Emerging chlamydial infections

Chlamydiae are important intracellular bacterial pathogens of vertebrates. In the last years, novel members of this group have been discovered: Parachlamydia acanthamoebae and Simkania negevensis seems to be emerging respiratory human pathogens, while Waddlia chondrophila might be a new agent of bovine abortion. Various species have been showed to infect also the herpetofauna and fishes, and some novel chlamydiae are endosymbionts of arthropods. In addition, molecular studies evidenced a huge diversity of chlamydiae from both environmental and clinical samples, most of such a diversity could be formed by novel lineages of chlamydiae. Experimental studies showed that free-living amoebae may support multiplication of various chlamydiae, then could play an important role as reservoir/vector of chlamydial infections. Here we reviewed literature data concerning chlamydial infections, with a particular emphasis on the novely described chlamydial organisms.     

Pathogenic potential of novel Chlamydiae and diagnostic approaches to infections due to these obligate intracellular bacteria

Novel chlamydiae are newly recognized members of the phylum Chlamydiales that are only distantly related to the classic Chlamydiaceae, i.e., Chlamydia and Chlamydophila species. They also exhibit an obligate biphasic intracellular life cycle within eukaryote host cells. Some of these new chlamydiae are currently considered potential emerging human and/or animal pathogens. Parachlamydia acanthamoebae and Simkania negevensis are both emerging respiratory human pathogens, Waddlia chondrophila could be a novel abortigenic bovine agent, and Piscichlamydia salmonis has recently been identified as an agent of the gill epitheliocystis in the Atlantic salmon. Fritschea spp. and Rhabdochlamydia spp. seem to be confined to arthropods, but some evidence for human exposure exists. In this review, we first summarize the data supporting a pathogenic potential of the novel chlamydiae for humans and other vertebrates and the interactions that most of these chlamydiae have with free-living amoebae. We then review the diagnostic approaches to infections potentially due to the novel chlamydiae, especially focusing on the currently available PCR-based protocols, mammalian cell culture, the amoebal coculture system, and serology.     

Emerging Chlamydial Infections

Chlamydiae are important intracellular bacterial pathogens of vertebrates. In the last years, novel members of this group have been discovered: Parachlamydia acanthamoebae and Simkania negevensis seems to be emerging respiratory human pathogens, while Waddlia chondrophila might be a new agent of bovine abortion. Various species have been showed to infect also the herpetofauna and fishes, and some novel chlamydiae are endosymbionts of arthropods. In addition, molecular studies evidenced a huge diversity of chlamydiae from both environmental and clinical samples, most of such a diversity could be formed by novel lineages of chlamydiae. Experimental studies showed that free-living amoebae may support multiplication of various chlamydiae, then could play an important role as reservoir/vector of chlamydial infections. Here we reviewed literature data concerning chlamydial infections, with a particular emphasis on the novely described chlamydial organisms.     

Increasing diversity within Chlamydiae

In recent years, 16S ribosomal DNA analyses has allowed the recognition of new chlamydia organisms, requiring the creation of new species, genera, and families within this unique, deep lineage of prokaryotes. The trachoma and psittaci groups chlamydiae are now recognized as separate genera, Chlamydia and Chlamydophila, respectively, and biovars of each group have been elevated to the species rank. Simkania and Parachlamydia have been associated with human respiratory infections, while Waddlia seems to be implicated in abortion in bovins. DNA amplification studies targeting the 16S rDNA have revealed a richer diversity within chlamydiae, identifying new lineages from both environmental and clinical samples. Further studies will be of interest to both examine the ecology and evaluate the clinical importance of these novel chlamydiae. Herein, we provide a summary of literature and our data about the novel chlamydial lineages.     

The many faces of chlamydiae

The application of modern research tools has broadened our understanding of the chlamydiae and their role in disease. Chlamydial genome analysis showed the presence of genes for ATP and peptidoglycan synthesis, contradicting the common belief that chlamydiae lack the ability to produce these compounds. Phylogenetic tree analysis suggests that chlamydiae could have evolved from an intracellular existence in amoebae. Newly discovered obligate intracellular organisms with chlamydia-like life-cycles have been classified as chlamydiae by rRNA homology with existing chlamydial species. A proposed new classification adds three new families to the order Chlamydiales as well as creates two genera and nine species within the family Chlamydiaceae. Chlamydiae are incriminated in an increasingly large spectrum of diseases both in humans and in animals. The emergence of multi-drug resistant C. trachomatis strains forewarns therapeutic problems with this organism. While C. pneumoniae remains a significant respiratory pathogen, the role it plays in the pathogenesis of atherosclerosis and ischaemic heart disease awaits definition.     

Parachlamydia acanthamoebae, an emerging agent of pneumonia

Parachlamydia acanthamoebae is a Chlamydia- like organism that easily grows within Acanthamoeba spp. Thus, it probably uses these widespread free-living amoebae as a replicative niche, a cosmopolite aquatic reservoir and a vector. A potential role of P. acanthamoebae as an agent of lower respiratory tract infection was initially suggested by its isolation within an Acanthamoeba sp. recovered from the water of a humidifier during the investigation of an outbreak of fever. Additional serological and molecular-based investigations further supported its pathogenic role, mainly in bronchiolitis, bronchitis, aspiration pneumonia and community-acquired pneumonia. P. acanthamoebae was shown to survive and replicate within human macrophages, lung fibroblasts and pneumocytes. Moreover, this strict intracellular bacterium also causes severe pneumonia in experimentally infected mice, thus fulfilling the third and fourth Koch criteria for a pathogenic role. Consequently, new tools have been developed for the diagnosis of parachlamydial infections. It will be important to routinely search for this emerging agent of pneumonia, as P. acanthamoebae is apparently resistant to quinolones, which are antibiotics often used for the empirical treatment of atypical pneumonia. Other Chlamydia -related bacteria, including Protochlamydia naegleriophila , Simkania negevensis and Waddlia chondrophila , might also cause lung infections. Moreover, several additional novel chlamydiae, e.g. Criblamydia sequanensis and Rhabdochlamydia crassificans , have been discovered and are now being investigated for their human pathogenicity.     

Tobacco smoke induces persistent infection of Chlamydophila pneumoniae in HEp-2 cells

We examined tobacco smoke exposure and its effect on the life cycle of Chlamydophila pneumoniae (C. pneumoniae) in HEp-2, a human respiratory epithelial cell line. Using noncytotoxic concentrations of smoke medium, chlamydiae were grown in tissue culture and infectious particles were quantitated indirectly by immunocytometry of infected indicator cells. Chlamydial genome copy number was assessed with real-time polymerase chain reaction, and ultrastructure was examined by transmission electron microscopy. There was a significant reduction (56-64%; p<0.05) in the number of infectious elementary bodies following smoke exposure compared to untreated cultures. Under the same conditions, at late time points, smoke-exposed cultures showed significantly fewer chlamydial DNA copies (p<0.04). Moreover, smoke exposure induced large aberrant bodies that predominated within the inclusion. Following in vitro smoke exposure, alterations in the developmental cycle of C. pneumoniae included: inhibition of productive infection, reduced bacterial cell division, and formation of aberrant bodies. Thus, using this novel system, we were able to induce chlamydial persistence. Tobacco smoke exposure may represent a risk for establishment of a chronic reservoir of C. pneumoniae infection within respiratory epithelium.     

Development of a new chlamydiales-specific real-time PCR and its application to respiratory clinical samples

Originally composed of the single family Chlamydiaceae, the Chlamydiales order has extended considerably over the last several decades. Chlamydia-related bacteria were added and classified into six different families and family-level lineages: the Criblamydiaceae, Parachlamydiaceae, Piscichlamydiaceae, Rhabdochlamydiaceae, Simkaniaceae, and Waddliaceae. While several members of the Chlamydiaceae family are known pathogens, recent studies showed diverse associations of Chlamydia-related bacteria with human and animal infections. Some of these latter bacteria might be of medical importance since, given their ability to replicate in free-living amoebae, they may also replicate efficiently in other phagocytic cells, including cells of the innate immune system. Thus, a new Chlamydiales-specific real-time PCR targeting the conserved 16S rRNA gene was developed. This new molecular tool can detect at least five DNA copies and show very high specificity without cross-amplification from other bacterial clade DNA. The new PCR was validated with 128 clinical samples positive or negative for Chlamydia trachomatis or C. pneumoniae. Of 65 positive samples, 61 (93.8%) were found to be positive with the new PCR. The four discordant samples, retested with the original test, were determined to be negative or below detection limits. Then, the new PCR was applied to 422 nasopharyngeal swabs taken from children with or without pneumonia; a total of 48 (11.4%) samples were determined to be positive, and 45 of these were successfully sequenced. The majority of the sequences corresponded to Chlamydia-related bacteria and especially to members of the Parachlamydiaceae family.     

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

This study describes the development and evaluation of a new commercial test, Chlamylege (Argene Inc.), which allows the simultaneous detection in respiratory samples of Chlamydophila pneumoniae, Mycoplasma pneumoniae, and most Legionella species, as well as PCR inhibitors, by using a multiplex PCR and microplate hybridization. The sensitivities of Chlamylege were 1 x 10(-3) IFU, 5 x 10(-2) color-changing units, and 1 CFU per reaction tube for C. pneumoniae, M. pneumoniae, and Legionella pneumophila, respectively. A cohort of 154 clinical samples from patients with documented respiratory infections was analyzed by the kit, including 2 samples from patients with C. pneumoniae infection, 9 samples from patients with M. pneumoniae infection, 19 samples from patients with Legionella species infection, and 114 samples that tested negative for the three pathogens. All the positive specimens were correctly detected and identified by the Chlamylege kit, and no false-positive result was observed with the negative samples. The kit was then evaluated in a pediatric prospective study that included 220 endotracheal aspirates, and the results were compared with those obtained by three single in-house PCR assays. Four specimens were found to be positive for C. pneumoniae and six were found to be positive for M. pneumoniae by using both strategies. The Chlamylege kit detected two additional samples positive for M. pneumoniae and one additional sample positive for a Legionella species other than L. pneumophila; these three samples were shown to be true positive by other techniques. These overall results demonstrate that the Chlamylege assay is sensitive, specific, and convenient for the rapid detection and identification of atypical pathogens in clinical samples from patients with respiratory infections.     

Detection of Chlamydia pneumoniae DNA and antigen in the circulating mononuclear cell fractions of humans and koalas

Chlamydia pneumoniae is a common respiratory pathogen of humans which, in addition to causing disease at the respiratory site, has recently been linked to disease at other body sites. If C. pneumoniae does contribute to disease at nonrespiratory sites, then it must have a mechanism by which it reaches these sites. We analyzed the peripheral blood mononuclear cell (PBMC) fractions from 60 healthy human blood donors for the presence of C. pneumoniae DNA (by ompA PCR) and chlamydial antigens (by genus- and species-specific monoclonal antibody staining). Ten of the sixty (16. 7%) blood donors were C. pneumoniae positive by PCR, and all 10 of these PCR-positive individuals' samples demonstrated specific staining with anti-C. pneumoniae monoclonal antibodies. The only other host naturally infected with C. pneumoniae is the koala, in which the bacterium also causes respiratory infections. We demonstrated the presence of C. pneumoniae DNA and antigens in the PBMC fractions of 30% of 20 koalas tested. Our finding of C. pneumoniae-infected PBMCs in koalas as well as humans suggests that the ability to infect PBMCs and to disseminate from the respiratory site is not restricted to the human biovar of C. pneumoniae but is a general characteristic of this chlamydial species.     

Evidence for numerous omp1 alleles of porcine Chlamydia trachomatis and novel chlamydial species obtained by PCR.

A nested PCR for genus-specific amplification of the Chlamydia omp1 locus was established. This PCR detected single template molecules in 200-microl specimen aliquots. Amplified chlamydial omp1 alleles were typed by heminested species PCRs and allele PCRs. We applied this method to 407 specimens from several host animals with various clinical conditions, and we detected prevalences of chlamydiae from 6 to 50%. Amplicons from peacock enteritis and equine infertility specimens were not typeable according to present omp1 allelic criteria for the chlamydial species. DNA sequencing revealed novel omp1 alleles which were 29.9 and 47.6% divergent in the deduced peptide sequences from the most closely related chlamydiae. Phylogenetic reconstruction indicated segregation of these alleles from the current four chlamydial species (90 and 97% bootstrap support), thus strongly suggesting the existence of additional chlamydial species. Allele typing of amplicons from swine with intestinal, urogenital, and respiratory infections demonstrated several unique omp1 allelic variants of Chlamydia trachomatis. These novel alleles had deduced peptide sequences which were 11.6 to 19% divergent from porcine C. trachomatis S45. Mutations were clustered in the C-terminal region of variable segment IV of the omp1 locus encoding subspecies and serovar determinants of the chlamydial major outer membrane protein, thus implying that there are numerous serovars of porcine C. trachomatis. These results demonstrate the need for routine application of sensitive genus-specific detection of chlamydiae in animal specimens and suggest a more prominent role than anticipated for chlamydiae in animal diseases.     

Chlamydial infections of the heart

Chlamydiae are common human pathogens, causing a broad spectrum of infectious diseases. Chlamydial infections involving the heart have been described in numerous previous reports. These organisms are documented to cause endocarditis, myocarditis and pericarditis. Furthermore,Chlamydia pneumoniae, the recently discovered respiratory pathogen, has also been implicated in coronary artery disease. For the first time the literature on involvement of the heart in chlamydial infections is reviewed. Information on the discovery ofChlamydia species is also included and the problem of the species determination ofChlamydia in interpretation of the older literature is mentioned.     

Detection of Simkania negevensis by culture, PCR, and serology in respiratory tract infection in Cornwall, UK

Respiratory tract infections are often treated empirically without investigation to detect the aetiological agent, which may be a virus or a bacterium, including atypical pathogens such as Chlamydophila pneumoniae or Mycoplasma pneumoniae. Recently, several types Chlamydia-like intracellular bacteria have been detected in environmental samples and clinical specimens. Little is known of their geographical distribution and potential pathogenicity. We describe the detection, by PCR and isolation in cell culture, of Simkania negevensis in nasopharyngeal aspirates of paediatric patients with bronchiolitis in Cornwall, UK. We also present serological evidence of exposure to the organism in 62% of adult patients and 46% of a sample of pregnant women. Empirical treatment of serious respiratory tract infection should consider the possible contribution of these organisms.     

Analysis of the humoral immune response to Chlamydia pneumoniae by immunoblotting and immunoprecipitation.

Chlamydia pneumoniae is a widely spread agent of respiratory tract infections in humans. A reliable serodiagnosis of the disease is hampered by the poor knowledge about immunodominant antigens in C. pneumoniae infections. We applied a novel strategy to identify immunogenic proteins of C. pneumoniae TW183 combining metabolic radiolabeling of de novo-synthesized chlamydial antigens with immunoprecipitation. By this technique C. pneumoniae antigens of approximately 160, 97 to 99, 60 to 62, 40, 27, and 15 kDa were detected in the vast majority of sera from patients with a current C. pneumoniae infection. By immunoblotting purified elementary bodies of C. pneumoniae TW183 with the same sera, only the 60- to 62-kDa antigen could be detected consistently. Sequential immunoprecipitation performed at different stages of the chlamydial developmental cycle revealed that the 60- to 62-kDa antigen is strongly upregulated after 24 to 48 h of host cell infection and is presented as a major immunogen in both C. pneumoniae-infected patients and mice. We conclude that, due to its high sensitivity and concurrent preservation of conformational epitopes, metabolic radiolabeling of chlamydial antigens combined with immunoprecipitation may be a useful method to reveal important immunogens in respiratory C. pneumoniae infection which might have been missed by immunoblot analysis.     

Effects of a probiotic strain of Enterococcus faecium on the rate of natural chlamydia infection in swine

Chlamydiae are obligately intracellular pathogens which cause infections associated with a broad range of diseases in both livestock and humans. In addition, a large proportion of animals may become persistently infected asymptomatic carriers and serve as reservoirs for other animals which also shed these potential zoonotic pathogens. Reducing the chlamydial load of animals is therefore of major importance, and since large-scale antibiotic treatment is neither desired nor feasible, alternative means of prevention are needed. Here we performed a study comparing the efficacy of a probiotic strain of Enterococcus faecium on the reduction of both the rate of natural infection and the shedding of chlamydiae in swine. The presence of Chlamydiaceae was detected by species-specific PCR of fecal samples of sows taken at three times prior to the birth of piglets. Piglets delivered from chlamydia-positive sows in either the control or the probiotic group were also examined for the frequency of chlamydiae at various ages. Eighty-five percent of the piglets from the control group were found to be chlamydia positive, whereas chlamydiae were found in only 60% of piglets from the probiotic group, results confirmed by fluorescence in situ hybridization and immunohistology, which showed higher rates of infection in the control group. In addition to the reduced frequency of chlamydia-positive piglets in the probiotic group, the time of appearance of positive samples was delayed. To our knowledge, these data show for the first time that a probiotic strain of E. faecium can reduce the rate of carryover infections of piglets by obligate intracellular pathogens.     

Enhanced identification of viral and atypical bacterial pathogens in lower respiratory tract samples with nucleic acid amplification tests

The advantages of nucleic acid amplification tests (NAT) over conventional methods for the detection of pathogens in lower respiratory tract samples have not been established. NAT for respiratory pathogens were performed on 439 endotracheal tube (ETT) and bronchoalveolar lavage (BAL) samples. A potential pathogen was detected in 87 samples. Of 22 samples that tested positive by conventional methods, 15 tested positive for the same pathogen by NAT, 1 tested positive for a different pathogen, 2 had co-infections identified only by NAT, and 4 tested negative by NAT. An additional 73 pathogens were detected by NAT in 65 samples including 30 pathogens that were missed by conventional methods (19 adenovirus, 6 respiratory syncytial virus, 3 parainfluenza virus 1-4, 2 influenza A), 41 pathogens not routinely identified by conventional methods in most laboratories (23 rhinovirus, 8 human coronavirus OC43, 5 human metapneumovirus (hMPV), 2 human coronavirus 229E, 2 human coronavirus NL63, 1 Chlamydophila pneumoniae) and 2 pathogens from samples where no respiratory virus testing was requested (1 influenza A, 1 parainfluenza virus). Four of 52 patients who had multiple BAL samples submitted on the same day had negative and positive results by NAT on different samples. NAT improves detection of potential pathogens from ETT and BAL samples.     

Hidden in Plain Sight: Chlamydial Gastrointestinal Infection and Its Relevance to Persistence in Human Genital Infection

Although the concept of persistence in chlamydial infections has been recognized for about 80 years, there is still very little known about the mechanism by which this occurs. In this review, we revisit an old paradigm, long known to chlamydiologists and veterinarians, that in virtually all hosts of chlamydiae, including mammals and birds, chlamydiae reside in the gastrointestinal tract for long periods of time in the absence of clinical disease. Thus, if gastrointestinal infection occurs in most hosts, then it is very likely that gastrointestinal infection occurs in humans as well. We demonstrate that gastrointestinal infection does indeed occur in humans and propose that this anatomical site is the source of persistent infection in humans. The data in ruminants and animal models demonstrate that the immune system is unable to clear chlamydiae from the gut, so they can remain indefinitely, with continual shedding in feces. Clearly, many women become reinfected from an untreated partner; however, we propose that women, cured of genital infection, remain at risk for autoinoculation from the lower gastrointestinal tract. Moreover, there are substantial data demonstrating treatment failure of chlamydial infections, particularly with azithromycin. New data in the mouse model have shown that azithromycin is far less effective against chlamydial gastrointestinal infection than against genital infections. Therefore, it is possible that women cured of genital infection by antibiotics remain infected in the gastrointestinal tract and can become reinfected by autoinoculation from that site.     

Single DNA sequence common to all chlamydial species employed for PCR detection of these organisms.

Chlamydial infection is responsible for a wide spectrum of diseases of the eye, genitourinary tract, and lung. This group of organisms is also implicated in the pathogenesis of coronary artery disease as well as arthritis. Since cross-species infection is widely reported (though probably underestimated), it is an advantage to have a rapid and reliable method to detect all forms of chlamydiae in patient samples. We have identified a 160/163-bp DNA fragment in Chlamydia which is highly conserved in all chlamydial species. A polymerase chain reaction method based on this sequence has been developed to detect, in clinical samples, chlamydiae which have been shown to be positive by fluorescent-staining immunoassay; this method can be utilized in combination with restriction endonuclease cleavage to identify individual chlamydial species. Thus we have developed a sensitive and rapid detection method and have used it on samples from patients with respiratory and genital infections.