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Psittacosis : molecular tools for detection and typing of Chlamydophila psittaci
Heddema, E.R.
Publication date 2007 Document Version Final published version
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Citation for published version (APA): Heddema, E. R. (2007). Psittacosis : molecular tools for detection and typing of Chlamydophila psittaci. Mercis.
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UvA-DARE is a service provided by the library of the University of Amsterdam (https://dare.uva.nl) Download date:26 Sep 2021 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. Heddema 1, Yvonne Pannekoek 1, Caroline E. Visser 1, Christina M.J.E. Vandenbroucke-Grauls 1,2
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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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 19 th 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).
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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).
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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).
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Figure 1. Chlamydial developmental-cycle (source www.chlamydiae.com).
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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.
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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 .
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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 Main clinical syndromes Typical hosts Zoonotic disease C. psittaci Respiratory infection, Birds Respiratory tract conjunctivitis infection C. abortus Abortion Sheep, goats Spontaneous abortion C. felis Conjunctivitis Cats Conjunctivitis C. caviae Conjunctivitis Guinea pigs C. pecorum Abortion Cattle C. pneumoniae Respiratory infection Humans
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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 Length (kb) GC % Plasmid Encoded proteins Source C. psittaci 6BC Unfinished Yes Parakeet C. abortus S26/3 1144 39 No 932 Sheep C. felis Fe/C-56 1166 39 Yes 1005 Cat C. caviae (GPIC) 1173 39 Yes 998 Guinea pig C. pecorum E58 Unfinished No Cow C. pneumoniae AR39 1229 40 No 1112 Human C. pneumoniae TW-183 1226 40 No 1113 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).
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Figure 3. Allignment of the ompA of six avian C. psittaci serovars (A- F) showing the conserved and variable domains of the gene. (GenBank accession numbers AY762608-AY762612 and AF269261 generated by Vector NTI v10.) 1 50 C. psittaci sero A (1) MKKLLKSALLFAATGSALSLQALPVGNPAEPSLLIDGTMWEGASGDPCDP C. psittaci sero B (1) MKKLLKSALLFAATGSALSLQALPVGNPAEPSLLIDGTMWEGASGDPCDP C. psittaci sero C (1) MKKLLKSALLFAATGSALSLQALPVGNPAEPSLLIDGTMWEGASGDPCDP C. psittaci sero D (1) MKKLLKSALLFAATGSALSLQALPVGNPAEPSLLIDGTMWEGASGDPCDP C. psittaci sero E (1) MKKLLKSALLFAATGSALSLQALPVGNPAEPSLLIDGTMWEGASGDPCDP C. psittaci sero F (1) MKKLLKSALLFAATGSALSLQALPVGNPAEPSLLIDGTMWEGASGDPCDP 51 100 C. psittaci sero A (51) CATWCDAISIRAGYYGDYVFDRVLKVDVNKT FSG MAAT PT QATGNASNTN C. psittaci sero B (51) CATWCDAISIRAGYYGDYVFDRVLKVDVNKT FSG MAAT PT QATGNASNTN C. psittaci sero C (51) CSTWCDAISIRAGYYGDYVFDRVLKVDVNKT FSG IGKKPT GSS----PND C. psittaci sero D (51) CATWCDAISIRAGYYGDYVFDRVLKVDVNKT FSG MA KSPT EATG --TASA C. psittaci sero E (51) CATWCDAISIRAGYYGDYVFDRVLKVDVNKT FSG MAAT PT QATGNASNTN C. psittaci sero F (51) CATWCDAISIRAGYYGDYVFDRVLKVDVNKT ISG MGAAPT GSA----AAD 101 150 C. psittaci sero A (101) QPEAN GRPNIAYG RHMEDAEWF SNAAFLALNIWDRFDIFCTLGASNGYFK C. psittaci sero B (101) QPEAN GRPNIAYG RHMQDAEWF SNAAFLALNIWDRFDIFCTLGASNGYFK C. psittaci sero C (97) FK NAED RPNVAYG RHLQDSEWF TNAAFLALNIWDRFDIFCTLGASNGYFK C. psittaci sero D (99) TTTAVDRTNLAYG KHLQDAEWF TNAAFLALNIWDRFDIFCTLGASNGYFK C. psittaci sero E (101) QPEAN GRPNIAYG RHMQDAEWF SNAAFLALNIWDRFDIFCTLGASNGYFK C. psittaci sero F (97) YKTP TD RPNIAYG KHLQDAEWF TNAAFLALNIWDRFDIFCTLGASNGYFK 151 200 C. psittaci sero A (151) ASSAAFNLVGLIG FSA ASSISTDLP TQLPNV GITQG VVEFYTDT SFSWSV C. psittaci sero B (151) SSSAAFNLVGLIG FSA TNSTSTDLP MQLPNV GITQG VVEFYTDT SFSWSV C. psittaci sero C (147) ASSAAFNLVGLIG VKGS----SLTND QLPNVAITQG VVEFYTDT TFSWSV C. psittaci sero D (149) ASSAAFNLVGLIG LK G----- TD FNN QLPNV AITQG VVEFYTDT TFSWSV C. psittaci sero E (151) SSSAAFNLVGLIG FSA TSSTST ELP MQLPNV GITQG VVEFYTDT SFSWSV C. psittaci sero F (147) ASSAAFNLVGLIG VKGT----SVAAD QLPNV GITQG IVEFYTDT TFSWSV 201 250 C. psittaci sero A (201) GARGALWECGCATLGAEFQYAQSNPKIE MLNV TSSP AQFV IHKPRGYKG A C. psittaci sero B (201) GARGALWECGCATLGAEFQYAQSNPKIE ILNV TSSP AQFV IHKPRGYKG A C. psittaci sero C (193) GARGALWECGCATLGAEFQYAQSNPKIE MLNV ISSP AQFV VHKPRGYKG T C. psittaci sero D (194) GARGALWECGCATLGAEFQYAQSNPKIE MLNV TSSP AQFV IHKPRGYKG T C. psittaci sero E (201) GARGALWECGCATLGAEFQYAQSNPKIE VLNV TSSP AQFV IHKPRGYKG A C. psittaci sero F (193) GARGALWECGCATLGAEFQYAQSNPKIE MLNV ISSP TQFV VHKPRGYKG T 251 300 C. psittaci sero A (251) SS NFPLP IT AGT TE ATDTKSAT IKYHEWQVGLALSYRLNMLVPYIGVNWS C. psittaci sero B (251) SS NFPLP IT AGT TE ATDTKSAT IKYHEWQVGLALSYRLNMLVPYIGVNWS C. psittaci sero C (243) SANFPLP ANAGT EA ATDTKSAT LKYHEWQVGLALSYRLNMLVPYIGVNWS C. psittaci sero D (244) GSNFPLP IDAGT EA ATDTKSAT LKYHEWQVGLALSYRLNMLVPYIGVNWS C. psittaci sero E (251) SS NFPLP IT AGT TE ATDTKSAT IKYHEWQVGLALSYRLNMLVPYIGVNWS C. psittaci sero F (243) GSNFPLP LTAGT DG ATDTKSAT LKYHEWQVGLALSYRLNMLVPYIGVNWS 301 350 C. psittaci sero A (301) RATFDAD TIRIAQPKL KSEI LN ITTWNP SLIGSTTAL PNN SGKDVLS DVL C. psittaci sero B (301) RATFDAD TIRIAQPKL KSEI LN ITTWNP SLLGSTTAL PNN SGKDVLS DVL C. psittaci sero C (293) RATFDAD TIRIAQPKL ASAVM NLTTWNP TLLGEA TMLDTS N---KFSDFL C. psittaci sero D (294) RATFDAD TIRIAQPKL ATAVLDLTTWNP TLLGKA TTVDGT N--- TYSDFL C. psittaci sero E (301) RATFDAD TIRIAQPKL KSEI LN ITTWNP SLLGSTTTLPNN GGKDVLS DVL C. psittaci sero F (293) RATFDAD SIRIAQPKL AAAVLN LTTWNP TLLGEA TAL DA SN---KFC DFL 351 366 C. psittaci sero A (351) QIASIQINKMKSRKAC C. psittaci sero B (351) QIASIQINKMKSRKAC C. psittaci sero C (340) QIASIQINKMKSRKAC C. psittaci sero D (341) QLASIQINKMKSRKAC C. psittaci sero E (351) QIASIQINKMKSRKAC C. psittaci sero F (340) QIASIQINKMKSRKAC
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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 Representative strain Host association A VS1 Psittacine birds B CP3 Pigeons E/B WS/RT/E30 Ducks C GD Ducks, geese D NJ1 Turkeys E MN Pigeons, turkeys F VS225 Psittacine birds M56 M56 Cattle WC WC 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.
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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 afore- mentioned 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.
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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 1988- 2005.
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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).
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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
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. Bedson, S. P. 1935. The use of the complement-fixation reaction in the diagnosis of human psittacosis. The Lancet 226 :1277-1280. 3. Bedson, S. P., G. T. Western, and S. Levy Simpson . 1930. Further observations on the aetiology of psittacosis. The Lancet 215 :345-346. 4. Bedson, S. P., G. T. Western, and S. L. Simpson . 1930. Observations on the aetiology of psittacosis. The Lancet 215 :235-236. 5. Bol J.J. 1957. Ornithosis. Ned.Tijdschr.Geneeskd. 101 :1068-1070. 6. 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. 7. Bruins Slot W.J. 1957. [Ornithosis.]. Ned.Tijdschr.Geneeskd. 101 :257-260. 8. Bush, R. M. and K. D. Everett . 2001. Molecular evolution of the Chlamydiaceae . Int.J.Syst.Evol.Microbiol. 51 :203-220. 9. Cartwright, K. A., E. O. Caul, and R. W. Lamb . 1988. Symptomatic Chlamydia psittaci reinfection. Lancet 1:1004. 10. CDC . 2004. Notifiable Diseases/Deaths in Selected Cities Weekly Information. MMWR 52 :1291-1299. 11. Chanudet, E., Y. Zhou, C. Bacon, A. Wotherspoon, H. K. Muller- Hermelink, P. Adam, H. Dong, J. D. de, Y. Li, R. Wei, X. Gong, Q. Wu, R. Ranaldi, G. Goteri, S. Pileri, H. Ye, R. Hamoudi, H. Liu, J. Radford, and M. Q. Du . 2006. Chlamydia psittaci is variably associated with ocular adnexal MALT lymphoma in different geographical regions. J.Pathol. 12. Cooper, S. M. and J. A. Ferriss . 1986. Reactive arthritis and psittacosis. Am.J.Med. 81 :555-557. 13. Corsaro, D., D. Venditti, and M. Valassina . 2002. New parachlamydial 16S rDNA phylotypes detected in human clinical samples. Res.Microbiol. 153 :563- 567. 14. Cotton, M. M. and M. R. Partridge . 1998. Infection with feline Chlamydia psittaci . Thorax 53 :75-76. 15. Daibata, M., Y. Nemoto, K. Togitani, A. Fukushima, H. Ueno, K. Ouchi, H. Fukushi, S. Imai, and H. Taguchi . 2006. Absence of Chlamydia psittaci in ocular adnexal lymphoma from Japanese patients. Br.J.Haematol. 132 :651- 652.
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16. Dekking, F. 1950. Universiteit van Amsterdam. Psittacosis en ornithosis in Nederland. 17. Dekking, F. 1949. Postduiven en psittacosis. Ned.Tijdschr.Geneeskd. 93 :4338- 4339. 18. Dijkstra, F. and O. F. J. Stenvers . 2006. Toename van individuele gevallen en clusters van psittacose in 2005. Dutch Infectious Diseases Bulletin 17 :5-7. 19. Eeckhout, E., A. Volckaert, A. Naessens, and W. Schandevyl . 1986. [Ornithosis as a general systemic disorder]. Ned.Tijdschr.Geneeskd. 130 :1487- 1489. 20. 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. 21. Ferreri, A. J., M. Guidoboni, M. Ponzoni, C. C. De, S. Dell'Oro, K. Fleischhauer, L. Caggiari, A. A. Lettini, C. E. Dal, R. Ieri, M. Freschi, E. Villa, M. Boiocchi, and R. Dolcetti . 2004. Evidence for an association between Chlamydia psittaci and ocular adnexal lymphomas. J.Natl.Cancer Inst. 96 :586-594. 22. 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. 23. Geens, T., A. Dewitte, N. Boon, and D. Vanrompay . 2005. Development of a Chlamydophila psittaci species-specific and genotype-specific real-time PCR. Vet.Res. 36 :787-797. 24. Gherman, R. B., L. L. Leventis, and R. C. Miller . 1995. Chlamydial psittacosis during pregnancy: a case report. Obstet.Gynecol. 86 :648-650. 25. Ginevra, C., C. Barranger, A. Ros, O. Mory, J. L. Stephan, F. Freymuth, M. Joannes, B. Pozzetto, and F. Grattard . 2005. Development and evaluation of Chlamylege, a new commercial test allowing simultaneous detection and identification of Legionella, Chlamydophila pneumoniae , and Mycoplasma pneumoniae in clinical respiratory specimens by multiplex PCR. J.Clin.Microbiol. 43 :3247-3254. 26. Gordon M.H. 1930. Virus studies concerning the aetiology of psittacosis. Lancet 215 :1174-1177.
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27. Grayston, J. T., C. C. Kuo, S. P. Wang, and J. Altman . 1986. A new Chlamydia psittaci strain, TWAR, isolated in acute respiratory tract infections. N.Engl.J.Med. 315 :161-168. 28. 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. 29. Harris, R. L. and T. W. Williams, Jr. 1985. "Contribution to the Question of Pneumotyphus": a discussion of the original article by J. Ritter in 1880. Rev.Infect.Dis. 7:119-122. 30. Hedberg, K., K. E. White, J. C. Forfang, J. A. Korlath, K. A. Friendshuh, C. W. Hedberg, K. L. MacDonald, and M. T. Osterholm. 1989. An outbreak of psittacosis in Minnesota turkey industry workers: implications for modes of transmission and control. Am.J.Epidemiol. 130 :569-577. 31. Herderschêe D . 1930. Gevallen van psittacosis in Amsterdam. Ned.Tijdschr.Geneeskd. 74 :873-879. 32. Herring, A. J., I. E. Anderson, M. McClenaghan, N. F. Inglis, H. Williams, B. A. Matheson, C. P. West, M. Rodger, and P. P. Brettle . 1987. Restriction endonuclease analysis of DNA from two isolates of Chlamydia psittaci obtained from human abortions. Br.Med.J.(Clin.Res.Ed) 295 :1239. 33. 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 :155- 166. 34. Iterson van G.L. 1957. [Two cases of presumptive ornithosis.]. Ned.Tijdschr.Geneeskd. 101 :1619-1620. 35. Johnson, S. R. and I. D. Pavord . 1996. Grand Rounds--City Hospital, Nottingham: A complicated case of community acquired pneumonia. BMJ 312 :899-901. 36. Kaltenboeck, B., K. G. Kousoulas, and J. Storz . 1993. Structures of and allelic diversity and relationships among the major outer membrane protein (ompA ) genes of the four chlamydial species. J.Bacteriol. 175 :487-502. 37. Kampinga, G. A., F. P. Schroder, I. J. Visser, J. M. Anderson, D. Buxton, and A. V. Moller . 2000. [Lambing ewes as a source of severe psittacosis in a pregnant woman]. Ned.Tijdschr.Geneeskd. 144 :2500-2504. 38. Levinthal W. 1935. Recent observations on psittacosis. Lancet 225 :1207- 1210. 39. Liu, Y. C., J. H. Ohyashiki, Y. Ito, K. I. Iwaya, H. Serizawa, K. Mukai, H. Goto, M. Usui, and K. Ohyashiki . 2006. Chlamydia psittaci in ocular adnexal lymphoma: Japanese experience. Leuk.Res.
27 Chapter 1
40. Longbottom, D. and L. J. Coulter . 2003. Animal chlamydioses and zoonotic implications. J.Comp Pathol. 128 :217-244. 41. 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. 42. 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. 43. Meijer, A., A. Brandenburg, J. de Vries, J. Beentjes, P. Roholl, and D. Dercksen . 2004. Chlamydophila abortus infection in a pregnant woman associated with indirect contact with infected goats. Eur.J.Clin.Microbiol.Infect.Dis. 23 :487-490. 44. 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. 45. 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. 46. Ministerie van de Vlaamse gemeenschap . 2004. Meldingen besmettelijke ziekten. Vlaams Infectieziektebulletin 10. 47. Ministerie van de Vlaamse gemeenschap . 2005. Meldingen besmettelijke ziekten. Vlaams Infectieziektebulletin 10. 48. Moulder, J. W. 1966. The relation of the psittacosis group ( Chlamydiae ) to bacteria and viruses. Annu.Rev.Microbiol. 20 :107-130. 49. 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. [Epub ahead of print] doi:10.1016/j.leukres.2005.12.003 50. Newman, C. P., S. R. Palmer, F. D. Kirby, and E. O. Caul . 1992. A prolonged outbreak of ornithosis in duck processors. Epidemiol.Infect. 108 :203-210. 51. Persson, K. and S. Haidl . 2000. Evaluation of a commercial test for antibodies to the chlamydial lipopolysaccharide (Medac) for serodiagnosis of acute infections by Chlamydia pneumoniae (TWAR) and Chlamydia psittaci . APMIS 108 :131-138. 52. Pinkhof, H. 1940. Argentinie - Epidemie van psittacosis. Nederlands Tijdschrift voor Geneeskunde 84 :1147.
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53. Potter, M. E., A. K. Kaufmann, and B. D. Plikaytis . 1983. Psittacosis in the United States, 1979. Morb.Mortal.Wkly.Rep.Surveill Summ. 32 :27SS-31SS. 54. Ritter, J. 1881. Beitrag zur Frage des Pneumotyphus. (Eine Hausepidemie in Uster [Schweiz] betreffend.). Deutches Archiv fur Klinische Medizin 25 :53- 96. 55. RIVM . 2004. Notified cases of infectious diseases in the Netherlands. Dutch Infectious Diseases Bulletin 15 . 56. RIVM . 2005. Notified cases of infectious diseases in the Netherlands. Dutch Infectious Diseases Bulletin 16 . 57. Robert Koch Institute . 2005. Meldepflichtige Infektionskrankheiten 2004. Epidemiologisches Bulletin 470. 58. Robert Koch Institute . 2005. Meldepflichtige Infektionskrankheiten 2005. Epidemiologisches Bulletin 14. 59. Rosado, M. F., G. E. Byrne, Jr., F. Ding, K. A. Fields, P. Ruiz, S. R. Dubovy, G. R. Walker, A. Markoe, and I. S. Lossos . 2006. Ocular adnexal lymphoma: a clinicopathologic study of a large cohort of patients with no evidence for an association with Chlamydia psittaci . Blood 107 :467-472. 60. Ruys, A. C. 1934. Psittacosis in Duitschland en Amerika. Nederlands Tijdschrift voor Geneeskunde 78 :2787. 61. Schachter, J., Ostler, H. B., and Meyer, K. F . 1969. Human infection with the agent of feline pneumonitis. Lancet 1:1063-1065. 62. Schachter, J., R. S. Stephens, P. Timms, C. Kuo, P. M. Bavoil, S. Birkelund, J. Boman, H. Caldwell, L. A. Campbell, M. Chernesky, G. Christiansen, I. N. Clarke, C. Gaydos, J. T. Grayston, T. Hackstadt, R. Hsia, B. Kaltenboeck, M. Leinonnen, D. Ocjius, G. McClarty, J. Orfila, R. Peeling, M. Puolakkainen, T. C. Quinn, R. G. Rank, J. Raulston, G. L. Ridgeway, P. Saikku, W. E. Stamm, D. T. Taylor-Robinson, S. P. Wang, and P. B. Wyrick . 2001. Radical changes to chlamydial taxonomy are not necessary just yet. Int.J.Syst.Evol.Microbiol. 51 :249, 251-249, 253. 63. Schaffner, W. 1997. Birds of a feather--do they flock together? Infect.Control Hosp.Epidemiol. 18 :162-164. 64. Schouten, J. A., J. M. Prins, M. J. Bonten, J. Degener, R. E. Janknegt, J. M. Hollander, R. E. Jonkers, W. J. Wijnands, T. J. Verheij, A. P. Sachs, and B. J. Kullberg . 2005. Revised SWAB guidelines for antimicrobial therapy of community-acquired pneumonia. Neth.J.Med. 63 :323-335. 65. Scientific committee on animal health and animal welfare . 2002. Avian chlamydiosis as a zoonotic risk and reduction strategies. http://europa.eu.int/comm/food/fs/sc/scah/out73_en.pdf
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66. Shapiro, D. S., S. C. Kenney, M. Johnson, C. H. Davis, S. T. Knight, and P. B. Wyrick . 1992. Brief report: Chlamydia psittaci endocarditis diagnosed by blood culture. N.Engl.J.Med. 326 :1192-1195. 67. 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. 68. 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. 69. Toyokawa, M., T. Kishimoto, Y. Cai, M. Ogawa, S. Shiga, I. Nishi, H. Hosotsubo, M. Horikawa, and S. Asari . 2004. Severe Chlamydophila psittaci pneumonia rapidly diagnosed by detection of antigen in sputum with an immunochromatography assay. J.Infect.Chemother. 10 :245-249. 70. 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. 71. Vanrompay, D., R. Ducatelle, and F. Haesebrouck . 1995. Chlamydia psittaci infections: a review with emphasis on avian chlamydiosis. Vet.Microbiol. 45 :93-119. 72. Vervoort H. and A. C. Ruys . 1940. The recognition of psittacosis. Antonie van Leeuwenhoek 6:11-21. 73. 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. 74. 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. 75. 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. 76. Yung, A. P. and M. L. Grayson . 1988. Psittacosis--a review of 135 cases. Med.J.Aust. 148 :228-233.
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 so- called “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 * 10 9 /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, pCO 2 3.1 kPa, pO 2 7.1 kPa, bi- carbonate 19.7 mmol/l, Base-Excess –1.2 mmol/l, O 2 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 2 nd and 7 th 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 :155- 166.
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7. Lynch, D. A. and J. D. Armstrong . 1991. A pattern-oriented approach to chest radiographs in atypical pneumonia syndromes. Clin.Chest Med. 12 :203- 222. 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 community- acquired 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.
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Development of an internally controlled real- time PCR assay for detection of Chlamydophila psittaci in the LightCycler 2.0 system
Edou R. Heddema 1, Marcel G.H.M. Beld 2, Bob de Wever 1, Ankie A.J. Langerak 1, Yvonne Pannekoek 1 and Birgitta Duim 1.
Department of Medical Microbiology 1 and Department of Clinical
Virology 2, Academic Medical Centre, University of Amsterdam, Amsterdam, the Netherlands.
Adapted from Clinical Microbiology and Infection 2006 Jun;12(6):571-5.
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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.
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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.
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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 pre- treatment. 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.
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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 uracil- N-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 MgCl 2. 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.
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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 real- time 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).
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Table 1. Lower limit of detection of the PCR assay for pPsittWT, IC and the combination. Copies per PCR pPsittWT a IC a pPsittWT with 80 copies of IC a 80 b 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 b For all the copy numbers presented, we assumed a 100% extraction and PCR efficiency .
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 real- time 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 phase- and 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.
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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 -/+ C. pecorum E58 a -/+ C. trachomatis Serovar D IC-CAL-8 a -/+ C. trachomatis Serovar E DK-20 a -/+ C. trachomatis Serovar F MRC-301 a -/+ a) , previously studied and characterized by Meyer et al. (15) ATCC, American Type Culture Collection QC, Dutch or UK quality control assessment strains
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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.
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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
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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 :422- 424. 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 :155- 166. 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
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obstructive pulmonary disease by micro-immunofluorescence and ELISA. J.Med.Microbiol. 46 :959-964. 22. 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 :2301- 2307. 23. 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. 24. 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. 25. 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. 26. Yung, A. P. and M. L. Grayson . 1988. Psittacosis--a review of 135 cases. Med.J.Aust. 148 :228-233.
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An outbreak of psittacosis due to Chlamydophila psittaci genotype A in a veterinary teaching hospital
Edou R. Heddema 1, Erik J. van Hannen 2, Birgitta Duim 1, Bartelt M. de Jongh 2, Jan A. Kaan 3, Rob van Kessel 4, Johannes T. Lumeij 5, Caroline E. Visser 1, Christina M.J.E. Vandenbroucke-Grauls 1,6
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):1571- 75.
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 14 th of December 2004 and had followed a post-graduate teaching course on the 30 th 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 5 th of January and not involved in the post-graduate teaching. A third patient was admitted on the 13 th 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 30 th 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 21 st 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).
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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).
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Figure 1 . Flow diagram of the outbreak
38 people included
3 admitted to hospital 29 PCR and convalescent 19 PCR and serologically serum samples available negative
3 PCR positive on 7 serologically proven 4 PCR negative on a sputum throat swab
2 serologically proven 3 PCR positive on a throat swab
1 ICU *: sepsis, MOF § 2 no symptoms 3 symptoms and treated 2 pneumonia 1 brief self-limiting illness 1 without symptoms
Hospital Veterinary teaching hospital
* ICU, intensive care unit
§ MOF, multi-organ failure
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Table 1. Patient characteristics
nd and 2 and st Gender Gender Age Clinical features PCR specimen PCR rELISA Complement Fixation test (days)period Incubation between 1 Days serum sample serum M 37 Sepsis Sputum + + + Na # 14 F 37 Pneumonia Sputum + + - 14 29 M 61 Pneumonia Sputum + - - Na 46 F 26 Fever, headache Throat swab - + + 12 21 F 27 Fever, headache Throat swab - + + 12 21 F 29 Fever, headache Throat swab + + + 11 21 M 28 none Throat swab - + + Na 41 M 35 none Throat swab + + - Na 21 F 25 none Throat swab + + - Na 28 F 30 Fever, headache Throat swab - + + 14 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. Vandenbroucke- Grauls, 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. Lopez- Hontagas, M. Salavert, and M. Gobernado . 1999. Value of the polymerase chain reaction assay in noninvasive respiratory samples for diagnosis of community-acquired pneumonia. Am.J.Respir.Crit Care Med. 159 :1868-1873. 11. Persson, K. and S. Haidl . 2000. Evaluation of a commercial test for antibodies to the chlamydial lipopolysaccharide (Medac) for serodiagnosis of acute infections by Chlamydia pneumoniae (TWAR) and Chlamydia psittaci . APMIS 108 :131-138.
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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 pneumoniae, or Chlamydia pneumoniae lower respiratory infection using the polymerase chain reaction on a single throat swab specimen. Diagn.Microbiol.Infect.Dis. 24 :7-14. 13. Schlossberg, D., J. Delgado, M. M. Moore, A. Wishner, and J. Mohn . 1993. An epidemic of avian and human psittacosis. Arch.Intern.Med. 153 :2594-2596. 14. Schneeberger, P. M., J. W. Dorigo-Zetsma, Z. A. van der, M. van Bon, and J. L. van Opstal . 2004. Diagnosis of atypical pathogens in patients hospitalized with community-acquired respiratory infection. Scand.J.Infect.Dis. 36 :269-273. 15. 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 16. 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. 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, J. M. van den Bosch, H. P. Van Helden, and H. A. Verbrugh . 1997. Diagnosis of Chlamydia pneumoniae infection in patients with chronic obstructive pulmonary disease by micro-immunofluorescence and ELISA. J.Med.Microbiol. 46 :959-964. 19. 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 :2301- 2307. 20. 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. 21. Yung, A. P. and M. L. Grayson . 1988. Psittacosis--a review of 135 cases. Med.J.Aust. 148 :228-233.
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Chapter 5
Prevalence of Chlamydophila psittaci in fecal droppings from feral pigeons in Amsterdam, The Netherlands
Edou R. Heddema 1*, Sietske ter Sluis 1, Jan A. Buys 2, Christina M.J.E. Vandenbroucke-Grauls 1,3 , Joop H. van Wijnen 2, Caroline E. Visser 1.
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 3 rd of February and the 8 th of March 2005, when breeding activity is low and on the 2 nd 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 real- time 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 MgCl 2. 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 (1- distance) 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).
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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 0/15 a 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 Total 8/160 (5%; 95% CI 18/171 (10%; 95% CI 2-10%) b 6-16%) 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 real- time 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 :155- 166. 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. Heddema 1*, Erik J. van Hannen 2, Birgitta Duim 1, Christina M.J.E. Vandenbroucke-Grauls 1,3 , Yvonne Pannekoek 1.
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 (A- F, 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.
83 Chapter 6
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 :422- 424. 6. Heddema, E. R., S. Sluis ter, J. A. Buijs, C. M. J. E. Vandenbroucke- Grauls, 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. Heddema 1, Yvonne Pannekoek 1, Caroline E. Visser 1, Christina M.J.E. Vandenbroucke-Grauls 1,2
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.
85 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.
86 Chapter 7
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
87 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
88 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
89 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.
90 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.
91 Chapter 7
Final conclusions and recommendations
Psittacosis, a notifiable disease, should be primarily diagnosed by real- time 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 :435- 461. 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 :53- 96. 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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Chapter 8 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
96 Chapter 8 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
97 Chapter 8 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
100 Chapter 8 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.
101 Chapter 8 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 arts- microbioloog 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.
102 Chapter 8 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. Vandenbroucke- Grauls, 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 : 1985- 1986.
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