolated (70). Humans are only
occasionally infected and are more or less accidental hosts.
Table 1a: Typical hosts of the six Chlamydophila species and their
most encountered clinical syndromes (adapted from Bush et al.(8)).
Species
C. psittaci
C. abortus
C. felis
C. caviae
C. pecorum
C. pneumoniae
Main clinical syndromes
Respiratory infection,
conjunctivitis
Abortion
Conjunctivitis
Conjunctivitis
Abortion
Respiratory infection
Typical hosts
Birds
Sheep, goats
Cats
Guinea pigs
Cattle
Humans
Zoonotic disease
Respiratory tract
infection
Spontaneous abortion
Conjunctivitis
14
Chapter 1
Genomes and major surface protein genes
The exact length of the Chlamydophila psittaci genome is not known,
although its annotation is currently in progress (The institute for
Genomic research, TIGR). However, the genomes of C. pneumoniae
AR39, CWL029, J138, TW-183, C. felis Fe/C-56, C. caviae (GPIC
strain) and C. abortus S26/3 have been fully sequenced and some
properties of major representatives of the Chlamydophila genus are
shown in table 1b.
Table 1b. Available Chlamydophila spp. genomes sequences.
Genome
C. psittaci 6BC
C. abortus S26/3
C. felis Fe/C-56
C. caviae (GPIC)
C. pecorum E58
C. pneumoniae AR39
C. pneumoniae TW-183
Length (kb)
Unfinished
1144
1166
1173
Unfinished
1229
1226
GC %
39
39
39
40
40
Plasmid
Yes
No
Yes
Yes
No
No
No
Encoded proteins
932
1005
998
1112
1113
Source
Parakeet
Sheep
Cat
Guinea pig
Cow
Human
Human
The Chlamydophila genome is therefore roughly 1200 kbp long. This is
one of the smallest prokaryotic genomes known. It encodes on average
approximately 1000 proteins. The ompA (omp1), omp2 and omp3 genes
encode respectively the major outer membrane protein (MOMP), and the
envB and the envA encode the methionine and cysteine rich outer
membrane envelope proteins. Chlamydiaceae also carry LPS that is
encoded by the kdtA (previously gseA) gene. A serologic test like ELISA
is based on this antigenic molecule. All 4 molecules are surface exposed.
The MOMP functions as a porin and is the basis for the serovar
classification of the Chlamydiaceae.
Typing
C. psittaci can be classified into serovars by use of monoclonal
antibodies (Mabs) directed against the MOMP. Currently, eight serovars
are recognized (A-F, WC, M56) (65). The MOMP consists of five
conserved domains (CD) and four variable domains (VD) (Figure 3). The
VD’s are the surface exposed parts of the protein. The VD’s of the
15
Chapter 1
MOMP are encoded by variable segments of the ompA gene. The
conserved segments of the ompA gene encode the CD’s of the MOMP
and are conserved throughout the Chlamydophila spp. or even genus
level. This makes these segments attractive genes for diagnostic PCR
assays as shown by Hewinson et al (33).
16
Chapter 1
Figure 3. Allignment of the ompA of six avian C. psittaci serovars (AF) showing the conserved and variable domains of the gene.
(GenBank accession numbers AY762608-AY762612 and AF269261
generated by Vector NTI v10.)
C.
C.
C.
C.
C.
C.
psittaci
psittaci
psittaci
psittaci
psittaci
psittaci
sero
sero
sero
sero
sero
sero
A
B
C
D
E
F
(1)
(1)
(1)
(1)
(1)
(1)
C.
C.
C.
C.
C.
C.
psittaci
psittaci
psittaci
psittaci
psittaci
psittaci
sero
sero
sero
sero
sero
sero
A
B
C
D
E
F
(51)
(51)
(51)
(51)
(51)
(51)
C.
C.
C.
C.
C.
C.
psittaci
psittaci
psittaci
psittaci
psittaci
psittaci
sero
sero
sero
sero
sero
sero
A
B
C
D
E
F
(101)
(101)
(97)
(99)
(101)
(97)
C.
C.
C.
C.
C.
C.
psittaci
psittaci
psittaci
psittaci
psittaci
psittaci
sero
sero
sero
sero
sero
sero
A
B
C
D
E
F
(151)
(151)
(147)
(149)
(151)
(147)
C.
C.
C.
C.
C.
C.
psittaci
psittaci
psittaci
psittaci
psittaci
psittaci
sero
sero
sero
sero
sero
sero
A
B
C
D
E
F
(201)
(201)
(193)
(194)
(201)
(193)
C.
C.
C.
C.
C.
C.
psittaci
psittaci
psittaci
psittaci
psittaci
psittaci
sero
sero
sero
sero
sero
sero
A
B
C
D
E
F
(251)
(251)
(243)
(244)
(251)
(243)
C.
C.
C.
C.
C.
C.
psittaci
psittaci
psittaci
psittaci
psittaci
psittaci
sero
sero
sero
sero
sero
sero
A
B
C
D
E
F
(301)
(301)
(293)
(294)
(301)
(293)
C.
C.
C.
C.
C.
C.
psittaci
psittaci
psittaci
psittaci
psittaci
psittaci
sero
sero
sero
sero
sero
sero
A
B
C
D
E
F
(351)
(351)
(340)
(341)
(351)
(340)
1
50
MKKLLKSALLFAATGSALSLQALPVGNPAEPSLLIDGTMWEGASGDPCDP
MKKLLKSALLFAATGSALSLQALPVGNPAEPSLLIDGTMWEGASGDPCDP
MKKLLKSALLFAATGSALSLQALPVGNPAEPSLLIDGTMWEGASGDPCDP
MKKLLKSALLFAATGSALSLQALPVGNPAEPSLLIDGTMWEGASGDPCDP
MKKLLKSALLFAATGSALSLQALPVGNPAEPSLLIDGTMWEGASGDPCDP
MKKLLKSALLFAATGSALSLQALPVGNPAEPSLLIDGTMWEGASGDPCDP
51
100
CATWCDAISIRAGYYGDYVFDRVLKVDVNKTFSGMAATPTQATGNASNTN
CATWCDAISIRAGYYGDYVFDRVLKVDVNKTFSGMAATPTQATGNASNTN
CSTWCDAISIRAGYYGDYVFDRVLKVDVNKTFSGIGKKPTGSS----PND
CATWCDAISIRAGYYGDYVFDRVLKVDVNKTFSGMAKSPTEATG--TASA
CATWCDAISIRAGYYGDYVFDRVLKVDVNKTFSGMAATPTQATGNASNTN
CATWCDAISIRAGYYGDYVFDRVLKVDVNKTISGMGAAPTGSA----AAD
101
150
QPEANGRPNIAYGRHMEDAEWFSNAAFLALNIWDRFDIFCTLGASNGYFK
QPEANGRPNIAYGRHMQDAEWFSNAAFLALNIWDRFDIFCTLGASNGYFK
FKNAEDRPNVAYGRHLQDSEWFTNAAFLALNIWDRFDIFCTLGASNGYFK
TTTAVDRTNLAYGKHLQDAEWFTNAAFLALNIWDRFDIFCTLGASNGYFK
QPEANGRPNIAYGRHMQDAEWFSNAAFLALNIWDRFDIFCTLGASNGYFK
YKTPTDRPNIAYGKHLQDAEWFTNAAFLALNIWDRFDIFCTLGASNGYFK
151
200
ASSAAFNLVGLIGFSAASSISTDLPTQLPNVGITQGVVEFYTDTSFSWSV
SSSAAFNLVGLIGFSATNSTSTDLPMQLPNVGITQGVVEFYTDTSFSWSV
ASSAAFNLVGLIGVKGS----SLTNDQLPNVAITQGVVEFYTDTTFSWSV
ASSAAFNLVGLIGLKG-----TDFNNQLPNVAITQGVVEFYTDTTFSWSV
SSSAAFNLVGLIGFSATSSTSTELPMQLPNVGITQGVVEFYTDTSFSWSV
ASSAAFNLVGLIGVKGT----SVAADQLPNVGITQGIVEFYTDTTFSWSV
201
250
GARGALWECGCATLGAEFQYAQSNPKIEMLNVTSSPAQFVIHKPRGYKGA
GARGALWECGCATLGAEFQYAQSNPKIEILNVTSSPAQFVIHKPRGYKGA
GARGALWECGCATLGAEFQYAQSNPKIEMLNVISSPAQFVVHKPRGYKGT
GARGALWECGCATLGAEFQYAQSNPKIEMLNVTSSPAQFVIHKPRGYKGT
GARGALWECGCATLGAEFQYAQSNPKIEVLNVTSSPAQFVIHKPRGYKGA
GARGALWECGCATLGAEFQYAQSNPKIEMLNVISSPTQFVVHKPRGYKGT
251
300
SSNFPLPITAGTTEATDTKSATIKYHEWQVGLALSYRLNMLVPYIGVNWS
SSNFPLPITAGTTEATDTKSATIKYHEWQVGLALSYRLNMLVPYIGVNWS
SANFPLPANAGTEAATDTKSATLKYHEWQVGLALSYRLNMLVPYIGVNWS
GSNFPLPIDAGTEAATDTKSATLKYHEWQVGLALSYRLNMLVPYIGVNWS
SSNFPLPITAGTTEATDTKSATIKYHEWQVGLALSYRLNMLVPYIGVNWS
GSNFPLPLTAGTDGATDTKSATLKYHEWQVGLALSYRLNMLVPYIGVNWS
301
350
RATFDADTIRIAQPKLKSEILNITTWNPSLIGSTTALPNNSGKDVLSDVL
RATFDADTIRIAQPKLKSEILNITTWNPSLLGSTTALPNNSGKDVLSDVL
RATFDADTIRIAQPKLASAVMNLTTWNPTLLGEATMLDTSN---KFSDFL
RATFDADTIRIAQPKLATAVLDLTTWNPTLLGKATTVDGTN---TYSDFL
RATFDADTIRIAQPKLKSEILNITTWNPSLLGSTTTLPNNGGKDVLSDVL
RATFDADSIRIAQPKLAAAVLNLTTWNPTLLGEATALDASN---KFCDFL
351
366
QIASIQINKMKSRKAC
QIASIQINKMKSRKAC
QIASIQINKMKSRKAC
QLASIQINKMKSRKAC
QIASIQINKMKSRKAC
QIASIQINKMKSRKAC
17
Chapter 1
Because of the variation in the VD’s, all known genotypes of C. psittaci
can be identified by sequence analysis of the ompA gene and this analysis
correlates closely with the known serovars. Recently it was shown that
ompA sequence analysis can identify all known genotypes including the
newly discovered genotype E/B (Table 2). This new genotype E/B could
not be reliably identified by Mabs or RFLP of the ompA. Therefore,
ompA sequencing is considered the most accurate typing method for
C. psittaci. (22).
Table 2. C. psittaci genotypes and their preferred animal host
(adapted from Geens, Bush and Scientific committee on animal health
and animal welfare (8,22,65)).
Genotype
A
B
E/B
C
D
E
F
M56
WC
Representative strain
VS1
CP3
WS/RT/E30
GD
NJ1
MN
VS225
M56
WC
Host association
Psittacine birds
Pigeons
Ducks
Ducks, geese
Turkeys
Pigeons, turkeys
Psittacine birds
Cattle
Musk rat, snowshoe hare
All genotypes of C. psittaci are considered transmissible to humans.
However, it is unknown which genotype is most prevalent in human
infections. As psittacine bird contact is thought to be responsible for most
of the human infections a major role for psittacine derived genotypes is
assumed. Up till now, genotyping of strains obtained from a series of
human clinical samples has to our knowledge not been published.
18
Chapter 1
Clinical syndromes in humans
The main clinical syndrome associated with C. psittaci infection, as
illustrated by Ritter and Herderschêe, is community-acquired pneumonia
(CAP). CAP is mainly caused by Streptococcus pneumoniae,
Haemophilus influenzae, Mycoplasma pneumonia, and Legionella
pneumophila. Viruses like Influenza A can also cause a significant
number of CAP’s (64). Some studies on the etiology of pneumonia have
included serological tests for detection of Chlamydophila spp. infections.
With serology, between 1 and 6 % of CAP cases is thought to be due to a
Chlamydophila spp. (64). However, cross-reactivity, often due to the use
of the genus-specific antigen LPS, greatly influences the interpretation of
these data. Even with extensive diagnostic testing, a pathogen is
identified in only 30-60% of the CAP’s (1). The lack of easy detection
methods for obligate intracellular pathogens such as C. psittaci hamper
their diagnosis.
C. psittaci can cause a systemic infection, and therefore manifest itself by
other clinical syndromes than pneumonia. A substantial part of the
infections may be subclinical or may present as a non-specific illness
with fever, malaise and a sore throat (30). Sometimes this non-specific
illness can be accompanied by severe headache. Besides the aforementioned lung involvement, cardiac, neurological and dermatological
disorders have been associated with C. psittaci infection. Cardiac
involvement consists of endocarditis, pericarditis and myocarditis (66).
Neurological abnormalities include cranial nerve palsy, myelitis,
encephalitis and seizures. Dermatologic involvement can present as
erythema nodosum or erythema multiforme (19). In the acute phase,
psittacosis can be complicated by diffuse intravascular coagulation and
multi-organ failure (24,35). After the initial infection, reactive arthritis
can occur within several weeks (12). Very recently, inconclusive results
concerning the association of C. psittaci and mucosa associated lymphoid
tissue (MALT) tumors of the orbit have been published
(11,15,21,39,49,59). Whether this association is clinically relevant and
consistent awaits further studies.
Most patients present with 1) fever with rigors, sweats and constitutional
symptoms but no localizing features or 2) cough, fever and occasionally
dyspnea or 3) severe headache and fever almost resembling meningitis
(76). Before the antibiotic era, during outbreaks, mortality ranged from
approximately 20 to 50% (52,60). With proper recognition and treatment
19
Chapter 1
mortality is nowadays less then 1 percent (76). Tetracycline derivates are
the treatment of choice, although newer agents like the quinolones and
macrolides are probably also effective.
Premature abortion in women who had contact with lambing sheep can
be caused by C. abortus (previously classified as abortive Chlamydia
psittaci serovar 1 strains) (32,37,43). Sporadically, conjunctivitis and
respiratory tract infections have been recognized as a result of infection
with C. felis, formerly known as feline Chlamydia psittaci strains
(13,14,61).
Clinical syndromes in birds
Birds are mainly infected via the respiratory route. In birds, C. psittaci
produces a systemic infection, which can be (sub) acute or chronic.
Subclinical infections are common (40). Typical signs include respiratory
distress, sneezing, purulent nasal discharge, conjunctivitis, dullness,
anorexia, diarrhea and polyuria. Yellow-green droppings are commonly
found (40,65,71). In racing pigeons the infection may lead to decreased
flying performance. Outbreaks of psittacosis in turkey farms may be
accompanied by an increase in mortality. Although chickens can be
infected with C. psittaci, they are relatively resistant to infection. Ducks
are quite often asymptomatically infected (50).
Diagnosis
To diagnose C. psittaci infection, culture, serology, antigen testing, and
polymerase chain reaction (PCR) can be used. Culture is performed on
cell lines (for example Vero, McCoy or Buffalo Green Monkey cells). It
is essential that the clinical specimen containing the infectious EB’s is
centrifuged on the cell monolayer in a shell vial culture system. After
approximately 48 hours of incubation, the monolayer is stained (for
example with Chlamydia spp. specific fluoresceinated monoclonal
antibodies) and by fluorescence microscopy, chlamydial inclusions are
searched for. Because of the biosafety concerns, many laboratories have
abandoned culturing of C. psittaci. Currently psittacosis is mainly
diagnosed by serology. Micro-immunofluorescence (MIF), complement
fixation (CF) and ELISA tests are commonly used. Although MIF is
often recommended, it still does not appear to be fully species-specific
(6,74,75). Furthermore, this test is time-consuming and difficult to
20
Chapter 1
interpret. The CF test is the oldest available test for detection of
C. psittaci infection and has been widely used in the past because it is a
very specific test at genus level (2). However, CF tests appear to be less
sensitive than the other serologic tests like MIF and ELISA (74). ELISA
is a very sensitive test but cross reactivity and thus lack of specificity is a
major problem (51). In general, serologic testing has to be performed on
two serum samples: one obtained in the early course of the illness and a
second during convalescence, as only a fourfold rise in antibody titer is
diagnostic. Therefore, serology provides only a retrospective diagnosis.
Commercial tests are available for detection of C. trachomatis antigen in
human cervical swab samples. These tests use group-specific murine
derived chlamydial LPS antibodies. Because of their cross-reactivity with
the Chlamydophila spp. they are widely used to detect C. psittaci in avian
samples (33). Although sporadic reports describe their use on respiratory
samples, in general they are not routinely employed for antigen detection
in human respiratory clinical samples (69).
Currently, there are no commercially available nucleic acid amplification
tests (NAAT) for detection of C. psittaci in human clinical samples. For
other Chlamydiaceae like C. trachomatis and C. pneumoniae
commercially available NAATs are available. One of these tests detects
C. pneumoniae together with two other fastidious respiratory pathogens,
M. pneumoniae and Legionella spp., in patients with pneumonia (25).
C. psittaci PCR’s have been described in the literature, but no protocols
have been developed for real-time PCR platforms with probe
hybridization. In addition, they have not been evaluated on human
clinical samples (for example sputum), are prone to contamination
(nested PCR), do not include the uracil-N-glycosylase system and lack an
internal control to monitor the process of DNA purification,
amplification and detection (33,41,45). Recently, a real-time PCR was
described , which includes an internal control and specific TaqMan probe
hybridization (23). However, this assay was evaluated on avian samples
and not on human respiratory samples. In general, real-time PCR assays
are promising because they are sensitive, potentially specific to the
species level and combine amplification and probe-hybridization without
the need for post-amplification activities.
21
Chapter 1
Epidemiology and public health considerations
Humans become infected when they come in contact with infected birds
or their secretions. Infection is acquired through inhalation of the agent
from desiccated droppings, feather dust or nasal secretions. In the USA
psittacine birds are the main source of exposure, followed by pigeons,
turkeys, geese and ducks (53). Many cases are linked to pet birds and
poultry, however increasing evidence points at wild free-ranging birds as
a potential source of infections (68,73). Human cases of psittacosis occur
worldwide, either sporadically or in outbreaks. There are two major risk
groups: 1) persons who keep birds for hobby or 2) persons who are
occupationally exposed to birds. However, in a substantial number of
cases, a direct link with birds cannot be established. The incubation
period in humans is 1- 4 weeks (76). After natural infection immunity is
only partial and re-infection has been described (9). To achieve early
recognition and to reduce further spread, psittacosis is a notifiable disease
in the Netherlands and many other countries. In the Netherlands 27 and
33 cases were reported in the year 2003 and 2004 respectively (Graph 1)
(55,56). In Germany, during the same years, 41 and 15 cases were
reported (57,58). In Belgium, Flanders, 14 and 9 cases were notified in
2003 and 2004 (46,47). In the USA, 15 cases were reported in 2003 (10).
These numbers are generally considered underestimates.
Graph 1. Reported cases of psittacosis in the Netherlands from 19882005.
22
Chapter 1
After a decline in the 1990’s, a steady increase in reported cases is seen
since 2003 in the Netherlands. Diagnostic PCR assays were introduced in
at least 2 hospital laboratories in the Netherlands in October 2004. It is
hypothesized that the introduction of this technique aided in more
accurate diagnosis of infection with C. psittaci and a better understanding
and recognition of the associated clinical syndromes. This has probably
led to the recognition of more clusters and sporadic cases of psittacosis
(18,28).
To reduce human morbidity due to psittacosis, European
recommendations have been issued on how to deal with the disease in
animals and to provide appropriate control measures (65). In the USA
similar recommendations have been issued (67).
23
Chapter 1
Aims and outline of this thesis
The diagnosis of psittacosis is hampered by lack of sensitive, specific and
fast methods and hence the incidence of this notifiable disease is
probably highly underestimated. The aim of the work presented in this
thesis is a specific, sensitive and fast method to detect C. psittaci in
human clinical samples and to recognize the source of infection. This
would influence antibiotic treatment and could expedite outbreak
management. We chose real-time PCR as the preferred technique for
diagnosis and genotyping as a method for source detection.
In chapter 2 we describe a case of psittacosis with the classical problem
of diagnostic difficulties due to lack of fast and specific methods to
confirm this infection. This case of psittacosis triggered us to develop a
C. psittaci specific PCR with a real-time format suited for human clinical
samples (chapter 3). This PCR, performed on sputum, appeared to be
very helpful for rapid diagnosis in hospitalized patients. With this PCR,
we investigated an outbreak of psittacosis in a veterinary teaching
hospital. Many birds were involved in this outbreak and therefore a
genotyping method was developed to distinguish the different isolates
and identify the source of the outbreak (chapter 4). Although most cases
of psittacosis are linked to pet birds and poultry, there is increasing
evidence that wild birds are responsible for a substantial number of cases.
Like in many other European cities, in Amsterdam, the Netherlands, the
feral pigeon (Colombia livia) is an abundant bird species. As these birds
often live in close contact with humans, shedding of C. psittaci by these
birds is a potential zoonotic reservoir. Therefore, we investigated the
prevalence of C. psittaci in fresh fecal samples obtained from feral
pigeons in Amsterdam and determined the genotype of C. psittaci in the
positive samples (chapter 5). At present, the distribution of the different
genotypes of C. psittaci causing infections in humans is unknown.
Strains of C. psittaci that caused infection in humans, were genotyped by
ompA sequence analysis of C. psittaci PCR positive human clinical
samples available in our laboratory (chapter 6).
24
Chapter 1
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30
Chapter 2
A woman with a lobar infiltrate due to
psittacosis detected by polymerase chain
reaction
Edou R. Heddema 1, Maarten C. Kraan 2, Herma E. C. M. Buys-Bergen 3,
Hilde E. Smith 3, Pauline M. E. Wertheim-van Dillen 1.
1
) The Netherlands, Amsterdam, Academic Medical Centre/ University
of Amsterdam, Department of Medical Microbiology, Division of
Clinical Virology.
2
) The Netherlands, Amsterdam, Academic Medical Centre/ University
of Amsterdam, Department of Internal Medicine, Division of Clinical
Immunology and Rheumatology.
3
) The Netherlands, Lelystad, ID-Lelystad: Institute for Animal Science
and Health.
Adapted from the Scandinavian Journal of Infectious Diseases 2003;
35(6-7):422-4.
31
Chapter 2
Summary
We report a case of community-acquired pneumonia due to
Chlamydophila psittaci presenting with a lobar infiltrate and diagnosed
by a newly developed ompA gene based PCR. This gene encodes a
specific C. psittaci major outer membrane protein. This kind of PCR
could reduce antibiotic consumption and expedite outbreak management.
32
Chapter 2
Introduction
Community-acquired pneumonia (CAP) is caused by a broad range of
pathogens. Besides conventional bacteria (Streptococcus pneumoniae,
Haemophilus influenzae), fastidious agents (Chlamydophila species,
Mycoplasma pneumoniae, Legionella spp.) should be considered as
etiological agents. Even when serious efforts are made only 30-60% of
the community acquired pneumonia’s yield an identifiable pathogen (2).
Formerly attempts have been made to distinguish clinically between socalled “typical and atypical pathogens”. Symptoms and radiographic
appearance however could not reliably distinguish between these two.
Therefore respected authorities have advised to treat CAP with a
macrolide in combination with a beta-lactam antibiotic or a quinolon to
cover almost all possible pathogens (1,8). This results in broad antibiotic
regimens and therefore selection pressure. A rapid diagnosis in an early
stage could narrow the antibiotic therapy. The case described here
demonstrates that psittacosis can present itself with a lobar pneumonia.
By the use of a newly developed polymerase chain reaction (PCR) assay,
Chlamydophila psittaci was afterwards identified as the causative agent.
33
Chapter 2
Case report
A 46-year-old woman presented to our hospital with high fever, chills,
dyspnea and headache for the past 5 days. There was a non-productive
cough. As medication she used methotrexate 7.5 milligram once weekly
for her rheumatoid arthritis, which was stable for several years. On
physical examination there was a fever of 39.1 degrees Celsius, a pulse
of 109 beats/min. And a tachypnea of 28/min, dullness to percussion and
bronchial breathing sounds in the lower third of the left chest. The
remainder of the physical examination was normal including the absence
of active arthritis. Laboratory results revealed an elevated ESR with 98
mm/1st hr., haemoglobin 14.5 g/dl (8.5 mmol/l), leukocytes 12.4 * 109
/ml, serum creatinine 76 mmol/l, C-reactive protein 317 mg/l. Liver
enzymes and other chemistry tests were within normal range. Arterial
blood gas analysis revealed: pH 7.54, pCO2 3.1 kPa, pO2 7.1 kPa, bicarbonate 19.7 mmol/l, Base-Excess –1.2 mmol/l, O2 saturation 93.6%. A
chest radiograph showed a lobar infiltrate in the left lower lobe with
signs of an air bronchogram (Fig. 1). Treatment was initiated with
amoxicillin for community-acquired pneumonia. Because of persistent
fever, increased dyspnea and hypoxaemia, treatment was switched to a
combination of cefotaxim and erythromycin 48 hours after admission.
Furthermore, additional investigations were performed to exclude
rheumatoid disease, methotrexate toxicity and opportunistic and other
infections. Bronchoalveolar lavage and subsequent microscopy and
culture did not reveal a causative micro-organism (including Legionella
species and Pneumocystis carinii). A thorough search for fastidious
organisms and viruses was done serologically on paired serum samples
obtained on the 2nd and 7th day of admission. A CT-scan showed a
massive consolidation of the left lower lobe with some pleural effusion.
After the change in the antibiotic treatment the headache disappeared
within 24 hours. Four days later she was discharged from the hospital
with a course of clarithromycin in good clinical condition. The acute and
convalescent sera tested for Chlamydia spp. antibodies (Medac
Diagnostika, Hamburg, Germany) showed a seroconversion for IgG and
IgA. IgM was negative in both. On further questioning it was revealed
that she owned a rabbit and a guinea pig and that her neighbour was a
pigeon breeder. A few weeks later a PCR on the ompA gene of the
C. psittaci genome was positive in the patient’s stored bronchoalveolar
lavage fluid (performed by the ID-Lelystad: Institute for Animal Science
34
Chapter 2
and Health). Her neighbour’s pigeons were screened for C. psittaci in
faecal samples by the same PCR but all appeared negative.
Figure 1. The chest radiograph showing consolidation in left lower lobe.
35
Chapter 2
Discussion
This case demonstrates the delay often encountered in diagnosing
Chlamydophila spp. as the etiological agent of CAP. Only two weeks
after the initial infection, 10 days after admission to the hospital and 4
days after discharge from the hospital, seroconversion for
Chlamydia spp. could be obtained. Confirmation by PCR a few weeks
later identified C. psittaci as the causative agent. Unfortunately, the
broad-spectrum antibiotic course was already finished and a delay in
outbreak management occurred.
Psittacosis is in most cases linked to owing pet birds or working in a pet
store, but a relation with gardening has been established as well (10).
Birds are the main reservoir either as a carrier or being overtly ill. They
shed C. psittaci mainly in their faeces and nasal discharges. Currently C.
psittaci is classified as one of the species within the genus
Chlamydophila. They are obligate intracellular bacteria. Two forms are
recognised: the elementary bodies and the reticulate bodies. The first
stage, the infective form, lacks the ability to replicate, but is suited for
survival in the environment. The reticulate bodies are the fragile,
intracellular, metabolically active forms and have the ability to replicate.
Man can be infected when the elementary bodies are inhaled. Psittacosis
is therefore considered to be a zoönosis. The clinical signs and chest
radiograph are often indistinguishable from other causes of pneumonia.
Headache is often a non-specific sign but, in case of a pneumonia, can
point to psittacosis. However it has been found in Legionnaires’ disease
as well (9). Our patient had never experienced such a severe headache
and the disappearance of it within 24 hours after start of adequate therapy
was the first sign of improvement. Chest radiographs have been used for
a pattern-oriented approach (7). Unfortunately, a broad range of
pathogens and even the same species could be responsible for different
patterns. The presence of a lobar infiltrate is not a common finding in a
C. psittaci infection but has been documented previously (5).
Currently, the diagnosis can be established by means of culture, serology
and PCR. Culture is difficult, time-consuming and requires extensive
safety precautions. Therefore diagnosis is often made on clinical signs
and serological evidence. Serum can be assayed by complement fixation,
micro-immunofluorescence and enzyme linked immuno specific assay.
In this situation cross-reactions, lack of sensitivity and specificity are
major problems (4). Furthermore, because convalescent sera are needed,
36
Chapter 2
diagnosis is often too late to be of use in the choice of treatment. A PCR
analysis is not yet widely available, but progress has been made recently
in developing these specific and sensitive assays. In our case the
diagnosis was confirmed by a newly developed PCR based on the ompA
gene encoding the major outer membrane protein of C. psittaci (3).
Previously, this gene was demonstrated to be a specific target for C.
psittaci and DNA from Chlamydophila pneumoniae,
Chlamydia trachomatis and C. pecorum was not amplified in this assay
(6). In conclusion, if rapid, sensitive and specific diagnostic tests for
C. psittaci were available, antibiotic consumption could be reduced,
delay in outbreak management prevented and subsequent treatment of
infected birds performed. Real-time PCR is one of the promising assays
studied at the moment. As an approach to the etiological diagnosis of
CAP it should be the aim for the near future to diagnose respiratory
Chlamydophila spp. infections within 24-48 hours.
References
1. Bartlett, J. G., R. F. Breiman, L. A. Mandell, and T. M. File, Jr. 1998.
Community-acquired pneumonia in adults: guidelines for management. The
Infectious Diseases Society of America. Clin.Infect.Dis. 26:811-838.
2. Bernstein, J. M. 1999. Treatment of community-acquired pneumonia--IDSA
guidelines. Infectious Diseases Society of America. Chest 115:9S-13S.
3. Buys-Bergen, H. E., F. Zijderveld van, and Smith K.A. 2002. A PCR
method to detect Chlamydia psittaci in faeces of infected birds. Nederlands
tijdschrift voor Medische Microbiologie 10.
4. Ekman, M. R., M. Leinonen, H. Syrjala, E. Linnanmaki, P. Kujala, and P.
Saikku. 1993. Evaluation of serological methods in the diagnosis of
Chlamydia pneumoniae pneumonia during an epidemic in Finland.
Eur.J.Clin.Microbiol.Infect.Dis. 12:756-760.
5. Goupil, F., D. Pelle-Duporte, S. Kouyoumdjian, B. Carbonnelle, and E.
Tuchais. 1998. [Severe pneumonia with a pneumococcal aspect during an
ornithosis outbreak]. Presse Med. 27:1084-1088.
6. Hewinson, R. G., P. C. Griffiths, B. J. Bevan, S. E. Kirwan, M. E. Field, M.
J. Woodward, and M. Dawson. 1997. Detection of Chlamydia psittaci DNA
in avian clinical samples by polymerase chain reaction. Vet.Microbiol. 54:155166.
37
Chapter 2
7. Lynch, D. A. and J. D. Armstrong. 1991. A pattern-oriented approach to
chest radiographs in atypical pneumonia syndromes. Clin.Chest Med. 12:203222.
8. Niederman, M. S., J. B. Bass, Jr., G. D. Campbell, A. M. Fein, R. F.
Grossman, L. A. Mandell, T. J. Marrie, G. A. Sarosi, A. Torres, and V. L.
Yu. 1993. Guidelines for the initial management of adults with communityacquired pneumonia: diagnosis, assessment of severity, and initial
antimicrobial therapy. American Thoracic Society. Medical Section of the
American Lung Association. Am.Rev.Respir.Dis. 148:1418-1426.
9. Schlossberg, D. 2000. Chlamydia psittaci, p. 2005. In Principles and practice
of infectious diseases. Churchill Livingstone, Edinburgh.
10. Williams, J., G. Tallis, C. Dalton, S. Ng, S. Beaton, M. Catton, J. Elliott,
and J. Carnie. 1998. Community outbreak of psittacosis in a rural Australian
town. Lancet 351:1697-1699.
38
Chapter 3
Development of an internally controlled realtime PCR assay for detection of
Chlamydophila psittaci in the LightCycler 2.0
system
Edou R. Heddema1, Marcel G.H.M. Beld2, Bob de Wever1, Ankie A.J.
Langerak1, Yvonne Pannekoek1 and Birgitta Duim1.
1
Department of Medical Microbiology and Department of Clinical
2
Virology , Academic Medical Centre, University of Amsterdam,
Amsterdam, the Netherlands.
Adapted from Clinical Microbiology and Infection 2006 Jun;12(6):571-5.
39
Chapter 3
Abstract
Psittacosis can be diagnosed with culture, serology and PCR. We
developed a real-time PCR with a DNA purification and inhibition
control (internal control (IC)) to detect Chlamydophila psittaci DNA in
human clinical samples. Novel C. psittaci specific primers targeting the
ompA gene were developed. The IC DNA contains the same primer
binding sites, has the same length and nucleotide content as the
C. psittaci DNA amplicon, but has a shuffled probe binding region. The
lower limit of detection was 80 target copies per PCR corresponding to
6250 copies/ml in a clinical sample. Specificity was tested using
reference strains of 30 bacterial species. No amplification was observed
in any of these samples. Eight respiratory samples from 8 patients were
positive with this PCR. Six of these patients were confirmed with
serologic testing. Two patients had increasing antibody titers but did not
fulfil previously proposed criteria for serologically proven Chlamydia
spp. infection. The described real-time PCR is a sensitive, specific and
fast method to detect C. psittaci DNA in human clinical respiratory
samples.
40
Chapter 3
Introduction
Chlamydophila psittaci is an obligate intracellular micro-organism that
causes psittacosis in humans. Psittacosis is characterized by fever, chills,
headache, dyspnoea and cough (26). The chest Radiograph often shows
an infiltrate. The disease is acquired through contact with infected birds,
bird droppings or feather dust (20). In the year 2003, in the USA and in
the Netherlands, 15 and 27 cases were reported respectively (6,18). The
diagnosis of C. psittaci infections can be made by culture, serology and
DNA detection. Culture is time-consuming and requires extensive safety
precautions. Laboratory-associated infections are well known (19).
C. psittaci should therefore be handled under biosafety level 3 conditions
(14). The “gold standard” for diagnosing psittacosis is the measurement
of a four-fold increase in serum antibodies using micro-immune
fluorescence (MIF) (20). Although MIF is the recommended method, it
still does not appear to be as species-specific as claimed by the
manufacturer (20,25). Furthermore the test is difficult to interpret and
needs convalescent sera. Most often serologic testing provides only a
retrospective diagnosis. A diagnostic PCR can overcome these problems.
In addition, it can help clinicians to narrow antibiotic treatment and it can
expedite outbreak management. Although some PCR assays have been
described to detect C. psittaci, they lack an internal control to monitor
DNA purification and possible inhibition of the PCR. Furthermore they
are not developed for a real-time PCR format (12,13,17). Real-time PCR
is a fast and sensitive format. Previously we already emphasized the need
for such an assay to detect C. psittaci in humans (11). Therefore, we
developed a real-time PCR assay with an internal control to detect C.
psittaci in clinical samples.
41
Chapter 3
Materials and methods
Respiratory specimens
Eight respiratory specimens from 8 individuals (sputum (4), broncho
alveolar lavage (BAL) fluid (1), throat swabs (3)) were included. One of
these 8 patients was already positive for C. psittaci DNA in BAL fluid in
a PCR assay conducted elsewhere and previously published as case
report (11). Ten respiratory specimens (6 throat washes and 4 sputum
specimens) from ten patients with respiratory infections due to other
bacteria (non-chlamydial) or viruses were tested. These infections were
caused by respectively Respiratory syncytial virus (n=2), Parainfluenza
virus (n=3), Enterovirus (n=1), Staphylococcus aureus (n=1),
Enterobacter cloacae (n=1) and Haemophilus influenzae (n=2). These
pathogens were detected by standard culture procedures or direct
immunofluorescence. As pigeons are one of the main reservoirs of C.
psittaci, a pigeon breeder provided nine nose swabs obtained from nine
pigeons with nasal discharge possibly due to C. psittaci infection.
Bacterial strains
Thirty ATCC or Quality control assessment strains were used for
specificity experiments. They represent a panel of commonly
encountered bacterial species in clinical (respiratory) specimens
including related Chlamydiaceae spp. (Table 2). Escherichia coli (E. coli
One shot®, Invitrogen B.V. Breda, The Netherlands) was used for
propagation of cloned plasmid constructs. Genomic C. psittaci DNA was
purified from C. psittaci Orni strain, isolated from a human case of
psittacosis (15,16). This DNA was used for construction of a C. psittaci
DNA control. C. psittaci 6BC (ATCC VR-125), C. abortus (C18/98), C.
felis (02DC0026) and C. caviae (GPIC strain) were also tested in our
PCR (kindly provided by prof. D. Vanrompay, Ghent University,
Belgium).
DNA extraction
Extraction of C. psittaci DNA from clinical samples was done with a
modified Boom-extraction (3). To liquefy sputum, 80% (vol/vol) sputum
solution was made by mixing sputum specimens with 10% acetylcysteine
42
Chapter 3
solution (50 mg/ml (local pharmacy)) and 10% bacterial lysis buffer (10
mM Tris-HCL (PH 8.3, 1 mM EDTA)(TE) containing 1% SDS (Merck,
Darmstadt, Germany), 5% Tween-20 (Bio-Rad Laboratories, Hercules,
CA) and 5% sarkosyl (Sigma Aldrich chemie, Steinheim, Germany).
Thorough mixing was performed in sterile tubes (Greiner bio-one,
Cellstar®, 50 ml PP-Test tubes, Omega Scientific, Tarzana, CA)
containing approximately 20 washed and autoclaved glass beads
(Emergo, Landsmeer, The Netherlands). After mixing, the tubes were
incubated at room temperature for a minimum of 30 minutes. Of the
liquefied sputum, 190 µl together with 10 µl IC solution (~80
copies/PCR) was subjected to the Boom-extraction (3). The pigeon and
human throat swabs, 200 µl of the human throat washes or BAL fluid and
100 µl of the bacterial suspensions were immediately suspended in 900
µl L6 lysis buffer used in the Boom-extraction procedure without pretreatment. DNA was eluted in 100 µl of TE.
Primers and probes
Primers were designed to amplify a conserved region of the C. psittaci
ompA gene. All known C. psittaci ompA gene sequences present in the
GenBank database were included in this design. The primers used for
amplification were CPsittF (5’-CGC TCT CTC CTT ACA AGC C -3’;
nucleotide position (nt) 411 - 429) and CPsittR (5’-AGC ACC TTC CCA
CAT AGT G -3’; nt 474 - 492). Nucleotide numbering was derived from
the C. psittaci 6BC ompA gene, GeneBank accession number X56980.
The TaqMan probes used for detection of the C. psittaci and IC
amplicons were respectively CPsitt Probe (5’ FAM -AGG GAA CCC
AGC TGA ACC AAG TTT-3’ TAMRA) and CPsitt IC Probe (5’ VIC TCG AGA CAG TGC AAC GTA AGC CTA-3’ TAMRA). Both primers
and the FAM-TAMRA labeled probe were obtained from Isogen
Bioscience B.V. (Maarsen, The Netherlands). The VIC-TAMRA labelled
probe was obtained from Applied Biosystems. This primer pair amplifies
an 82-bp DNA fragment of the C. psittaci ompA gene as well as the IC
which has the same length and nucleotide content as the C. psittaci DNA
amplicon but a shuffled probe binding region.
43
Chapter 3
Construction of the C. psittaci DNA control
C. psittaci DNA was purified from the C. psittaci Orni strain and an
amplicon was generated using the CPsitt primer pair. The amplicon was
cloned into a PCR 2.1 plasmid, thereby creating pPsittWT (PCR 2.1;
Invitrogen B.V., Breda, The Netherlands). The pPsittWT was
propagated in E. coli and purified using the Wizard Plus Miniprep
isolation kit (Promega, Leiden, The Netherlands). The sequence of the
DNA insert of the pPsittWT was checked by dideoxynucleotide
sequencing (BigDye® Terminator v1.1 cycle sequencing kit, Applied
Biosystems). The concentration and purity of the isolated plasmid
construct was measured with a spectrophotometer at 260 and 280 nm
respectively, and stored in TE at -20 ˚C.
Construction of the internal control
The IC was constructed using 2 oligonucleotides: IC-CPsitt-1 (5’- CGC
TCT CTC CTT ACA AGC CTT GCC TGT TCG AGA CAG TGC AAC
GTA AGC CTA -3’) and IC-CPsitt-2 (5’- AGC ACC TTC CCA CAT
AGT GCC ATC GAT TAA TTA GGC TTA CGT TGC ACT GTC TCG
A -3’) as previously described (2). In short, two nanogram of both
oligonucleotides were annealed, extended and amplified using the above
mentioned CPsitt primer pair, thus creating an amplicon which contained
the two CPsitt primer sites, the same length and nucleotide content as the
C. psittaci amplicon but a shuffled probe binding region compared to the
target amplicon. This amplicon was cloned into a PCR 2.1 plasmid (PCR
2.1; Invitrogen B.V., Breda, The Netherlands) thereby creating the IC.
The IC was subsequently propagated in E. coli. DNA sequence analysis
of the IC, purification, quantification and storage of the IC plasmids was
done in the same manner as described for the pPsittWT.
Dilution series of pPsittWT and IC
Dilutions series of the pPsittWT and IC were used to determine the lower
limit of detection. Decreasing amounts were diluted in a stabilizing lysis
buffer (5.25 M GuSCN, 50 mM Tris-HCL (pH 6.4), 20 mM EDTA)
supplemented with 20 ng calf thymus DNA per µl and were stored at - 20
˚C. Extraction of the dilution series, corresponding to 80, 40, 20 and 10
copies / PCR, was performed in six fold. This was done in a background
44
Chapter 3
of 190 µl C. psittaci DNA negative pooled and liquefied sputum by the
Boom procedure (3).
Real-time PCR assay
Reactions were performed in the LightCycler 2.0 system (Roche
Diagnostics, Penzberg, Germany) using two TaqMan probes. The uracilN-glycosylase (UNG (Applied biosystems)) system was used to prevent
false positive reactions due to amplicon carry over. The final reaction
volume (20 µl) included 8 µl eluate, and contained 2 µl (10x)
LightCycler Faststart DNA Master Hybridization Probes Mix (Roche
Diagnostics, Penzberg, Germany), 0.2 U of UNG and a final
concentration of 0.3 µM of each probe, 0.7 µM of each primer and 4.5
mM MgCl2. The real-time PCR steps were as follows: 1) 50 ˚C for 10’,
2) 95 ˚C for 10’, 3) 49 cycles of 95 ˚C for 10’’, 62 ˚C for 5’’, 72 ˚C for
10’’ and 4) 30 ˚C for 30’’. Fluorescence values for the FAM and VIC
probe signal, used for detection of C. psittaci DNA or IC, were detected
in channel 530/610 back and 560/610 back nm, respectively. In the final
internally controlled PCR assay a colour compensation file, according to
the manufacturer’s instructions, was used to prevent crosstalk of the two
fluorescent probes signals.
45
Chapter 3
Results
Optimization of the real-time PCR for use with the TaqMan probes
The real-time PCR was optimized to achieve maximum sensitivity of the
assay. During the LightCycler assay the accumulation of amplicon is
shown by an increase in fluorescence emitted by the FAM-reporter dye.
Emission takes place after hydrolysis of the TaqMan probe. Initially,
analysis of 10 µl of the amplified products showed large amounts of the
80 bp amplicon on an 2,5 % agarose gel that was disproportionate to the
detected fluorescence signal (data not shown). The LightCycler’s default
temperature transition rate setting (20 ˚C / sec) seemed insufficient for an
adequate fluorescence signal. Therefore we optimized the real-time PCR
by varying the temperature transition rate. Using a temperature transition
rate of 1 ˚C / sec during the annealing step allowed optimal binding of
the TaqMan probes and adequate fluorescence signals as shown by the
lower limit of detection.
Determination of the lower limit of detection of the C. psittaci realtime PCR
In a background of C. psittaci negative pooled sputum with decreasing
pPsittWT amounts, we were always able to detect 80 copies of pPsittWT
per PCR (6/6 runs positive, “100% hit rate”) (Table 1). The lowest
detection limit for pPsittWT was 10 copies per PCR (1/6 positive).
Almost identical results were obtained for decreasing amounts of IC. We
were also able to detect 80 copies IC per PCR (6/6 runs positive). The
lowest detection limit for IC was 10 copies per PCR (3/6 positive).
Therefore we used 80 copies of IC in the internally controlled PCR assay.
In the internally controlled real-time PCR assay with decreasing amounts
of pPsittWT and 80 copies of IC we were always able to detect 80 copies
of pPsittWT as well (6/6 runs positive). When 80 until 80,000 copies
pPsittWT were tested in the internally controlled real-time PCR assay
with 80 copies IC per PCR, the result was always positive. This suggests
that there is no significant competition between the pPsittWT and IC
when at least 80 copies of pPsittWT are present in a clinical sample (data
not shown). Detection of 80 copies per PCR would correspond to a
minimum sensitivity of 6250 copies/ml in a clinical sample (sputum).
46
Chapter 3
Table 1. Lower limit of detection of the PCR assay for pPsittWT, IC and
the combination.
pPsittWT with 80 copies of ICa
Copies per PCR
pPsittWT a IC a
80b
6/6
6/6
6/6
40
4/6
3/6
2/6
20
2/6
2/6
1/6
10
1/6
3/6
0/6
0
0/2
0/2
0/2
a
The data represent number of samples positive versus the number of samples tested
For all the copy numbers presented, we assumed a 100% extraction and PCR
efficiency.
b
Specificity of the real-time PCR assay
No amplification was observed when DNA of 30 bacterial species,
including related Chlamydiaceae spp., was tested in the PCR (Table 2).
All IC signals were positive indicating a true negative result. Although
this PCR is not developed as a quantitative test, mean Ct value (crossing
point) for the IC signal in the 30 bacterial samples was 34.1 cycles
(standard deviation of the mean (stdev) 1.3). DNA obtained from the
avian type strain C. psittaci 6BC (ATCC VR-125), C. abortus (C18/98),
C. felis (02DC0026) and C. caviae (GPIC strain) were also tested in our
PCR. They amplified as expected based on the sequence homology (9).
Respiratory specimens
Four sputa, 1 BAL fluid and three throat swabs were positive in our realtime PCR. These 8 PCR positive samples were tested six fold in separate
PCR runs. Mean Ct values for these positive samples when tested six fold
were between 27.2-35.9 cycles (stdev ranged from 0.3-1.5). There was no
clear association between the obtained Ct values and the different
respiratory samples (ic. BAL, sputum or throat swab). In our institution
we use an ELISA as serological tool for the diagnosis of C. psittaci
infections (Chlamydia IgG/A/M rELISA, Medac Diagnostika, Hamburg,
Germany). The serological diagnosis is often determined in acute phaseand convalescent sera by a three fold rise in Chlamydia – IgG, a twofold
or greater change in the IgM or a twofold increase in the IgG titer in
combination with a twofold increase in the IgA antibody titer (21,22). Six
out of these 8 cases were serologically confirmed when applying the
47
Chapter 3
above rules. However two cases did not completely apply to these
proposed criteria. One case had positive IgA and IgM titres that did not
change and a twofold rise in IgG serum antibodies and the other case had
a twofold rise in IgA together with increasing IgG serum antibodies
however not reaching a double titre. Three out of the 9 nose swabs
obtained from 9 pigeons were positive for C. psittaci. The 10 respiratory
samples from 10 patients with other respiratory infections were negative.
All PCR negative samples were truly negative, because the IC was
positive.
48
Chapter 3
Table 2. Bacterial species used for specificity testing in the C. psittaci
PCR.
Species
Source
PCR result/ IC result
H. influenzae
ATCC 49247
-/+
P. aeruginosa
ATCC 27853
-/+
S. aureus
ATCC 29213
-/+
S. agalactiae
ATCC 624
-/+
N. meningitides
ATCC 13090
-/+
S. pneumoniae
ATCC 49619
-/+
B. cepacia
ATCC 25416
-/+
C. trachomatis L2
ATCC VR 902b
-/+
C. pneumoniae AR39
ATCC 53592
-/+
C. pneumoniae CWL 029 ATCC VR-1310
-/+
C. pneumoniae TW-183
ATCC VR-2282
-/+
M. pneumoniae
ATCC 15492
-/+
E. cloacae
ATCC 700323
-/+
P. vulgaris
ATCC 6380
-/+
E. coli
ATCC 35218
-/+
S. pyogenes
QC
-/+
F. varium
QC
-/+
C. ulcerans
QC
-/+
N. gonnorhoea
QC
-/+
S. mucilaginosus
QC
-/+
A. hemolyticum
QC
-/+
R. equi
QC
-/+
B. pertussis
QC
-/+
K. pneumoniae
QC
-/+
L. pneumophila
QC
-/+
M. catarrhalis
QC
-/+
a
C. pecorum
E58
-/+
C. trachomatis Serovar D IC-CAL-8 a
-/+
a
C. trachomatis Serovar E
DK-20
-/+
a
C. trachomatis Serovar F
MRC-301
-/+
a)
, previously studied and characterized by Meyer et al. (15)
ATCC, American Type Culture Collection
QC, Dutch or UK quality control assessment strains
49
Chapter 3
Discussion
The real-time PCR described in this study is a sensitive and specific
format for diagnosing psittacosis. This is the first report of an internally
controlled real-time PCR assay to detect C. psittaci DNA in the
LightCycler 2.0 system using 2 TaqMan probes. Each extracted sample
included an IC mimicking the C. psittaci target, except for the shuffled
probe binding site. The use of this IC enabled the detection of false
negative results, and when the IC amplified well, it ensured an optimal
performance of the real-time PCR. In fact, it monitors the process of
nucleic acid purification and amplification for each individual sample.
When the described method to liquefy sputum was followed, the Boom
extraction seemed to be a powerful tool for purification of DNA from
sputum samples, as it was always possible to detect 80 copies of
pPsittWT or IC per PCR. Specificity of the real-time PCR assay was
tested with a set of typed bacterial species. No amplification was
observed with any of these bacteria tested including related
Chlamydiaceae spp. The negative results for the respiratory samples
from the patients with evidence for other respiratory infections, that
contain numerous commensal throat bacteria, confirm the specificity of
the real-time PCR.
In the recently revised new taxonomic classification C. psittaci has been
subdivided in 4 Chlamydophila spp. : C. abortus, C. psittaci, C. felis and
C. caviae (9,10). This new classification included new available
sequence data and showed the relatedness of Chlamydophila spp. in
typical hosts (for example cats, birds and guinea pigs). The new
classification is mainly based on minor sequence differences in the 16S
rRNA gene, 23S rRNA gene and internal ribosomal spacer region (9).
When we developed the C. psittaci primers and probes using sequences
available in the GenBank database, we observed that our primers and
probe have the ability to amplify and detect the other 3 species as well.
Detection of DNA of these closely related bacterial species by our PCR
confirms this observation. The sequence homology in the ompA gene did
not allow us to design primers that could distinguish these 4 species on a
LightCycler format. For clinical purposes this is not important since all 4
species are considered potentially infectious for humans (7,8). However,
sequence analyses on the ompA gene can be performed for strain or
serovar speciation (5). The incidence of confirmed cases of psittacosis is
low (6,18), but the number may be underestimated as accurate methods
50
Chapter 3
for diagnosis of psittacosis are not always available. In addition, many
patients with this disease are unable to produce sputum and receive broad
antibiotic treatment without invasive sampling (broncho-alveolar lavage).
Given these facts, the number of samples for clinical evaluation of the
PCR is limited. In this study we were able to detect C. psittaci DNA in 8
respiratory samples obtained from 8 patients. The 8 PCR positive patients
we report represent approximately 30% of the annual reported cases in
the Netherlands (8/27) (18). One of these 8 patient samples was already
tested positive in another PCR with a different primer set and was
published in detail (11,12). Six out of 8 cases were serologically
confirmed using the above mentioned criteria. Two cases showed
increasing titres of Chlamydia spp specific IgG serum antibodies but did
not meet the proposed criteria. In general serologic test for the diagnosis
of C. psittaci infection are hampered by lack of sensitivity, specificity
(genus and/or species) and some have poor reproducibility (1,4,24,25).
These two PCR positive, but serologically negative cases highlight this
problem. A false positive result is unlikely as we defined the specificity
of this PCR using a panel of 30 bacterial species, including related
Chlamydiaceae spp. and 10 respiratory samples. But it is always difficult
to validate a sensitive and specific newly developed PCR against a poor
“gold standard”. Although the positive pigeon samples lacked a
comparison with a gold standard, we tested these samples in order to
determine if we could detect C. psittaci DNA in highly suspected animal
reservoirs. The detection of C. psittaci DNA in 3 out of 9 pigeons with a
possible clinical picture of C. psittaci infection is highly suggestive of
true disease. The availability of this test may persuade clinicians to
obtain adequate respiratory samples. Results of this PCR can be obtained
in a few hours and avoids waiting for serologic confirmation. As C.
psittaci outbreaks have been described, this PCR can be applied for rapid
identification and management of psittacosis outbreaks (19,23). This can
substantially expedite outbreak management. In conclusion, this PCR is a
valuable addition to the diagnostic tools available for patients suspected
of having psittacosis. It avoids waiting for serologic testing and bypasses
the need for culture. This real-time PCR assay is a fast, specific and
sensitive method to detect C. psittaci DNA in human respiratory
specimens. It can help clinicians to narrow the antibiotic treatment and it
can expedite outbreak management. The inclusion of an IC in this PCR
excludes the occurrence of false negative PCR results.
51
Chapter 3
Acknowledgements
We thank H.C.J.G. (Herry) Peters, pigeon breeder, for collecting the 9
pigeon nose swabs, Naomi E. Vrede for the construction of the internal
control and prof. D. Vanrompay (Ghent university, Belgium) for
providing the C. abortus, C. caviae and C. felis strains.
References
1. Bas, S., P. Muzzin, B. Ninet, J. E. Bornand, C. Scieux, and T. L. Vischer.
2001. Chlamydial serology: comparative diagnostic value of immunoblotting,
microimmunofluorescence test, and immunoassays using different recombinant
proteins as antigens. J.Clin.Microbiol. 39:1368-1377.
2. Beld, M., R. Minnaar, J. Weel, C. Sol, M. Damen, van der Avoort H., P.
Wertheim-van Dillen, A. van Breda, and R. Boom. 2004. Highly sensitive
assay for detection of enterovirus in clinical specimens by reverse
transcription-PCR with an armored RNA internal control. J.Clin.Microbiol.
42:3059-3064.
3. Boom, R., C. J. Sol, M. M. Salimans, C. L. Jansen, P. M. Wertheim-Van
Dillen, and van der Noordaa J. 1990. Rapid and simple method for
purification of nucleic acids. J.Clin.Microbiol. 28:495-503.
4. Bourke, S. J., D. Carrington, C. E. Frew, R. D. Stevenson, and S. W.
Banham. 1989. Serological cross-reactivity among chlamydial strains in a
family outbreak of psittacosis. J.Infect. 19:41-45.
5. Bush, R. M. and K. D. Everett. 2001. Molecular evolution of the
Chlamydiaceae. Int.J.Syst.Evol.Microbiol. 51:203-220.
6. CDC. 2004. Notifiable Diseases/Deaths in Selected Cities Weekly
Information. MMWR 52:1291-1299.
7. Corsaro, D., D. Venditti, and M. Valassina. 2002. New parachlamydial 16S
rDNA phylotypes detected in human clinical samples. Res.Microbiol. 153:563567.
8. Cotton, M. M. and M. R. Partridge. 1998. Infection with feline Chlamydia
psittaci. Thorax 53:75-76.
9. Everett, K. D., R. M. Bush, and A. A. Andersen. 1999. Emended description
of the order Chlamydiales, proposal of Parachlamydiaceae fam. nov. and
Simkaniaceae fam. nov., each containing one monotypic genus, revised
taxonomy of the family Chlamydiaceae, including a new genus and five new
species, and standards for the identification of organisms. Int.J.Syst.Bacteriol.
49 Pt 2:415-440.
52
Chapter 3
10. Garrity G.M., Bell J.A., and Lilburn T.G. 2003. Bergey's Manual of
Systematic Bacteriology, p. 300-301. Springer-Verlag.
11. Heddema, E. R., M. C. Kraan, H. E. Buys-Bergen, H. E. Smith, and P. M.
Wertheim-Van Dillen. 2003. A woman with a lobar infiltrate due to
psittacosis detected by polymerase chain reaction. Scand.J.Infect.Dis. 35:422424.
12. Hewinson, R. G., P. C. Griffiths, B. J. Bevan, S. E. Kirwan, M. E. Field, M.
J. Woodward, and M. Dawson. 1997. Detection of Chlamydia psittaci DNA
in avian clinical samples by polymerase chain reaction. Vet.Microbiol. 54:155166.
13. Madico, G., T. C. Quinn, J. Boman, and C. A. Gaydos. 2000. Touchdown
enzyme time release-PCR for detection and identification of Chlamydia
trachomatis, C. pneumoniae, and C. psittaci using the 16S and 16S-23S spacer
rRNA genes. J.Clin.Microbiol. 38:1085-1093.
14. Mahony J.B., Coo, Coombes B.K., and Chernesky M.A. 2003. Chlamydia
and Chlamydophila, p. 991-1004. In P. R. Murray, E. J. Baron, Jorgensen J.H.,
Pfaller M.A., and Yolken R.H. (eds.), ASM Press, Washington, D.C.
15. Meijer, A., G. J. Kwakkel, A. De Vries, L. M. Schouls, and J. M.
Ossewaarde. 1997. Species identification of Chlamydia isolates by analyzing
restriction fragment length polymorphism of the 16S-23S rRNA spacer region.
J.Clin.Microbiol. 35:1179-1183.
16. Meijer, A., S. A. Morre, A. J. van den Brule, P. H. Savelkoul, and J. M.
Ossewaarde. 1999. Genomic relatedness of Chlamydia isolates determined by
amplified fragment length polymorphism analysis. J.Bacteriol. 181:4469-4475.
17. Messmer, T. O., S. K. Skelton, J. F. Moroney, H. Daugharty, and B. S.
Fields. 1997. Application of a nested, multiplex PCR to psittacosis outbreaks.
J.Clin.Microbiol. 35:2043-2046.
18. RIVM. 2004. Notified cases of infectious diseases in the Netherlands. Dutch
Infectious Diseases Bulletin 15.
19. Sewell, D. L. 1995. Laboratory-associated infections and biosafety.
Clin.Microbiol.Rev. 8:389-405.
20. Smith, K. A., K. K. Bradley, M. G. Stobierski, and L. A. Tengelsen. 2005.
Compendium of measures to control Chlamydophila psittaci (formerly
Chlamydia psittaci) infection among humans (psittacosis) and pet birds, 2005.
J.Am.Vet.Med.Assoc. 226:532-539.
21. Verkooyen, R. P., N. A. Van Lent, S. A. Mousavi Joulandan, R. J. Snijder,
J. M. van den Bosch, H. P. Van Helden, and H. A. Verbrugh. 1997.
Diagnosis of Chlamydia pneumoniae infection in patients with chronic
53
Chapter 3
22.
23.
24.
25.
26.
obstructive pulmonary disease by micro-immunofluorescence and ELISA.
J.Med.Microbiol. 46:959-964.
Verkooyen, R. P., D. Willemse, S. C. Hiep-van Casteren, S. A. Joulandan,
R. J. Snijder, J. M. van den Bosch, H. P. Van Helden, M. F. Peeters, and
H. A. Verbrugh. 1998. Evaluation of PCR, culture, and serology for diagnosis
of Chlamydia pneumoniae respiratory infections. J.Clin.Microbiol. 36:23012307.
Williams, J., G. Tallis, C. Dalton, S. Ng, S. Beaton, M. Catton, J. Elliott,
and J. Carnie. 1998. Community outbreak of psittacosis in a rural Australian
town. Lancet 351:1697-1699.
Wong, K. H., S. K. Skelton, and H. Daugharty. 1994. Utility of complement
fixation and microimmunofluorescence assays for detecting serologic
responses in patients with clinically diagnosed psittacosis. J.Clin.Microbiol.
32:2417-2421.
Wong, Y. K., J. M. Sueur, C. H. Fall, J. Orfila, and M. E. Ward. 1999. The
species specificity of the microimmunofluorescence antibody test and
comparisons with a time resolved fluoroscopic immunoassay for measuring
IgG antibodies against Chlamydia pneumoniae. J.Clin.Pathol. 52:99-102.
Yung, A. P. and M. L. Grayson. 1988. Psittacosis--a review of 135 cases.
Med.J.Aust. 148:228-233.
54
Chapter 4
An outbreak of psittacosis due to
Chlamydophila psittaci genotype A in a
veterinary teaching hospital
Edou R. Heddema1, Erik J. van Hannen2, Birgitta Duim1, Bartelt M. de
Jongh2, Jan A. Kaan3, Rob van Kessel4, Johannes T. Lumeij5, Caroline E.
Visser1, Christina M.J.E. Vandenbroucke-Grauls1,6
1
) Department of Medical Microbiology, Academic Medical Centre,
University of Amsterdam, Amsterdam, the Netherlands.
2
) Department of Medical Microbiology and Immunology, St. Antonius
Hospital, Nieuwegein, the Netherlands.
3
) Department of Medical Microbiology and Immunology, Diakonessen
Hospital, Utrecht, the Netherlands.
4
) Department of Infectious Diseases and Hygiene, Municipal Health
Service, Utrecht, the Netherlands
5
) Department of Clinical Sciences of Companion Animals, Faculty of
Veterinary Medicine, Division of Avian and Exotic Animal Medicine,
University of Utrecht, Utrecht, the Netherlands.
6
) Department of Medical Microbiology & Infection Control, VU
University Medical Centre, Amsterdam, the Netherlands.
Adapted from Journal of Medical Microbiology 2006 nov;55(11):157175.
55
Chapter 4
Summary
An outbreak of psittacosis in a veterinary teaching hospital was
recognized in December 2004. Outbreak management was instituted to
evaluate the extent of the outbreak and to determine the avian source.
Real-time PCR, serologic testing and sequencing of the ompA gene of
Chlamydophila psittaci were performed. Sputum samples from patients,
throat swab samples from exposed students and staff and faecal
specimens from parrots and pigeons were tested. In this outbreak 34 %
(10/29) of the tested individuals were infected. The clinical features of
the infection ranged from none to sepsis with multi-organ failure
requiring intensive care unit admission. C. psittaci genotype A was
identified as the outbreak strain. Parrots, recently exposed to a group of
cockatiels coming from outside the teaching facility, which were used in
a practical teaching session appeared to be the source of the outbreak.
One of the tested pigeons harboured an unrelated C. psittaci genotype B
strain.
The microbiological diagnosis by real-time PCR on clinical specimens
allowed for rapid outbreak management; subsequent genotyping of the
isolates identified the avian source. Recommendations are made to
reduce the incidence and extent of future outbreaks.
56
Chapter 4
Introduction
Psittacosis is a disease caused by infection with Chlamydophila psittaci,
an obligate intracellular bacterium. It is a zoonosis since the main
reservoir of C. psittaci is birds, which can transmit the bacterium to man.
Symptoms in birds range from none to overt disease. Both carriers and ill
animals can shed the bacterium from many sites including nasal- and
faecal secretions. Two distinct forms of this pathogen are recognised: the
infectious elementary bodies and the fragile, metabolically active
reticulate bodies. C. psittaci is classified into eight serovars (A-F, WC
and M56). Infection in man occurs when elementary bodies are inhaled.
Fever, chills, headache, dyspnoea and cough usually characterize the
disease in humans. The chest Radiograph often shows an infiltrate
(13,16,21). Serologic tests are mainly used for diagnosis. The main
drawback of most commonly used serologic tests like enzyme-linked
immunosorbent assay (ELISA), microimmunofluorescence (MIF) or
complement-fixing antibody (CF) tests is that they all give only a
retrospective diagnosis. In this study we describe an outbreak of
psittacosis in a veterinary teaching hospital. Real-time PCR allowed for
rapid outbreak management and together with serologic testing and
genotyping we evaluated the extent of the outbreak and determined the
avian source.
57
Chapter 4
Methods
Background
On the fifth of January 2005 the Department of Medical Microbiology of
the Academic Medical Centre in Amsterdam was informed of an
outbreak of psittacosis in a veterinary teaching hospital. Two people were
already admitted to two different hospitals for presumed psittacosis. One
of them was a veterinarian who was admitted on the 14th of December
2004 and had followed a post-graduate teaching course on the 30th of
November 2004 provided by the veterinary teaching hospital (index
case). The second person was a staff member of the veterinary hospital
admitted on the 5th of January and not involved in the post-graduate
teaching. A third patient was admitted on the 13th of January when the
outbreak was already recognised. Sputa of the three patients were tested
positive for C. psittaci with an in-house-real-time PCR assay in the St.
Antonius hospital, Nieuwegein, the Netherlands. The suspected source
was a flock of nine cockatiels that were used in a post-graduate teaching
session on the 30th of November. These cockatiels were untraceable
when the outbreak was recognized. The cockatiels were used only once
in the post-academic teaching session together with nine Amazon parrots
and 144 pigeons from the teaching hospital. In the past, the Amazon
parrots were tested negative several times for C. psittaci by immunoassay
(QuickVue, Quidel, Marburg, Germany). The exposed parrots and
pigeons were used again in a practical for veterinary students on the 21st
and 23 rd of December in the veterinary teaching hospital. Some of these
parrots became overtly ill in the first week of January 2005. The extent of
this outbreak was investigated by offering all students and staff the
possibility for serologic testing and PCR for C. psittaci on sputum or a
throat swab. In addition we obtained faeces or cloacal swabs from the
available birds involved in this outbreak, to establish the source.
Inclusion
The following cases for whom PCR on a throat swab and serologic
testing on two consecutive serum samples could be performed, were
included: the index case, all students and staff working at the Division of
Avian and Exotic Animal Medicine, where the parrots were
accommodated, and all students who participated in the practical. We
58
Chapter 4
obtained faecal specimens from the nine parrots and cloacal swab
specimens from a subset of 23 out of 144 pigeons. These 23 pigeon
samples were randomly obtained from the 144 pigeons that were held in
7 cages. From each cage at least 2 pigeons were sampled.
Investigations and case definition
Serological testing was performed on two consecutive sera drawn at least
two weeks apart (Chlamydia IgG/A/M rELISA, Medac Diagnostika,
Hamburg, Germany). A psittacosis case was considered serologically
proven by a three fold rise in Chlamydia spp. – specific IgG, a twofold or
greater change in the specific IgM or a twofold increase in the specific
IgG titre in combination with a twofold increase in the specific IgA
antibody titre (11,18,19). The complement fixation test (CF) was
performed with a commercially available Chlamydia group antigen
(Virion, Zurich, Switzerland) on all rELISA positive sera. A fourfold rise
in CF titres was considered a true positive result. PCR was performed on
faeces (parrots), cloacal swabs (pigeons) or throat washes, sputum and
throat swabs (humans) with a recently developed and validated real-time
protocol (6). Briefly, this real-time PCR assay targets an 82 bp fragment
of the ompA gene of C. psittaci as well as an internal control plasmid
(IC). Throat wash samples (20 ml) were only obtained in a subset of the
participating students. DNA was extracted according to the
guanidiniumthiocyanate-silica-procedure (1,2). All participants received
a questionnaire in which information was asked on gender, age, day of
disease onset, antibiotic use and symptoms (headache, fever, muscle
aches, dyspnoea and chills). A person was considered as infected with C.
psittaci if a positive PCR or serologic evidence for Chlamydia spp.
antibodies could be obtained. C. psittaci PCR positive samples were
genotyped by sequencing of the ompA gene as previously described (7).
MEGA3 was used for editing and aligning the individual sequences and
for phylogenetic analysis (9). A similarity index was calculated based on
the translation of a 921 bp fragment of the ompA gene. Reference ompA
genotype sequences available in the GenBank database (GeneBank
accession numbers AY762608-12, AF269261) were included in this
analysis (4,5).
59
Chapter 4
Results and discussion
Initially 38 exposed students and staff members participated (figure 1).
For 29 individuals (8 male, 21 female), PCR and convalescent sera were
available. Mean age was 37 years (range 19-61).
In total, we identified 10 cases of psittacosis (Table 1). Of the tested
individuals 34 % (10/29) were therefore infected. Three individuals were
admitted to three different hospitals, the index case and two staff
members of the faculty. They were C. psittaci PCR positive in sputum.
One of them presented with sepsis and multi-organ failure and was
admitted to the intensive care unit. The other two presented with
community-acquired pneumonia. In these the patients, bloodcultures,
sputum cultures and in-house PCR’s for detection of Legionella spp. ,
Mycoplasma pneumoniae and Chlamydophila pneumoniae DNA were all
negative except for one sputum culture that was rejected because of bad
quality and one sputum culture that grew Staphylococcus aureus. This
pathogen was however not considered as the causative agent in that
patient.
Of the remaining 26 students and staff members three were C. psittaci
PCR positive on a throat swab and showed seroconversion in the
rELISA. They remained asymptomatic or had a brief self-limiting illness
(fever, headache). None of them received antibiotic treatment. A PCR on
a second throat swab, three weeks later, was negative for two of these
three students. One student remained PCR positive when sampled three
weeks and two months later. Throat wash samples were obtained from 16
students, but none of these samples was PCR positive. One throat wash
sample was obtained from a student who was PCR positive on a throat
swab sample.
Four students were PCR negative, but serologically confirmed (Table 1).
One of these four students did not exactly meet the definition for a
positive rELISA result because she had a 2.7 fold (instead of 3) increase
in IgG. She did not reach a threefold increase in IgG , mainly because her
first serum sample (drawn 8 days after symptom onset) was already
positive for IgG. The CF test on these sera showed a four fold increase.
Therefore she was included in the analysis. Three out of these 4 PCR
negative, but serologically confirmed cases had symptoms and received
doxycycline treatment. Inhibition of the PCR in these 4 cases was
excluded as the IC amplified correctly. The serologic assays used in this
research are genus- and not species specific. Therefore, these tests do not
60
Chapter 4
differentiate between antibodies directed against C. psittaci,
C. pneumoniae or Chlamydia trachomatis. Some authors have
recommended the use of the micro-immunefluorescence test (MIF)
which uses C. psittaci elementary bodies as antigen. It should be more
specific then the rELISA and CF test which we have used. However,
several reports have shown that the use of MIF still results in
considerable cross-reactivity between the different Chlamydia- and
Chlamydophila spp. (3,20). In addition, MIF is difficult to interpret for
people who do not use this test on a regular base and reproducibility is
poor. For these reasons we did not test the serum samples of these four
cases with MIF. But the combination of the clinical picture and obvious
contact with infected birds almost excludes the other two pathogens.
The reported symptoms were muscle aches (3/3), severe headache (3/3),
fever (3/3), dyspnoea (2/3) and chills (2/3). All symptoms resolved
rapidly with doxycycline treatment. As the day of disease onset and the
date of the practical were known, it was possible to establish the
incubation periods for these three students. These were 12, 12 and 14
days respectively. One student received doxycycline from his general
practitioner for suspected psittacoses, while subsequent PCR on a throat
swab and serologic testing remained negative.
Six out of nine parrots were C. psittaci PCR positive in faecal samples.
Only one of the 23 pigeon samples was PCR positive. All nine parrots
and 144 pigeons received doxycycline treatment. The ompA gene could
be amplified and sequenced directly from the clinical specimens obtained
from one of the hospital patients, three students, three parrots and the
pigeon. All sequences obtained from the human respiratory samples and
the parrot faeces were identical to the C. psittaci ompA genotype A
reference strain. The sequence obtained from the pigeon was similar to
the reference genotype B sequence and thus unrelated to the outbreak.
The sequences of the ompA gene of the outbreak strain and the unrelated
pigeon isolate were submitted to GenBank and designated C. psittaci
OSV and C. psittaci CLV (accession no. DQ230095 and DQ230096).
61
Chapter 4
Figure 1. Flow diagram of the outbreak
38 people included
3 admitted to hospital
29 PCR and convalescent
serum samples available
19 PCR and serologically
negative
3 PCR positive on
sputum
7 serologically proven
4 PCR negative on a
throat swab
2 serologically proven
3 PCR positive on a
throat swab
1 ICU*: sepsis, MOF§
2 pneumonia
2 no symptoms
1 brief self-limiting illness
Hospital
Veterinary teaching hospital
3 symptoms and treated
1 without symptoms
* ICU, intensive care unit
§ MOF, multi-organ failure
62
Chapter 4
Gender
Age
Clinical features
PCR specimen
PCR
rELISA
Complement Fixation test
Incubation period (days)
Days between 1st and 2nd
serum sample
Table 1. Patient characteristics
M
F
M
F
F
F
M
M
F
F
37
37
61
26
27
29
28
35
25
30
Sepsis
Pneumonia
Pneumonia
Fever, headache
Fever, headache
Fever, headache
none
none
none
Fever, headache
Sputum
Sputum
Sputum
Throat swab
Throat swab
Throat swab
Throat swab
Throat swab
Throat swab
Throat swab
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Na#
14
Na
12
12
11
Na
Na
Na
14
14
29
46
21
21
21
41
21
28
15
#
not applicable
We describe a psittacosis outbreak in humans and birds where real-time
PCR was used for detection of C. psittaci and sequencing of the ompA
gene for genotyping of the isolates. Six people, six parrots and one
pigeon were C. psittaci PCR positive. Genotyping of the isolates on the
ompA gene identified the parrots as the source of the outbreak. The
outbreak strain appeared to be a genotype A strain. The identification of
an unrelated genotype B of C. psittaci in contact pigeons emphasizes the
need for strain identification in human and animal outbreaks to gain a
better understanding of the epidemiology of psittacosis in humans and
birds.
In this outbreak 34 % of the tested population was infected. Huminer and
Schlossberg described outbreaks where 81 % (n=37) and 54 % (n=24) of
their tested population was infected with C. psittaci (8,13). The spectrum
of symptoms ranged from none to sepsis with multi-organ failure
63
Chapter 4
requiring intensive care unit admission. This diversity of symptoms is in
agreement with what is described by others (8). During this outbreak
three people were admitted to three different hospitals, three students
were treated by their general practitioner for psittacosis and another 4
students were infected but did not require antibiotic treatment. The
hospitalized patients therefore represent only a small fraction of all
infected persons. In nine of the 10 affected people, two or three tests
were positive. One person had a positive PCR, a twofold increase in IgG
and a negative CF test. Soon after hospital admission, this patient
received treatment with doxycycline and this could have diminished the
antibody response. PCR performed on sputum was very helpful for rapid
diagnosis in the hospitalized patients. The central laboratory facility of
the different hospitals helped in identifying this outbreak. Subsequent
investigation of the outbreak and outbreak management was therefore
possible.
PCR on throat swabs in symptomatic students was of limited value in
detecting C. psittaci infection. PCR on throat swabs is often used to
diagnose pneumonia (10,12,14). As demonstrated earlier, we confirmed
the observation that C. psittaci can be detected for prolonged period of
time in a throat swab sample (8). Possibly, throat swabs might not be
representative material for lower respiratory tract infection due to C.
psittaci. A positive PCR on a throat swab could indicate an asymptomatic
carrier and in contrast, a negative PCR result on a throat swab does not
rule out psittacosis. Therefore data validating the use of throat swabs for
diagnosing the aetiology of pneumonia are needed.
C. psittaci is classified into eight serovars (A-F, WC and M56). Six
serovars are endemic in birds. Currently, at least nine genotypes are
known. Recently it was stated that identification of all known genotypes
and the newly discovered genotype E/B is only possible by sequencing of
the ompA gene (5). By sequencing of the ompA gene directly from
clinical samples, we bypassed the need for culture. It should be
mentioned that genotyping of the ompA gene can not definitely prove that
this is a clonal outbreak, but the clinical data together with the ompA
sequence analysis are highly suggestive. Certain genotypes appear to be
associated with specific groups of birds (17). We found genotype A to be
responsible for the outbreak. This genotype is most often found among
psittacine birds such as parrots and cockatiels. The most prevalent
C. psittaci genotype in human infections is currently unknown. In our
study the primary source of the outbreak turned out to be the nine parrots
64
Chapter 4
that were used in practical teaching sessions, but outbreak management
among the contact birds was hampered by lack of records of bird
identification and bird transactions. The cockatiels used in a practical
teaching session four weeks prior to the outbreak together with the
parrots, were untraceable at the moment of the outbreak. It is very likely
that these cockatiels infected the parrots and the index case, since the
parrots from the teaching hospital were checked on several occasions by
immunoassay and tested negative.
In many countries, psittacosis is a notifiable disease. In Europe and the
USA measures to control C. psittaci infections among humans and birds
have been issued (15,16). Maintenance of accurate records of bird related
transactions for at least one year are recommended. These records should
include species of bird, bird identification, source of birds and any
identified illnesses or deaths among birds. Newly acquired birds, should
be tested or prophylactically treated before adding them to a group. For
pet birds they recommend that only healthy, PCR negative birds should
be sold by bird retailers.
Applying (some of) the recommendations to these birds could have
prevented or limited this outbreak and could have made it possible to
trace the cockatiels. The veterinary teaching hospital has changed its
policy regarding the use of birds for teaching purposes and will only use
birds from their in-house population. The relative poor control of the
disease in birds and the broad spectrum of clinical syndromes of people
infected with C. psittaci raise the question whether this micro-organism
may be a much more frequent pathogen than previously considered. For
accurate diagnosis, we therefore recommend the detection of C. psittaci
infections by PCR. Real-time PCR can specifically identify the pathogen
and expedite the diagnosis of psittacosis. Sequencing of the ompA gene
for genotyping is a helpful tool for identification of the avian source and
improves our understanding of the epidemiology of this disease in birds,
humans and outbreak settings.
Acknowledgements
We thank J.M. Defoer and G. Koen, both working at the Department of
Virology of the Academic Medical Centre, Amsterdam, The Netherlands,
for performing the rELISA and Complement fixation tests.
65
Chapter 4
References
1. Boom, R., C. Sol, J. Weel, K. Lettinga, Y. Gerrits, A. van Breda, and P.
Wertheim-van Dillen. 2000. Detection and quantitation of human
cytomegalovirus DNA in faeces. J.Virol.Methods 84:1-14.
2. Boom, R., C. J. Sol, M. M. Salimans, C. L. Jansen, P. M. Wertheim-Van
Dillen, and van der Noordaa J. 1990. Rapid and simple method for
purification of nucleic acids. J.Clin.Microbiol. 28:495-503.
3. Bourke, S. J., D. Carrington, C. E. Frew, R. D. Stevenson, and S. W.
Banham. 1989. Serological cross-reactivity among chlamydial strains in a
family outbreak of psittacosis. J.Infect. 19:41-45.
4. Bush, R. M. and K. D. Everett. 2001. Molecular evolution of the
Chlamydiaceae. Int.J.Syst.Evol.Microbiol. 51:203-220.
5. Geens, T., A. Desplanques, M. Van Loock, B. M. Bonner, E. F. Kaleta, S.
Magnino, A. A. Andersen, K. D. Everett, and D. Vanrompay. 2005.
Sequencing of the Chlamydophila psittaci ompA Gene Reveals a New
Genotype, E/B, and the Need for a Rapid Discriminatory Genotyping Method.
J.Clin.Microbiol. 43:2456-2461.
6. Heddema, E. R., M. Beld, Wever de B, Langerak A.A.J., Pannekoek Y,
and Duim B. 2006. Development of an internally controlled real-time PCR
assay for detection of Chlamydophila psittaci in the LightCycler 2.0 system.
Clinical Microbiology and Infection 12:574-576.
7. Heddema, E. R., S. Sluis ter, J. A. Buijs, C. M. J. E. VandenbrouckeGrauls, J. H. Van Wijnen, and C. E. Visser. 2006. Prevalence of
Chlamydophila psittaci in fecal droppings from feral pigeons in Amsterdam,
The Netherlands. Applied and Environmental Microbiology 72:4423-4425.
8. Huminer, D., Z. Samra, Y. Weisman, and S. Pitlik. 1988. Family outbreaks
of psittacosis in Israel. Lancet 2:615-618.
9. Kumar, S., K. Tamura, and M. Nei. 2004. MEGA3: Integrated software for
Molecular Evolutionary Genetics Analysis and sequence alignment.
Brief.Bioinform. 5:150-163.
10. Menendez, R., J. Cordoba, C. P. de La, M. J. Cremades, J. L. LopezHontagas, M. Salavert, and M. Gobernado. 1999. Value of the polymerase
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11. Persson, K. and S. Haidl. 2000. Evaluation of a commercial test for
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12. Ramirez, J. A., S. Ahkee, A. Tolentino, R. D. Miller, and J. T.
Summersgill. 1996. Diagnosis of Legionella pneumophila, Mycoplasma
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14. Schneeberger, P. M., J. W. Dorigo-Zetsma, Z. A. van der, M. van Bon, and
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chlamydiosis as a zoonotic risk and reduction strategies. [Online]
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16. Smith, K. A., K. K. Bradley, M. G. Stobierski, and L. A. Tengelsen. 2005.
Compendium of measures to control Chlamydophila psittaci (formerly
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17. Vanrompay, D., P. Butaye, C. Sayada, R. Ducatelle, and F. Haesebrouck.
1997. Characterization of avian Chlamydia psittaci strains using omp1
restriction mapping and serovar-specific monoclonal antibodies.
Res.Microbiol. 148:327-333.
18. Verkooyen, R. P., N. A. Van Lent, S. A. Mousavi Joulandan, R. J. Snijder,
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R. J. Snijder, J. M. van den Bosch, H. P. Van Helden, M. F. Peeters, and
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67
Chapter 5
Prevalence of Chlamydophila psittaci in fecal
droppings from feral pigeons in Amsterdam,
The Netherlands
Edou R. Heddema1*, Sietske ter Sluis1, Jan A. Buys2, Christina M.J.E.
Vandenbroucke-Grauls1,3, Joop H. van Wijnen2, Caroline E. Visser1.
1
) Department of Medical Microbiology, Academic Medical Center,
University of Amsterdam, Amsterdam, the Netherlands.
2
) Cluster Environment and Public Health, Municipal Health Service,
Amsterdam, the Netherlands
3
) Department of Medical Microbiology & Infection Control, VU
University Medical Center, Amsterdam, the Netherlands.
Adapted from Applied and Environmental Microbiology 2006
Jun;72(6):4423-5.
69
Chapter 5
Abstract
In many cities the feral pigeon is an abundant bird species that can harbor
Chlamydophila psittaci. We determined the prevalence and genotype of
C. psittaci in fresh fecal samples from feral pigeons in Amsterdam, The
Netherlands. The prevalence was overall 7.9% (26/331;95%CI 5-11).
Ten genotyped PCR positive samples were all genotype B.
70
Chapter 5
Introduction
In many European cities the feral rock dove (Columbia livia) is an
abundant bird species that often lives in close contact with humans. It is
known that pigeons, like many other bird species, can harbor
Chlamydophila psittaci. This bacterium is a pathogen of birds, but can
cause zoonotic disease (5). Birds can shed this bacterium in the
environment either overtly ill or without any symptoms. In birds the
bacterium can be isolated from feces, the cloacae and respiratory and
conjunctiva secretions. In this study we determined the prevalence of
C. psittaci shedding in feces from feral pigeons in Amsterdam, the
Netherlands, and the genotype in the PCR positive samples. C. psittaci in
these specimens was determined with a recently developed real-time
PCR (6).
Setting and sampling
The city of Amsterdam consists of 14 town councils. Pigeon samples
were obtained on 9 locations in 8 town councils. These locations were
geographically widely distributed in Amsterdam, all were situated in the
public area and chosen based on previous research of assembling
locations of feral pigeons in Amsterdam (3). On these locations, pigeons
were attracted with food and their fresh fecal droppings were sampled
with sterile cotton swabs (MW&E, UK). As shedding occurs
intermittently and can be activated by stress factors such as breeding,
samples were taken on the 3rd of February and the 8th of March 2005,
when breeding activity is low and on the 2nd of May 2005 when breeding
is frequent(12).
DNA extraction and PCR
The cotton swabs were placed in a 1.5 ml tube in 300 µl Baker water
(Boom B.V. Meppel, The Netherlands) and vortexed thoroughly. 50 µl of
this fecal suspension was used as input for the DNA extraction procedure
(1). C. psittaci PCR was performed as previously described (6). Briefly,
this real-time PCR targets an 82 bp fragment of the ompA gene of C.
psittaci as well as an internal control plasmid (IC) using primers CpsittF
(5’-CGCTCTCTCCTTACAAGCC-3’) and CPsittR (5’AGCACCTTCCCACATAGTG -3’). The IC, added to each sample, has
71
Chapter 5
the same primer sites, length and nucleotide content as the C. psittaci
amplicon but a shuffled probe binding region. To prevent false-positive
reactions due to amplicon carry-over, we used the uracil-N-glycosylase
system, and a unidirectional work-flow combined with separation of PCR
mix preparation and DNA extraction from all (post-)amplification
activities.
Genotyping
PCR positive samples were genotyped by ompA sequence analysis. The
gene was amplified with the primers CPsittGenoFor (5’–
GCTACGGGTTCCGCTCT–3’; nucleotide position (nt) 400-416) and
CPsittGenoRev (5’–TTTGTTGATYTGAATCGAAGC–3’; nt 1420-1441
). Nucleotide position according to the C. psittaci 6BC ompA gene
(GeneBank X56980), resulting in a 1041 bp amplicon. These primers are
located in the conserved regions of the ompA enclosing the four variable
domains. Genotype PCR was performed in the LightCycler 2.0 (Roche
Diagnostics, Germany). The final reaction volume (20 µl) included 8 µl
eluate and was essentially the same as described previously (6). The realtime PCR steps were as follows: 50˚C for 10’, 95˚C for 10’, 45 cycles of
95˚C for 10’’, 62˚C for 5’’, 72˚C for 50’’ and 30˚C for 30’’. Ten µl of the
PCR product was analyzed by 1% agarose gel electrophoresis (AGE).
The expected amplicon was excised from the gel, purified with a
simplified guanidiniumthiocyanate extraction procedure (2,5 µl silica;
wash cycles with L2, ethanol and acetone) and eluted in 15 µl TE buffer
(10 mM Tris-HCL,1 mM EDTA[PH 8.0]) (2). To obtain sufficient
product for sequence analysis, re-amplification for only 20 cycles was
performed in a GeneAmp 9700 (Perkin-Elmer). The reaction mixture for
re-amplification (50 µl) included 2 µl of eluate, 5 µl (10x)PCR II buffer,
5 µg BSA, 0.25 U Amplitaq Gold, 0.16 µM of each primer and 4.5 mM
MgCl2. The PCR steps were as follows: 95˚C for 10’, 20 cycles of 95˚C
for 1’, 55˚C for 1’, 72˚C for 2’ and 72˚C for 10’. When a single band of
approximately 1041 bp was obtained with AGE, the PCR product was
subjected to sequence analysis (BigDye® Terminator sequencing kit,
Applied Biosystems). Overlapping sequences were obtained with four
sequencing primers; the above mentioned genotype primers and two
inner primers CPsittFinner (5’-CGCTCTCTCCTTACAAGCC-3’) and
CPsittRinner (5’-GATCTGAATCGAAGCAATTTG-3’). We used the C.
psittaci ORNI (genotype A) strain and a C. abortus strain as positive
72
Chapter 5
controls. To prevent amplicon carry-over, the same measures as
described for the real-time PCR were taken. The resulting sequences
were aligned and similarity index based on the resulting amino acids was
calculated on an 894 bp fragment of the ompA gene. Similarity (1distance) was calculated using the pairwise distance method generated by
MEGA3 (8). Reference ompA genotype sequences A-F available in the
GenBank database (accession numbers AY762608-12, AF269261) were
included in this analysis (4).
73
Chapter 5
Results
In total 331 fecal samples were obtained, 160 samples before and 171 in
the breeding period (Table 1). On each location at least 15 samples were
collected. In the low-breeding period 5% (8/160; 95% CI 2-10) of all
samples was PCR positive. In samples obtained during the breeding
period 10% (18/171; 95% CI 6-16) was positive, hence the prevalence of
positive samples during the breeding period was twice the prevalence in
the low-breeding period (Fisher’s exact test: p=0.07 (GraphPad Software,
San Diego, CA, USA)). The overall prevalence was 7.9 % (26/331; 95%
CI 5-11). All the negative samples were truly negative since all internal
controls amplified correctly, thus excluding inhibition of the PCR. It was
possible to genotype 10 of the 26 PCR positive samples. The obtained
sequences were all 100% similar to reference genotype B. Similarity
based on amino acid sequence was respectively 98% (genotype A), 56%
(C), 43% (D), 99% (E) and 51% (F). The positive controls (C. psittaci
ORNI and C. abortus) amplified as expected and could be subsequently
sequenced.
Table 1. The number of C. psittaci PCR positive fecal samples in feral
pigeons by sampling location in Amsterdam, the Netherlands.
Town Council
Low-breeding
Breeding period
period
Oost Watergraafsmeer 0/15a
6/15
Oud Zuid
3/15
0/20
Binnenstad (Dam)
0/20
2/27
Binnenstad (Leidse
2/25
3/27
plein)
Zeeburg
0/15
3/15
Zuider Amstel
0/15
3/15
Geuzenveld
0/15
0/15
Bos en Lommer
0/20
0/15
Oud West
3/20
1/22
18/171 (10%; 95% CI
Total
8/160 (5%; 95% CI
6-16%)
2-10%) b
a
) Number of positive samples/ samples per council tested.
b
) 95% CI; 95% confidence interval.
74
Chapter 5
Discussion
This study shows that between 5 and 10% of our sample of the urban
feral pigeons in Amsterdam shed C. psittaci in their feces. Only genotype
B was found in these isolates. We were unable to genotype all PCR
positive samples. For genotyping, a 1041 bp fragment had to be
amplified; this PCR is less sensitive than the optimized diagnostic realtime PCR which amplifies a fragment of only 82 bp. Therefore, samples
with relatively low C. psittaci loads could not be amplified with the
genotype PCR. The major advantage of this study was the use of an
internally controlled real-time PCR assay. PCR is a sensitive and specific
test compared to ELISA and tissue-culture available for C. psittaci
detection in birds (7,9). Salinas reported one of the largest series on the
prevalence of C. psittaci in feral pigeons (10). In that study C. psittaci
was found by culture in 18% (7/39; 95%CI 9-33) of fecal samples, a
prevalence that is similar to our results obtained by PCR. Recently,
Tanaka found C. psittaci in 106 out of 463 (22.9%; 95%CI 19-27) fecal
samples obtained from feral pigeons. However, they did not use
exclusively fresh fecal samples and applied a nested PCR protocol, which
is known to be particularly prone to contamination (13).
A previous study indicated that in 2001 the pigeon population size in
Amsterdam averaged approximately 30,000 (3). Combined with our
results the number of feral pigeons shedding C. psittaci in their feces
would be on average about 2400 (95% CI 1500 - 3300). Our isolates
were all identical to genotype B. Currently, at least nine genotypes are
known. Each genotype is more or less associated with a specific group of
birds from which it is most commonly isolated. Geens and Vanrompay
also found genotype B to be particularly associated with the pigeon host
(4,14). However, this genotype has been recovered from many bird
species, like turkeys, parakeets and ducks (11,15). Whether shedding of
C. psittaci by feral pigeons in Amsterdam poses a substantial zoonotic
risk for humans has to be determined. Besides the zoonotic potential,
there is also the risk of infection of domesticated birds, like pet birds and
poultry, which live in closer contact with human beings. Diagnosing
C. psittaci infections has been hampered by a lack of sensitive and
specific methods. Culture is only performed in some selected
laboratories, serologic tests do not fully differentiate infection with the
various Chlamydia spp. and PCR is not routinely performed. However
PCR can provide a definite diagnosis of psittacosis. We recommend that
75
Chapter 5
psittacosis in humans should be diagnosed by detection of the agent by
PCR in combination with or without serologic testing instead of serologic
testing alone. Subsequent ompA gene sequence analysis can identify the
responsible genotype. This approach could lead to a better understanding
of the epidemiology of the different genotypes of C. psittaci in infected
bird populations, human psittacosis cases and the relation between these
two.
GeneBank accession numbers
The ompA sequences of the positive control strains and the genotype B
sequence obtained from the fecal pigeon samples were submitted to
GenBank. (DQ267973, DQ435299, DQ435300).
Acknowledgements
Dr. Y. Pannekoek, PhD, University of Amsterdam, Amsterdam provided
the C. psittaci ORNI strain. Prof. D. Vanrompay, Ghent University,
Belgium provided the C. abortus strain.
References
1. Boom, R., C. Sol, J. Weel, K. Lettinga, Y. Gerrits, A. van Breda, and P.
Wertheim-van Dillen. 2000. Detection and quantitation of human
cytomegalovirus DNA in faeces. J.Virol.Methods 84:1-14.
2. Boom, R., C. J. Sol, M. M. Salimans, C. L. Jansen, P. M. Wertheim-Van
Dillen, and van der Noordaa J. 1990. Rapid and simple method for
purification of nucleic acids. J.Clin.Microbiol. 28:495-503.
3. Buijs, J. A. and J. H. Van Wijnen. 2001. Survey of feral rock doves
(Columba livia) in Amsterdam, a bird-human association. Urban Ecosystems
5:235-241.
4. Geens, T., A. Desplanques, M. Van Loock, B. M. Bonner, E. F. Kaleta, S.
Magnino, A. A. Andersen, K. D. Everett, and D. Vanrompay. 2005.
Sequencing of the Chlamydophila psittaci ompA Gene Reveals a New
Genotype, E/B, and the Need for a Rapid Discriminatory Genotyping Method.
J.Clin.Microbiol. 43:2456-2461.
5. Haag-Wackernagel, D. and H. Moch. 2004. Health hazards posed by feral
pigeons. J.Infect. 48:307-313.
76
Chapter 5
6. Heddema, E. R., Beld, M., Wever de B, Langerak A.A.J., Pannekoek Y,
and Duim B. Development of an internally controlled real-time PCR assay for
detection of Chlamydophila psittaci in the LightCycler 2.0 system. Clinical
Microbiology and Infection . 2005. In Press
7. Hewinson, R. G., P. C. Griffiths, B. J. Bevan, S. E. Kirwan, M. E. Field, M.
J. Woodward, and M. Dawson. 1997. Detection of Chlamydia psittaci DNA
in avian clinical samples by polymerase chain reaction. Vet.Microbiol. 54:155166.
8. Kumar, S., K. Tamura, and M. Nei. 2004. MEGA3: Integrated software for
Molecular Evolutionary Genetics Analysis and sequence alignment.
Brief.Bioinform. 5:150-163.
9. McElnea, C. L. and G. M. Cross. 1999. Methods of detection of Chlamydia
psittaci in domesticated and wild birds. Aust.Vet.J. 77:516-521.
10. Salinas, J., M. R. Caro, and F. Cuello. 1993. Antibody prevalence and
isolation of Chlamydia psittaci from pigeons (Columba livia). Avian Dis.
37:523-527.
11. Sayada, C., A. A. Andersen, C. Storey, A. Milon, F. Eb, N. Hashimoto, K.
Hirai, J. Elion, and E. Denamur. 1995. Usefulness of omp1 restriction
mapping for avian Chlamydia psittaci isolate differentiation. Res.Microbiol.
146:155-165.
12. Scientific committee on animal health and animal welfare. 2002. Avian
chlamydiosis as a zoonotic risk and reduction strategies. [Online]
http://europa.eu.int/comm/food/fs/sc/scah/out73_en.pdf
13. Tanaka, C., T. Miyazawa, M. Watarai, and N. Ishiguro. 2005.
Bacteriological survey of feces from feral pigeons in Japan. J.Vet.Med.Sci.
67:951-953.
14. Vanrompay, D., A. A. Andersen, R. Ducatelle, and F. Haesebrouck. 1993.
Serotyping of European isolates of Chlamydia psittaci from poultry and other
birds. J.Clin.Microbiol. 31:134-137.
15. Vanrompay, D., P. Butaye, C. Sayada, R. Ducatelle, and F. Haesebrouck.
1997. Characterization of avian Chlamydia psittaci strains using omp1
restriction mapping and serovar-specific monoclonal antibodies.
Res.Microbiol. 148:327-333.
77
Chapter 6
Genotyping of Chlamydophila psittaci strains
in human clinical samples by ompA sequence
analysis
Edou R. Heddema1*, Erik J. van Hannen2, Birgitta Duim1, Christina
M.J.E. Vandenbroucke-Grauls1,3, Yvonne Pannekoek1.
1
) Department of Medical Microbiology, Academic Medical Center,
University of Amsterdam, Amsterdam, the Netherlands.
2
) Department of Medical Microbiology and Immunology, St. Antonius
Hospital, Nieuwegein, the Netherlands.
3
) Department of Medical Microbiology & Infection Control, VU
University Medical Center, Amsterdam, the Netherlands.
Adapted from Emerging Infectious Diseases 2006 dec;12(12):1985-86.
79
Chapter 6
Abstract
Chlamydophila psittaci genotypes A, B, C and a new genotype most
similar to the 6BC type strain were found in ten human psittacosis cases
by ompA sequencing. Genotypes B (n=3) and C (n=1) are endemic in
non-psittacine European birds. These birds may represent an important
part of the zoonotic reservoir.
80
Chapter 6
Psittacosis is a zoonosis caused by infection with Chlamydophila psittaci,
an obligate intracellular bacterium. C. psittaci is divided in 8 serovars (AF, M56, WC) and at least 9 genotypes. Sequence analysis of the ompA
gene is currently the most accurate method to identify all known
genotypes (3). All genotypes are more or less associated with specific
bird groups from which they are predominantly isolated (9,10). At
present, the prevalence of the different genotypes of C. psittaci causing
infection in humans is unknown. In this study we therefore genotyped all
C. psittaci PCR positive human clinical samples available in our
laboratory.
Ten human clinical samples, positive for C. psittaci DNA in our
previously described real-time PCR assay, were characterized by ompA
sequencing (4). These samples were collected between 2002 and 2005
and included four sputa, four broncho-alveolar lavage fluids (BAL), one
throat swab and one serum. All samples were obtained from symptomatic
patients with psittacosis admitted to hospitals in the Netherlands. All
patients had pneumonia of which six required admission to the intensive
care unit (ICU). The DNA of one outbreak strain, infecting at least ten
people, was included only once. One of the samples was obtained from a
patient who has been described previously (5)
DNA purification was done by the guanidiniumthiocyanate-silica
extraction procedure (1). Genotyping was performed essentially as
previously described (6). Briefly, a part of the ompA gene was amplified
with the primers CPsittGenoFor (5’ – GCT ACG GGT TCC GCT CT –
3’) and CPsittGenoRev (5’ – TTT GTT GAT YTG AAT CGA AGC –
3’). These primers are located in the conserved regions of the ompA
enclosing the four variable domains (VD). Based on the published ompA
sequence of the C. psittaci 6BC type strain (GenBank accession no
X56980), we calculated that the resulting amplicon should have a size of
1041 bp. PCR products were analyzed by agarose gel electrophoresis and
the expected 1041 bp amplicon was excised from the gel. DNA was
extracted from the gel and re-amplified for 20 cycles and amplicons were
controlled for size by agarose gel electrophoresis. C. psittaci ORNI
(genotype A) strain and a Chlamydophila abortus strain were used as
positive controls. Calf thymus DNA was used as negative control. If we
were unable to amplify the ompA gene with the above mentioned
procedure, a nested PCR with the primers CPsittFinner and CpsittRinner
(see below) was applied. The amplified product (n=8) or the nested PCR
product (n=2) was subjected to sequence analysis (BigDye® Terminator
81
Chapter 6
v1.1 cycle sequencing kit, Applied Biosystems). Overlapping sequences
were obtained with six sequencing primers; CPsittGenoFor and
CPsittGenoRev, two inner primers CPsittFinner (5’-CGC TCT CTC CTT
ACA AGC C -3’) and CPsittRinner (5’ – GAT CTG AAT CGA AGC
AAT TTG - 3’) and two primers situated approximately halfway the
ompA gene CPsittHFor (5’ – TCT TGG AGC GTR GGT GC- 3’) and
CPsittHRev ( 5’ – GCA CCY ACG CTC CAA GA - 3’). The resulting
sequences were aligned and a similarity index based on the translation of
the 984 bp long gene fragment was calculated. Similarity (1- distance)
was calculated using the pairwise distance method generated by MEGA3
(7). Reference ompA genotype sequences A-F and the ompA sequence of
the C. psittaci 6BC type strain available in GenBank (accession numbers
AY762608-AY762612, X56980 and AF269261) were included in this
analysis (2,3).
All ten isolates could be genotyped. The ompA sequence of five
isolates was identical to the sequence of reference genotype A, three
isolates were identical to genotype B and the ompA sequence of one
isolate was identical to genotype C. One isolate carried a novel ompA
sequence type that was 99.4% similar to the genotype A reference, but
even more similar to the C. psittaci 6BC type strain (99.7%). Two
nonsynonymous mutations compared to genotype A were present in this
sequence. A substitution of thymine for an adenine in VD 1 resulted in
Ser instead of Thr on amino acid position 92 of the ompA amino acid
sequence, identical to what is found in genotype C. A substitution of
cytosine to guanidine, also located in VD 1, resulted in Gln instead of
Glu on amino acid position 117, as found in genotype B and strain 6BC
(numbering according to the ompA amino acid sequence of the C. psittaci
6BC strain, GenBank accession no. X56980). We designated this new
variant C. psittaci 05/02 and deposited the sequence in GenBank
(accession no DQ324426). Two genotype B, three genotype A and the
novel genotype 05/02 strain were obtained from patients admitted to the
ICU.
To our knowledge, this is the first report of a series of genotyped
C. psittaci strains isolated from clinical samples obtained from
symptomatic, hospitalized patients. These ten samples reflect
approximately one third of all cases notified each year in the Netherlands
(8). From the genotypes that we identified we may infer the zoonotic
reservoirs of C. psittaci in the Netherlands. The different genotypes of
C. psittaci are associated, although not exclusively, with different groups
82
Chapter 6
of birds from which they are mostly isolated. Genotype A is mainly
found in psittacine birds and is the most prevalent genotype in our
clinical samples (3,10). C. psittaci 05/02 was most related to C. psittaci
6BC and the reference genotype A (strain VS1). Both reference strains
have been classified as serovar A strains. Based on two distinct
restriction fragment length polymorphism patterns, Sayada et al.
suggested that serovar A should be divided into two distinct genogroups
(9). Our isolate 05/02 is a new ompA sequence variant within this
probably heterogeneous group.
Genotype B has been mainly isolated from feral pigeons and
several other bird species and this genotype is considered endemic in
European non-psittacine birds (10,11). Genotype C has been mainly
isolated from ducks, we detected this genotype in one of our human
samples. We did not find genotype D, most prevalent among poultry,
especially turkeys, nor genotypes E and F. These latter two genotypes are
rare and found occasionally in birds (3,11). In the past, imported
psittacine birds, which carry mainly genotype A, have been proposed as
the major source for human psittacosis cases (12). In our study four out
of ten isolates were genotype B and C. These genotypes are rarely found
in psittacine birds. This suggests that non-psittacine birds may represent
an underestimated source for human psittacosis cases.
In conclusion, in a series of ten C. psittaci positive clinical samples we
detected isolates of genotype A, B, C and a new genotype most similar to
the C. psittaci 6BC strain. Genotypes B and C are endemic in European
non-psittacine birds and these birds may therefore represent an important
part of the zoonotic reservoir for human psittacosis cases.
References
1. Boom, R., C. J. Sol, M. M. Salimans, C. L. Jansen, P. M. Wertheim-Van
Dillen, and van der Noordaa J. 1990. Rapid and simple method for
purification of nucleic acids. J.Clin.Microbiol. 28:495-503.
2. Bush, R. M. and K. D. Everett. 2001. Molecular evolution of the
Chlamydiaceae. Int.J.Syst.Evol.Microbiol. 51:203-220.
3. Geens, T., A. Desplanques, M. Van Loock, B. M. Bonner, E. F. Kaleta, S.
Magnino, A. A. Andersen, K. D. Everett, and D. Vanrompay. 2005.
Sequencing of the Chlamydophila psittaci ompA Gene Reveals a New
Genotype, E/B, and the Need for a Rapid Discriminatory Genotyping Method.
J.Clin.Microbiol. 43:2456-2461.
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4. Heddema, E. R., M. Beld, Wever de B, Langerak A.A.J., Pannekoek Y,
and Duim B. 2006. Development of an internally controlled real-time PCR
assay for detection of Chlamydophila psittaci in the LightCycler 2.0 system.
Clinical Microbiology and Infection 12:574-576.
5. Heddema, E. R., M. C. Kraan, H. E. Buys-Bergen, H. E. Smith, and P. M.
Wertheim-Van Dillen. 2003. A woman with a lobar infiltrate due to
psittacosis detected by polymerase chain reaction. Scand.J.Infect.Dis. 35:422424.
6. Heddema, E. R., S. Sluis ter, J. A. Buijs, C. M. J. E. VandenbrouckeGrauls, J. H. Van Wijnen, and C. E. Visser. 2006. Prevalence of
Chlamydophila psittaci in fecal droppings from feral pigeons in Amsterdam,
The Netherlands. Applied and Environmental Microbiology 72:4423-4425.
7. Kumar, S., K. Tamura, and M. Nei. 2004. MEGA3: Integrated software for
Molecular Evolutionary Genetics Analysis and sequence alignment.
Brief.Bioinform. 5:150-163.
8. RIVM. 2004. Notified cases of infectious diseases in the Netherlands. Dutch
Infectious Diseases Bulletin 15.
9. Sayada, C., A. A. Andersen, C. Storey, A. Milon, F. Eb, N. Hashimoto, K.
Hirai, J. Elion, and E. Denamur. 1995. Usefulness of omp1 restriction
mapping for avian Chlamydia psittaci isolate differentiation. Res.Microbiol.
146:155-165.
10. Vanrompay, D., A. A. Andersen, R. Ducatelle, and F. Haesebrouck. 1993.
Serotyping of European isolates of Chlamydia psittaci from poultry and other
birds. J.Clin.Microbiol. 31:134-137.
11. Vanrompay, D., P. Butaye, C. Sayada, R. Ducatelle, and F. Haesebrouck.
1997. Characterization of avian Chlamydia psittaci strains using omp1
restriction mapping and serovar-specific monoclonal antibodies.
Res.Microbiol. 148:327-333.
12. Wreghitt, T. G. and C. E. Taylor. 1988. Incidence of respiratory tract
chlamydial infections and importation of psittacine birds. Lancet 1:582.
84
Chapter 7
Summarizing discussion: molecular tools for
the detection and typing of Chlamydophila
psittaci strains causing human and avian
infections
Edou R. Heddema1, Yvonne Pannekoek1, Caroline E. Visser1, Christina
M.J.E. Vandenbroucke-Grauls1,2
1
) Department of Medical Microbiology, Academic Medical Center,
University of Amsterdam, Amsterdam, The Netherlands.
2
) Department of Medical Microbiology & Infection Control, VU
University Medical Center, Amsterdam, The Netherlands.
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Chapter 7
Summarizing discussion
Psittacosis is an infection caused by Chlamydophila psittaci, a pathogen
acquired via birds. Culture and serologic tests have been the best
available diagnostic tools for years, but the diagnosis of this infection is
troublesome because these methods are neither fast nor sensitive nor
specific. Without appropriate antibiotic treatment, the infection can be
life threatening. The infection can occur in outbreaks and therefore
detection is important for the public health as removal or treatment of the
avian source can prevent further cases. In this study we have developed a
rapid, sensitive and specific test to detect this pathogen and thus diagnose
this infection. A typing method was developed to gain better insight in
the genotypes causing human and avian infections.
In chapter 2 a case description of psittacosis was given and the problem
of accurate diagnostic options was addressed. This case led to the
development of the real-time PCR assay for the detection of C. psittaci
described in chapter 3. This assay appeared to be a sensitive, specific and
rapid method to detect C. psittaci DNA in human clinical respiratory
samples. In chapter 4 an outbreak of psittacosis in a veterinary teaching
hospital was documented by this PCR. A genotyping method based on
sequence analysis of the outer membrane protein A gene (ompA) was
developed to exactly identify the outbreak strain. In chapter 5 we
describe an abundant source of C. psittaci: overall 7.9% of feral urban
pigeons were shown to shed C. psittaci in the environment with their
fecal droppings. Most were C. psittaci genotype B strains. In chapter 6
ten strains of C. psittaci that caused infection in humans were genotyped;
these were mainly genotype A or B. The other isolates were one
genotype C and one new genotype. Half of the isolates were genotype A
strains. This genotype is mainly associated with psittacine (parrot like)
birds. The other genotypes, B and C, have been mainly isolated from
birds endemic in Europe, especially pigeons and ducks.
Broader use of molecular diagnostic tools for detection and subsequent
genotyping of C. psittaci isolates has improved our knowledge of an old
but probably underestimated infection. This knowledge can influence
future decisions on how to deal with psittacosis as a notifiable disease.
Molecular diagnostic methods provide the tools for a different approach
to this zoonotic infection.
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Recognition of the disease
Since the first description of psittacosis by Ritter in 1881, medicine has
changed dramatically (9). Nowadays clinicians do not have the
opportunity like Ritter to observe infectious diseases for more than 4
weeks. Early treatment with antibiotics is often instituted before the
complete clinical picture is obvious. Infectious diseases, almost
untreatable at that time, are now one of the best treatable diseases.
Antibiotics have made psittacosis from an often deadly disease to a very
well treatable infection if recognized in time and treated with appropriate
anti-microbial agents (8,9,11,14). However, public health considerations
are still important with this infection. As shown in chapter 4, one case
diagnosed in hospital can be an indication of several more patients
consulting their general practitioner or physician and in fact represent an
outbreak. During this research, the use of real-time PCR and thus the
early recognition of the disease taught us a lot of the clinical signs and
symptoms of psittacosis. Severe headache was often a very prominent
symptom (chapter 2 and 4). Although one single symptom cannot
distinguish the different bacterial pathogens involved in respiratory
infections, a combination of several signs and certain details often seems
very helpful (12). For example, severe headache, fever, respiratory
symptoms and obvious bird contact is highly suspicious. In case of
pneumonia, history taking should therefore always include questions
concerning these issues. When real-time PCR becomes more widely
available, the prompt diagnosis of psittacosis will influence treatment and
improve future recognition of the clinical picture of psittacosis. Recently,
this was observed in the Netherlands when psittacosis was identified by
PCR and subsequently published in a case report (3). Probably a trend
similar to that observed as when the urinary antigen test for the diagnosis
of legionnaire’s disease became widely available will be seen. This
resulted in an approximately six fold increase in notified legionellosis
cases in the Netherlands.
Accurate diagnostic options
Real-time PCR for detection of C. psittaci, a new diagnostic assay, is
very specific, fast and sensitive. This assay, with the inclusion of an
internal control (IC), provides specific hybridization with Taqman probes
and excludes false-negative results due to the technical procedure. In
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Chapter 7
chapter 3-6 we used respectively sputum, broncho-alveolar lavage fluid,
throat swab samples, parrot feces, pigeon feces, and serum as input for
our assay. Although we only determined the lower limit of detection in
sputum, we proved that this PCR assay can be used for all these
materials, because we monitored the DNA extraction procedure by
including the IC and were always able to detect it by PCR. This means
that the PCR was never less sensitive than what we determined on
sputum, as shown in chapter 3. Real-time PCR is a fast technique and,
especially in outbreak settings, a valuable tool compared with serologic
testing, as waiting for convalescent serum samples is not needed (chapter
4). In addition, it avoids the troublesome interpretation of Chlamydophila
spp. serology.
Two of the drawbacks of this PCR are its price and the need for deep
respiratory samples (sputum or bronch-alveolar lavage (BAL) fuids).
Serologic testing is currently cheaper than real-time PCR, but the extra
costs of PCR can diminish uncertainty concerning the diagnosis and, if
diagnosis is fast, switch to a cheap antibiotic like doxycycline can be
achieved. The results presented in chapter 4 show that with throat swabs
it is not possible to detect all symptomatic cases while PCR was positive
on sputum from all patients admitted to the hospital. In general, patients
with pneumonia do often not expectorate sputum, this can still remain an
obstacle for correct diagnosis. In these cases BAL should be considered.
Acute phase serum or plasma may be an alternative sample since we
detected C. psittaci DNA in such a sample in one patient (chapter 6).
From an epidemiological point of view, real-time PCR can aid in
obtaining more precise incidence and prevalence numbers and
monitoring of the frequency of this disease will therefore be more
straightforward.
Genotyping
Genotyping of C. psittaci can help to identify avian sources of human
psittacosis cases and monitor the incidence of the different genotypes to
infer the most likely avian sources. In chapters 4-6 genotyping was used
to identify the outbreak strain, determine the most prevalent genotype in
feral pigeons and to infer the most likely avian sources of human cases.
Genotype B was detected in feral pigeons in Amsterdam and in a pigeon
during the described outbreak of psittacosis in a veterinary teaching
hospital. The case of psittacosis presented in chapter 2 was identified as
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Chapter 7
caused by a C. psittaci genotype B strain, as described in chapter 6.
Although her neighbors’ pigeons were tested for C. psittaci and were
tested negative, the genotype B suggests pigeons as the most likely
source for her infection. In chapter 4 genotype A was responsible for a
large outbreak in a veterinary teaching hospital and in chapter 6 it was
the most prevalent genotype found in sporadic human psittacosis cases
admitted to hospitals in the Netherlands. We detected a novel genotype
and a genotype C during this study, but we did not find C. psittaci
genotypes D, E, E/B and F. Continuous genotypic monitoring of human
C. psittaci infections will show whether these genotypes also cause
human infections thus have zoonotic relevance.
C. psittaci is endemic in many different bird species. Currently,
there is evidence that C. psittaci can infect at least 469 bird species (5).
Therefore eradication of C. psittaci from the environment is not feasible
and close monitoring of the prevalence and incidence of psittacosis is the
second best method to control the disease in humans. Genotyping of
avian and human strains will identify the avian sources that are the most
relevant in view of the zoonotic potential. Genotyping, and diagnostic
options for laboratories that do not have these tests for routine use, is
probably most effectively performed in one central laboratory facility
that provides national genotyping expertise for C. psittaci infections. The
need for such a department was already proposed by Dekking in his
thesis in 1950 (1). In addition, this would facilitate collection of data on
symptoms, and signs and identification of avian reservoirs of the disease.
Identification of the avian source
Currently, everyone who buys a bird is at risk of ending up with an
infected bird and of a subsequent zoonotic C. psittaci infection. Although
psittacosis has been included in the “infectieziektenwet” (infectious
diseases law) and protocols have been issued on how to deal with this
disease, outbreak management among birds relies completely on
cooperative bird owners and vendors (6). As described in chapter 4, the
most likely source of the outbreak were cockatiels that could not be
traced when the outbreak was recognized as transaction records were
lacking. Bird and pet shops owners can refuse to cooperate with public
health officials without any consequence. It would be helpful if rules
would be issued that it is compulsory to cooperate with public health
officials if psittacosis is suspected in a bird flock. If this is unfeasible
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Chapter 7
other actions like disclosure of uncooperative pet shops or bird vendors
could be tried (for example on the internet). Besides pet bird contact,
psittacosis can also be acquired from environmental birds (2,4,13). The
excretion of C. psittaci genotype B by feral pigeons in Amsterdam is a
large potential reservoir for zoonotic disease. Although the impact of this
large C. psittaci reservoir in the capital city of the Netherlands as direct
or indirect reservoir for zoonotic disease has still to be determined. That
we identified this genotype B as the second most prevalent genotype in
human clinical samples points to pigeons as a substantial large zoonotic
reservoir.
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Chapter 7
Psittacosis as a notifiable disease: proposal for a new approach
In the Netherlands, like in many other countries, psittacosis is a notifiable
disease to monitor the prevalence and incidence of the infection to
provide knowledge for infection control measures. In the Netherlands,
notifiable diseases are clustered in three groups. Group A consists of the
most contagious and serious infectious diseases and diseases within this
group have to be notified also when they are only suspected. Group B
includes infectious diseases which should be notified when they are
proven. Group C holds a number of infectious diseases that are notified
by the head of the laboratory. If necessary, the public health authorities
can request precise information for source identification and probable
place of acquisition. Psittacosis is classified as a group C disease (7).
Each year a few dozen cases of psittacosis are notified (10). It is
generally assumed that more cases occur each year but many of them go
unnoticed. Outbreaks of psittacosis can involve large numbers of people,
as shown in the outbreak described in chapter 4. In that outbreak only
two people presented with pneumonia, which is considered the classical
presentation of the disease. The other patients presented with a flu-like
illness or with symptoms of severe sepsis without pneumonia.
Underestimation is probably the result of the lack of accurate diagnostic
techniques and the subsequent unfamiliarity of clinicians with the diverse
clinical symptoms of the disease. In my opinion, this leads to loss of
interest in the disease by clinicians, microbiologists and public health
physicians and subsequently to more undiagnosed (and not notified)
cases of psittacosis. In a setting without proper diagnostic techniques and
lack of recognition, notification of psittacosis is surrounded by
uncertainties.
To improve notification at least four aspects need attention: 1)
accurate diagnostic options, like the described real-time PCR, should be
available at reasonable cost, 2) clinicians should be aware of their
responsibility to recognize and diagnose the infection for the sake of
public health 3) genotyping of C. psittaci strains should be available for
source identification, for monitoring circulating genotypes to infer the
most probable avian sources and to improve our knowledge of the
epidemiology of the infection and 4) for proven human cases of
psittacosis, identification of the avian source is important and
cooperation of the bird owner(s) should be obligatory.
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Chapter 7
Final conclusions and recommendations
Psittacosis, a notifiable disease, should be primarily diagnosed by realtime PCR instead of serologic testing. The described PCR assay allows
for specific, sensitive and rapid detection of C. psittaci in human clinical
samples.
Genotyping of avian and human isolates should be performed to identify
the source of the zoonotic infection and to determine and monitor the
most prevalent genotypes in human psittacosis cases to infer the most
likely avian reservoirs.
Such an approach leads to more precise estimations of the incidence and
prevalence of psittacosis in humans and to a better understanding of the
epidemiology of the different genotypes of C. psittaci in infected bird
populations, human psittacosis cases and the relation between these two.
References
1. Dekking, F. 1950. Universiteit van Amsterdam. Psittacosis en ornithosis in
Nederland.
2. Haag-Wackernagel, D. and H. Moch. 2004. Health hazards posed by feral
pigeons. J.Infect. 48:307-313.
3. Haas, L. E., D. H. Tjan, M. A. Schouten, and A. R. van Zanten. 2006.
[Severe pneumonia from psittacosis in a bird-keeper]. Ned.Tijdschr.Geneeskd.
150:117-121.
4. Henry, K. and K. Crossley. 1986. Wild-pigeon-related psittacosis in a family.
Chest 90:708-710.
5. Kaleta, E. F. and E. M. Taday. 2003. Avian host range of Chlamydophila
spp. based on isolation, antigen detection and serology. Avian Pathol. 32:435461.
6. LCI. 1994. Ornithose/psittacose.
7. Ministerie van VWS. 1998. Infectieziektewet. Staatsblad 1-11.
8. Pinkhof, H. 1940. Argentinie - Epidemie van psittacosis. Nederlands
Tijdschrift voor Geneeskunde 84:1147.
9. Ritter, J. 1881. Beitrag zur Frage des Pneumotyphus. (Eine Hausepidemie in
Uster [Schweiz] betreffend.). Deutches Archiv fur Klinische Medizin 25:5396.
10. RIVM. 2005. Notified cases of infectious diseases in the Netherlands. Dutch
Infectious Diseases Bulletin 16.
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11. Ruys, A. C. 1934. Psittacosis in Duitschland en Amerika. Nederlands
Tijdschrift voor Geneeskunde 78:2787.
12. Sopena, N., M. Sabria-Leal, M. L. Pedro-Botet, E. Padilla, J. Dominguez,
J. Morera, and P. Tudela. 1998. Comparative study of the clinical
presentation of Legionella pneumonia and other community-acquired
pneumonias. Chest 113:1195-1200.
13. Telfer, B. L., S. A. Moberley, K. P. Hort, J. M. Branley, D. E. Dwyer, D. J.
Muscatello, P. K. Correll, J. England, and J. M. McAnulty. 2005. Probable
psittacosis outbreak linked to wild birds. Emerg.Infect.Dis. 11:391-397.
14. Yung, A. P. and M. L. Grayson. 1988. Psittacosis--a review of 135 cases.
Med.J.Aust. 148:228-233.
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Nederlandse samenvatting
95
Chapter 8
Psittacose, ook wel papegaaienziekte genoemd, wordt veroorzaakt door
de bacterie Chlamydophila psittaci. Vogels zoals papegaaien, maar ook
parkieten, duiven, eenden, kalkoenen, kanaries en andere vogels kunnen
met de bacterie geïnfecteerd zijn. De dieren hoeven niet ziek te zijn. De
bacterie kan gevonden worden in vogelpoep, snot en oogvocht van
geïnfecteerde vogels. Na contact met vogels of hun uitwerpselen kan
door verstuiving de bacterie via de lucht mensen infecteren. Dit soort
situaties kan ontstaan bij het opruimen van vogelpoep, intiem contact met
vogels die als huisdier gehouden worden, bezoek aan dierenwinkels waar
besmette vogels verkocht worden of in het kader van werk (bv poelier) of
hobby (duivenmelker). Soms kunnen mensen zich geen contact met
vogels herinneren terwijl ze de ziekte toch hebben opgelopen.
De ziekte werd voor het eerst beschreven in 1881 door Jacob Ritter die
zijn broer verloor aan deze infectieziekte. Aanvankelijk dacht men dat de
ziekte veroorzaakt werd door een virus. Pas in 1966 werd duidelijk dat
het om een bacterie ging die niet kan overleven zonder zich te delen in
een gastheercel. Men noemt dit obligaat intracellulaire bacteriën. Vroeger
was de ziekte vaak dodelijk, maar met de komst van antibiotica zoals de
tetracyclinen (tetracycline, doxycycline) bleek de infectie goed te
behandelen.
Omdat de bacterie een gastheercel nodig heeft om in te groeien, is kweek
alleen mogelijk op cellijnen of bevruchte kippeneieren. Dit is
arbeidsintensief, een niet erg gevoelige techniek en kan het risico geven
van laboratorium besmettingen. Kweek wordt daarom nog maar zelden
gedaan. Serologisch onderzoek met behulp van 2 serum monsters is
momenteel de meest gebruikte methode om de ziekte te diagnosticeren.
Het eerste serummonster wordt vergeleken met een tweede
serummonster enkele weken later om zodoende een stijging in antistoffen
tegen C. psittaci aan te tonen. De methode is echter traag en niet
specifiek.
In dit proefschrift beschrijven we een snelle, specifieke en gevoelige
methode om deze bacterie aan te tonen. Vervolgens is er een manier
ontwikkeld om de bacterie met behulp van DNA analyse tot in detail te
typeren. Dit wordt genotyperen genoemd. Het genotype van C. psittaci
correleert nauw met bepaalde groepen vogels waaruit het genotype
voornamelijk geïsoleerd wordt.
In hoofdstuk 2 wordt een beschrijving gegeven van een typisch geval van
psittacose. De diagnostische mogelijkheden worden besproken en
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uiteindelijk wordt de ziekte met behulp van een moleculaire techniek
(polymerase kettingreactie; PCR) met zekerheid aangetoond. De
beschreven casus leidde tot de ontwikkeling van een real-time PCR om
de bacterie en dus de ziekte, in de toekomst met zekerheid te kunnen
aantonen (hoofdstuk 3). In hoofdstuk 4 beschrijven we hoe deze techniek
gebruikt kon worden om een uitbraak van psittacose in een academische
dierenkliniek te detecteren en verder in kaart te brengen. Met behulp van
een genotyperingstechniek, waarbij de DNA sequentie van het gen wat
codeert voor het buitenste membraan eiwit van C. psittaci geanalyseerd
wordt, kon de bron van de uitbraak vastgesteld worden. In hoofdstuk 5
beschrijven we een grote bron van C. psittaci in de omgeving. Bij
onderzoek van ontlasting van Amsterdamse stadsduiven bleek 7,9% van
alle monsters positief te zijn voor C. psittaci. In alle positieve monsters
die we konden typeren bleek er sprake te zijn van C. psittaci genotype B.
In hoofdstuk 6 beschrijven we de typering van 10 C. psittaci PCR
positieve monsters afgenomen bij 10 sporadische gevallen van psittacose
die opgenomen werden in Nederlandse ziekenhuizen. Met name C.
psittaci genotypen A en B werden gevonden. Tevens vonden we een
genotype C en een nieuw genotype. Genotype A wordt over het vooral
gevonden bij papegaaiachtige (zgn. psittacine- ) vogels. Genotype B
wordt met name gevonden bij duiven. Deze 2 vogelsoorten zijn de meest
waarschijnlijke bronnen van psittacose bij mensen. Het genotype C is
eerder gevonden bij watervogels zoals eenden en ganzen. Het is dus
aannemelijk dat ook deze vogels een besmettingsbron van psittacose
vormen voor mensen.
Met behulp van het onderzoek in dit proefschrift hebben wij geprobeerd
om de herkenning, diagnostiek en typering van C. psittaci infecties bij
mensen te verbeteren. Herkenning van de ziekte verbetert als de
diagnostiek makkelijker, sneller en preciezer kan. Betere diagnostiek kan
er toe leidden dat meer gevallen van psittacose herkend zullen worden.
Omdat psittacose in Nederland een aangifteplichtige ziekte is, kan men
uit de aangifte cijfers afleiden of dit het geval is. De laatste 2 jaar (2004
en 2005) is er een stijging te zien van het aantal aangegeven psittacose
gevallen. Of PCR diagnostiek hier een belangrijke rol in speelt is de
verwachting, maar moet nog met zekerheid aangetoond worden. De
aangifteplicht is ingesteld omdat oorzakelijke vogelbronnen op die
manier opgespoord en behandeld kunnen worden. Typering van de
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humane isolaten kan richting geven aan bronopsporing en de meest
waarschijnlijke vogelsoort identificeren.
Conclusies en aanbevelingen
Psittacose is een aangifteplichtige ziekte en moet bij voorkeur
gediagnosticeerd worden met behulp van PCR onderzoek. De in dit
proefschrift beschreven PCR is een snelle, gevoelige en specifieke
manier om dat doel te bereiken.
Typering van aviaire en humane isolaten zou moeten worden verricht om
de vogelbron vast te stellen en om de belangrijkste reservoirs van
humane psittacose gevallen hieruit af te leiden.
Op deze manier kan een betere schatting gemaakt worden van de
incidentie en prevalentie van humane psittacose gevallen en het verbetert
ons begrip van de epidemiologie van de verschillende genotypen van C.
psittaci in geïnfecteerde vogels, humane psittacose gevallen en de relatie
tussen deze twee.
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Dankwoord
Volgens de van Dale betekent Dankwoord “formele dankbetuiging van
iemand jegens een gehoor”. Ik hoop dat ik de afgelopen jaren ook
informeel wel eens impliciet of expliciet mijn dank heb getoond voor
verleende hulp, maar om verwijten hieromtrent te voorkomen volgt dan
hier het “Dankwoord”.
Christina Vandenbroucke-Grauls, mijn promotor, Caroline Visser en
Yvonne Pannekoek, mijn co-promotoren wil ik bedanken omdat ze het
wel zagen zitten met dit psittacose onderzoek. Caroline, zonder jou
daadkracht (“even een afspraakje maken met Christina”) was ik
waarschijnlijk niet tot dit proefschrift gekomen. Yvonne, zonder jou
Chlamydophiele belangstelling en je enorme bacterie verzameling was
dit proefschrift waarschijnlijk ook niet tot stand gekomen. Christina, ik
heb jou bijdragen erg gewaardeerd, met name je gave om de “grote lijn”
in de gaten te houden.
De Moleculaire Bacteriologische Unit waaronder Birgitta, Bob, Robin en
Lambert. Op jullie afdeling heb ik veel vrijheid gekregen om dit
onderzoek in alle rust uit te voeren. Birgitta bedankt voor het overleg
waarin we filosofeerden over de mogelijkheden en onmogelijkheden van
dit onderzoek. Bob bedankt voor al het informele overleg, waarbij we
geprobeerd hebben om moleculaire problemen op te lossen of in ieder
geval te omzeilen. Robin, je relatie met een diergeneeskunde student
heeft me een hoofdstuk in dit proefschrift opgeleverd. Bedankt! Niet
helemaal onderdeel van deze afdeling, maar wel noemenswaard: Ankie
Langerak. Jij hebt me praktisch flink op weg geholpen. Totdat jij je
ermee ging bemoeien zeiden termen als “plasmiden isolatie,
transformatie en kloneren” me in de praktijk erg weinig.
De afdeling Klinisch Virologie heeft direct en indirect een grote invloed
uitgeoefend op het tot stand komen van dit proefschrift. Pauline
Wertheim en Jan Weel, klinisch virologen, jullie hebben me gemotiveerd
en waren altijd enthousiast. Ook de vrijheid die jullie mij gaven om
klinisch diagnostische problemen uit te zoeken was leerzaam. Dit
resulteerde o.a. in hoofdstuk 2 van dit proefschrift. In het bijzonder wil ik
Gerrit Koen en Pien Defoer nog noemen. Jullie waren altijd bereid om
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(een) serum monster(s) “mee te nemen” in de serologische testen. Dit is
onmisbaar geweest voor het onderzoek en dit proefschrift.
Moleculaire diagnostiek leer je als art-assistent medische microbiologie
in het AMC pas echt als je op de afdeling Klinische moleculaire virologie
bent geweest! Marcel Beld, je kennis en prettige manier van
communiceren, heeft veel voor me betekend. Zonder jou kennis en het
gebruik van je R&D lab was dit proefschrift waarschijnlijk ook niet
ontstaan.
Jan Kaan, Erik van Hannen en Bartelt de Jongh van respectievelijk het
Mesos medisch centrum in Utrecht en de laatste 2 van het Antonius
ziekenhuis in Nieuwegein. De samenwerking met jullie was vanaf het
begin zowat vanzelfsprekend. Erg bijzonder. Ik loop lang genoeg rond in
Academische ziekenhuizen om te weten dat samenwerken, zonder
voorwaarden vooraf, niet altijd vanzelfsprekend is. We bleken allemaal te
werken aan het verbeteren van Chlamydophila psittaci diagnostiek met
behulp van PCR en de samenwerking heeft ons allemaal, denk ik,
voordelen opgeleverd. Maar mij waarschijnlijk het meest!
Sietske ter Sluis, HLO stagiair en inmiddels afgestudeerd. Ja ook jij in dit
rijtje! Ondanks dat je “in de belangstelling staan” nooit zo leuk vindt, wil
ik hier toch melden dat je ongelooflijk veel werk gedaan hebt. Je hebt een
groot deel van de feces monsters van de Amsterdamse stadsduiven
verzameld en getest en bent ook betrokken geweest bij het uitbraak
onderzoek in Utrecht. Zonder jou inzet was dit onderzoek nooit in zo’n
stroomversnelling gekomen. Ik heb je hier vermeld onder de mensen van
het Antonius in Nieuwegein, je nieuwe werkplek. Ik hoop dat je het bij
Bartelt en Erik op de afdeling naar je zin hebt.
Sjeng Lumeij, dierenarts verbonden aan de polikliniek bijzondere vogels
& gezelschapsdieren van de universiteit van Utrecht, bedankt voor de
snel opgezette samenwerking tijdens de uitbraak van psittacose in jullie
dierenkliniek. Het contact met een dierenarts tijdens dit onderzoek was
vaak verhelderend. Ik zal niet gauw vergeten dat ik een nieuw cloaca
monster vroeg van een geïnfecteerde vogel voor verder onderzoek
(genotypering) en dat je antwoordde: “je kunt ook de hele vogel krijgen
als je wilt”. Nou ja, dat wilde ik dus niet…., maar het ultieme verschil
tussen humane en veterinaire diergeneeskunde kwam in enkele seconden
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voorbij. Rest me nog te vermelden dat de vogel behandeld is met
antibiotica en het naar omstandigheden goed maakt.
Jan Buys en Joop van Wijnen van de GGD Amsterdam, dienst ongedierte
bestrijding. Bedankt voor jullie inzet en samenwerking. Ook bij jullie
geen moeilijke “mitsen en maren”, maar gewoon aan de slag! Dit
resulteerde, naar mijn mening, in een van de leukste artikelen in dit
proefschrift.
Mijn uiteindelijk volledig herziene populatie kamergenoten tijdens dit
onderzoek: Daan, Marjolein, Peter, Rob, Bas, Rogier J, Rogier v D,
Danny en in de laatste fase Caspar. Mijn onhebbelijkheid om mijn directe
kamergenoten deelgenoot te maken van alle frustraties die ik tegenkom
in mijn dagelijkse werk, moet af en toe vermoeiend zijn geweest.
Maja en Bart, met jullie als paranimfen moet het gaan lukken. Met een
ergotherapeut en een kindercardioloog denk ik dat er bij dit
promotieonderwerp toch weer een sterk secondanten team wordt
opgesteld!
Heit en Mem, ik weet dat jullie dit proefschrift leuk voor me vinden,
maar belangrijker voor mij is dat het jullie eigenlijk helemaal niets kan
schelen wat voor titels ik allemaal probeer te vergaren. Jullie vinden me
zonder net zo goed.
Natascha, jij bent niet zo onder de indruk van al dat laboratorium
onderzoek. Aan jou niet besteed. Deze relativering houdt mij met beide
benen op de grond. Wat wij (jij, ik, Indra en Imke) samen hebben is veel
mooier en zelfs van een hele andere orde dan de vreugde dat dit
proefschrift nu afgerond is.
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Curriculum vitae
Edou Redbad Heddema werd geboren op 3 juli 1971 te Wolvega. Hij
doorliep de HAVO en daarna het VWO aan het toenmalige Nassau
College te Heerenveen. In 1990 begon hij met de studie Geneeskunde
aan de VU te Amsterdam. Hij verhuisde van zijn toenmalige woonplaats
De Knipe, een dorp met ca. 1000 inwoners naar het grote Amsterdam (ca.
700.000 inwoners). Aanvankelijk met het doel om tropenarts te worden,
maar dit streven bekoelde na een wetenschappelijke stage in Ghana in
1994-1995. Ondanks de mooie tijd die hier doorgebracht werd, leek een
opleiding tot tropenarts toch niet de goede keus. In 1998 behaalde hij zijn
artsendiploma en ging vervolgens aan de slag als arts-assistent Interne
Geneeskunde in het Kennemer Gasthuis, locatie DEO te Haarlem. Hier
leerde hij de van oorsprong Limburgse Natascha Peters kennen waarmee
hij in 2003 trouwde. In 2000 werd begonnen met de opleiding tot artsmicrobioloog in het Academisch medisch centrum (AMC) te Amsterdam.
Gedurende deze opleiding kregen zij op 6 augustus 2004 hun eerste kind,
dochter Indra Famke. In mei 2005 volgde inschrijving in het medisch
specialisten registratie systeem als arts-microbioloog. Tot 1 december
2005 werkte hij verder aan zijn promotie onderzoek. Hierna is hij
begonnen als arts-microbioloog in het VU medisch centrum te
Amsterdam. Op 15 December 2006 werd hij voor de tweede maal vader,
ditmaal van dochter Imke Anna.
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Publicaties
Heddema, E. R., J. P. Teengs, and P. W. Kunst. 2002. Een patiënt met
een longabces, primair behandeld met drainage en aanvullend met
antibiotica. Ned.Tijdschr.Geneeskd. 146:77-79.
Wever, P. C., E. R. Heddema, M. G. van Vonderen, J. T. van der
Meer, M. D. de Jong, and van Gool T. 2003. Detection of
pneumococcemia by quantitative buffy coat analysis.
Eur.J.Clin.Microbiol.Infect.Dis. 22:450-452.
Heddema, E. R., M. C. Kraan, H. E. Buys-Bergen, H. E. Smith, and
P. M. Wertheim-van Dillen. 2003. A woman with a lobar infiltrate due
to psittacosis detected by polymerase chain reaction. Scand.J.Infect.Dis.
35:422-424.
Heddema, E. R., M. G. Beld, B. de Wever, A. A. Langerak, Y.
Pannekoek, and B. Duim. 2006. Development of an internally
controlled real-time PCR assay for detection of Chlamydophila psittaci in
the LightCycler 2.0 system. Clin.Microbiol.Infect. 12:571-575.
Heddema, E. R., S. ter Sluis, J. A. Buys, C. M. VandenbrouckeGrauls, J. H. van Wijnen, and C. E. Visser. 2006. Prevalence of
Chlamydophila psittaci in fecal droppings from feral pigeons in
Amsterdam, The Netherlands. Appl.Environ.Microbiol. 72:4423-4425.
Al Naiemi N., E. R. Heddema, A. Bart, E. de Jonge, C. M.
Vandenbroucke-Grauls, P. H. Savelkoul, and B. Duim. 2006.
Emergence of multidrug-resistant Gram-negative bacteria during
selective decontamination of the digestive tract on an intensive care unit.
J.Antimicrob.Chemother. 58:853-6.
Mulder, M. M., E. R. Heddema, Y. Pannekoek, K. Faridpooya, M. E.
Oud, E. Schilder-Tol, P. Saeed, and S. T. Pals. 2006. No evidence for
an association of ocular adnexal lymphoma with Chlamydia psittaci in a
cohort of patients from the Netherlands. Leuk.Res. 30:1305-1307.
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Heddema,E. R., E. J. van Hannen, B. Duim, B. M. de Jongh, J. A.
Kaan, R. van Kessel, J. T. Lumeij, C. E. Visser, and C. M. J. E.
Vandenbroucke-Grauls. 2006. An outbreak of psittacosis due to
Chlamydophila psittaci genotype A in a veterinary teaching hospital. J
Med Microbiol 55: 1571-1575.
Heddema,E. R., E. J. van Hannen, B. Duim, C. M. J. E.
Vandenbroucke-Grauls, and Y. Pannekoek. 2006. Genotyping of
Chlamydophila psittaci in human samples. Emerg Infect Dis 12: 19851986.
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