Pathogenic Potential of Novel Chlamydiae and Diagnostic Approaches to Infections Due to These Obligate Intracellular Bacteria

INTRODUCTION

Chlamydiae are obligate intracellular parasites of vertebrates, of some arthropod species, and of several free-living amoebae (reviewed in references 14, 32, and 68). They exhibit a peculiar two-stage developmental cycle that includes an extracellular infectious elementary body and an intracellular vegetative reticulate body. A further infective stage, the crescent body, was recently described for the Parachlamydiaceae (41), a new family within the order Chlamydiales.

The family Chlamydiaceae comprises two genera, Chlamydia and Chlamydophila (29). While Chlamydia species seem to infect only mammals such as humans, rodents, and swine, host specificity for Chlamydophila species is less strict, including for amphibians, reptiles, birds, and mammals (14). Both Chlamydia and Chlamydophila species comprise important human pathogens (88). Chlamydia trachomatis is a common cause of urogenital infection in humans and the agent of trachoma, one of the leading infectious causes of blindness worldwide. Chlamydophila pneumoniae is another important human pathogen, causing mainly respiratory infections. C. pneumoniae might also be involved in the pathogenesis of atherosclerotic cardiovascular diseases (8) and neurodegenerative syndromes (91). The other species are mainly veterinary pathogens, though some of them may cause rare but severe anthropozoonotic infections, such as psittacosis due to Chlamydophila psittaci or zoonotic abortion due to Chlamydophila abortus (14, 68, 83).

Historically, the term “chlamydia-like organisms” has been applied to refer to any intracellular microorganism that, like Chlamydiaceae, exhibited a two-stage developmental cycle. Since phylogenetic molecular analyses performed on some of these chlamydia-like organisms have showed this group to be polyphyletic, the term “chlamydia-like organisms” is only descriptive and retains no taxonomic value. Other authors (55, 56) proposed the term “environmental chlamydiae” to refer to chlamydial organisms that fall outside the family Chlamydiaceae, with the latter being designated “pathogenic chlamydiae.” However, such a distinction seems inadequate, since a growing body of evidence supports the pathogenic role of some of these chlamydiae (32, 40). By analogy to the term “amoeba-resistant microorganisms” (43), the term “amoeba-resisting chlamydiae” could also be applied to the species that may infect and survive within amoebae, i.e., Parachlamydiaceae, Simkania negevensis, and Waddlia chondrophila (see below). Indeed, most of these new species exhibited symbiotic or lytic interaction with amoebae. Nevertheless, since not all new species of chlamydiae have been tested for their ability to resist destruction by free-living amoebae, the more general term “novel chlamydiae” should be preferred to designate all the chlamydiae not belonging to the Chlamydiaceae.

These chlamydiae, which have recently been discovered and assigned to new families, are currently being investigated for their role as emerging pathogens. Simkania negevensis (family Simkaniaceae) (32) and Parachlamydia acanthamoebae (family Parachlamydiaceae) (40) could represent important respiratory pathogens in humans, while Waddlia chondrophila (family Waddliaceae) seems to be a new agent of abortion in ruminants (21, 53).

Molecular studies performed on human and animal samples have demonstrated the wide biodiversity and broad host range of chlamydiae. Thus, additional Parachlamydiaceae species, such as Neochlamydia sp., and a large variety of new 16S rRNA gene phylotypes have been detected in humans (15, 17), cats (93), Australian marsupials (7, 20), reptiles (6, 90), and fishes (24), as well as in various environmental samples (16, 18).

Moreover, new members of the Chlamydiales infecting invertebrates have recently been characterized. These include Fritschea bemisiae and Fritschea eriococci (family Simkaniaceae), which infect homopteran insects (28, 92), and Rhabdochlamydia porcellionis (64) and Rhabdochlamydia crassificans (19), which infect the woodlouse Porcellio scaber (Crustacea: Isopoda) and the cockroach Blatta orientalis (Insecta: Blattodea), respectively. The presence of chlamydiae in arthropods is interesting, since arthropods were not previously considered to play a role in the epidemiology of chlamydial infections, with the only exception being flies as a vector of the agent of trachoma (25, 26). To date, if we exclude cases of possible Rhabdochlamydia-related uveitis in humans (see below), there is no hint that these invertebrate-associated chlamydiae may be pathogenic to humans.

Evidence for a much larger biodiversity within Chlamydiales was afforded by several molecular studies performed on humans, animals, and environmental samples (Table 1). Here we report the present knowledge on the pathogenicity of all Chlamydiales except Chlamydiaceae, and we review the diagnostic methods currently available to diagnose human infections potentially due to these emerging pathogens.

PATHOGENIC POTENTIAL OF NOVEL CHLAMYDIAE

INTERACTIONS BETWEEN CHLAMYDIAE AND AMOEBAE

Free-living amoebae such as Acanthamoeba and Hartmannella play a key role as reservoirs (Fig. 1) and/or vectors for a variety of amoeba-resisting microorganisms (reviewed in reference 43), including the chlamydiae. Acanthamoeba is a suitable host for most Parachlamydiaceae (1, 76) and S. negevensis (59, 61), whereas Hartmannella, Balamuthia, Dictyostelium, and Naegleria (Table 2) are also able to sustain infection by some Parachlamydiaceae (57, 89), S. negevensis (75), and Waddlia chondrophila (78). Various interactions between chlamydiae and amoebae may take place, depending on the chlamydial and the amoebal species and strain and on environmental conditions. Thus, P. acanthamoebae is endosymbiotic at 25 to 30°C and lytic at 32 to 37°C for Acanthamoeba spp. (45). Since the temperature of the human nasal mucosa is generally lower (about 30°C) than that of the lower respiratory tract (generally between 35 and 37°C), P. acanthamoebae could reside symbiotically within amoebae present on the nasal mucosa and induce the lysis of the amoebae that reach the lower respiratory tract, inducing their own release and possibly starting an infectious process. Conversely, the virulence of the amoebae could be increased when harboring chlamydiae. Fritsche et al. (35) reported that the cytopathic effect on human embryonic tonsillar fibroblasts was enhanced by 66 to 70% when Acanthamoeba contained the Parachlamydia-related symbiont UWE25, which was recently designated as a new genus and species, Protochlamydia amoebophila (11).

Additionally, the growth rate of the amoebae may be differentially affected by their parachlamydial symbionts. Thus, Hartmannella vermiformis infected by Neochlamydia hartmannellae grew more rapidly, while an Acanthamoeba sp. infected by Protochlamydia amoebophila (strain UWE25) grew more slowly, with respect to the aposymbiotic amoebae (12). Interestingly, this amoeba- parachlamydia couple (Acanthamoeba sp. strain UWC1/Protochlamydia amoebophila UWE25) was the same that showed the stronger cytopathic effect enhancement in the study by Fritsche et al. (35). Similarly, amoebal development stages, such as encystment (Acanthamoeba), enflagellation (Naegleria), or body fruiting (Dictyostelium), may be differentially affected depending on the infecting chlamydial strain (57, 75, 76, 78, 89). Notably, only Hartmannella strains were susceptible to infection at the first challenge with Waddlia chondrophila, whereas Waddlia grown in hartmannellae acquired the ability to infect several other free-living amoebae (78).

Whether chlamydiae and amoebae in some way influence each other's virulence deserves additional studies. However, it is likely that the ability of P. acanthamoebae to resist the microbicidal effector mechanisms of macrophages (44) has been acquired during their long history of coevolution with amoebae (43).

PROCESSING OF MICROBIOLOGICAL SPECIMENS

Little information is available on the optimal procedures for collecting and processing the novel chlamydiae. Handling of clinical samples for detection of chlamydiae should be performed in a biosafety level 2 security laboratory, given their pathogenic potential and the fact that various chlamydiae share the same ecological niche (the amoebae) with other amoeba-resisting bacteria of established pathogenicity, such as Legionella pneumophila.

The obligate intracellular nature of chlamydiae implies that an adequate specimen must contain infected host cells and/or a sufficient amount of extracellular elementary bodies. Since extracellular elementary bodies are unlikely to be present during persistent infection, the presence in the specimen of infected host cells is mandatory for both culture and nucleic acid amplification. For mucosal specimens, swabbing seems appropriate for the recovery of a sufficient amount of infected cells. Alginate swabs should be avoided since they may produce artifacts when Giemsa staining is performed. Cotton swabs are preferred. Sampling error may be reduced by scraping of mucosal surfaces, which increases the cellular yield compared to simple swabbing. However, this somewhat invasive procedure may induce bleeding and should be used only for selected indications.

Specific transport media such as the 2-sucrose phosphate or the sucrose-glutamate phosphate medium, originally developed for rickettsiae, may be used. Commonly available viral transport media generally contain antimicrobial agents and should therefore be avoided, since macrolides and tetracyclines inhibit the growth of both Parachlamydia (72) and Simkania (32) and tetracyclines are active against Waddlia species (10, 85). Bacterial overgrowth may be prevented by adding gentamicin (10 μg/ml) and/or vancomycin (100 μg/ml) to the transport medium. Although amphotericin B (1 to 5 μg/ml) is frequently used to prevent fungal overgrowth in cell culture, this compound should be avoided for amoebal coculture given the susceptibility of most free-living amoebae to amphotericin B.

Parachlamydiaceae, Simkania negevensis, and Waddlia spp. have been isolated and propagated in cell cultures or amoebae by using transport media and/or culture media containing penicillins (1, 10, 21, 53, 60). However, the use of penicillins should ideally be avoided given the susceptibility of Chlamydiaceae and the potential susceptibility of the other chlamydiae to penicillins and, by analogy with what is known for Chlamydiaceae, given the risk of induction of persistent nonmultiplying aberrant forms.

Microbiological specimens should be stored at 4 to 8°C and processed as soon as possible. If the time between collection and processing is >24 h, specimens should be frozen at −70°C. Freezing may result in a loss of chlamydial viability. This loss has been estimated at up to 20% for Chlamydiaceae and may be reduced by the addition of fetal bovine serum (2 to 10%) (2). No data are available for novel chlamydiae.

NUCLEIC ACID AMPLIFICATION

Nucleic acid amplification by PCR and related techniques is widely used in clinical microbiology laboratories in order to detect and/or identify any microorganisms that may be present in clinical samples. These molecular techniques are especially useful in epidemiological studies and as diagnostic tools, e.g., when used for the identification of microorganisms that are difficult to grow in culture. Additionally, PCR approaches that target phylogenetically informative genes such as the 16S rRNA gene contribute to the identification of new strains and species.

PCR techniques that target mainly the ribosomal operon have recently been developed to study the novel chlamydiae. However, sequences of other genes of additional diagnostic and phylogenetic value, such as the ATP/ADP translocase-encoding gene (42), are becoming available. At present, whole genome sequences are available for various Chlamydia and Chlamydophila strains, for Protochlamydia amoebophila strain UWE25 (55), and for S. negevensis (see the website http://www.tigr.org/tdb/mdb/mdbinprogress.html), whereas a 16.6-kbp genome portion, including the ribosomal operon and nine other genes, has been determined for Fritschea bemisiae (92). PCR tests that have been developed mainly for diagnostic purposes are summarized in the following section.

CELL CULTURE

The isolation and propagation of intracellular bacteria such as chlamydiae in a cell culture system are fundamental in demonstrating the presence of a viable organism in the clinical sample and characterizing the strain beyond its gene sequence. Considering the huge diversity within chlamydiae that is suggested by molecular studies, efforts should be made to adapt cell culture protocols to allow the recovery of these new species. Chlamydiaceae have been cultivated into embryonated eggs, but this culture system was relatively rapidly replaced by cell cultures, which are easier and more sensitive (2). Embryonated eggs are used today mainly for massive production of antigens or for propagation of fastidious strains (2). Egg culture has not yet been tested for any novel chlamydia.

The possibility of Chlamydiaceae entering into a persistent/cryptic state is a key pathogenic feature explaining the wide range of chronic diseases that most Chlamydiaceae seem to induce in their vertebrate hosts (3, 54). However, persistence complicates the laboratory diagnosis of chlamydial infection, at least when using culture-based approaches, potentially leading to false-negative results. It is yet unknown whether Parachlamydiaceae, Simkaniaceae, and Waddliaceae may also show a persistent state and which cell lines or amoebal strains may especially help in resuscitating such viable but nonculturable bacteria.

SEROLOGY

In addition to molecular approaches and culture, serology is increasingly used to assess the pathogenic roles of the novel chlamydiae, even though serology provides only an indirect hint of pathogenicity (5, 31, 48, 66, 71).

An enzyme-linked immunosorbent assay (ELISA) has been developed for the diagnosis of Simkania negevensis infection (60, 67). This ELISA, which may detect immunoglobulin A (IgA) and IgG antibodies (but not IgM), was initially designed to work with sera diluted 1:30 to 1:100 for IgA and IgG, respectively (67). With this ELISA, the presence of IgA was empirically considered as an indicator of recent infection, whereas that of IgG suggested recent or past infection. Using this ELISA at the mentioned serum dilutions, as many as 39 to 68% of healthy adults living in different parts of the world (Israel, Canada, the United States, and Denmark) were found to be seropositive (32). Of note, using immunofluorescence and a cutoff for positivity of as low as 1:8, Yamaguchi et al. did not find such a high prevalence, with a 7.5% seroprevalence in adults and a maximum seropositivity of 15% in elderly subjects (94). Thus, the clinical significance of antibody reactivity against Simkania should be interpreted with caution, especially when using ELISA. Yamaguchi et al. also showed limited cross-reactivity between Simkania and C. pneumoniae (94).

Immunofluorescence, which is considered the gold standard for Chlamydiaceae (23), has been used for Parachlamydiaceae in different studies, supporting the role of these organisms in community-acquired pneumonia (4, 5, 71) and in aspiration pneumonia (48). Whole heat-inactivated bacteria were used as the antigen. Cutoffs for seropositivity used in these studies ranged from an antibody titer of >1:50 (71) to ≥1:128 (4).

To use the same dilutions as those used for the diagnosis of Chlamydia-related infections (23), it seems preferable in the future to use serial dilutions starting at 1:8 and not at 1:25 for the diagnosis of infections due to the new chlamydiae. Since among 100 sera taken from healthy blood donors, none exhibited an antibody titer of ≥1:50, we suggest considering an anti-IgG titer of ≥1/64 as evidence of prior infection. Based on our own experience and the criteria used by Marrie et al. (71), we suggest considering the following serological results as indicative of a recent infection: (i) a single IgM titer of ≥1:32, (ii) seroconversion (IgG increase from 0 to ≥1:64 in paired specimens), and (iii) a ≥4-fold rise in the IgG titer between acute- and convalescent-phase sera. Since no definite cutoffs have been established for immunofluorescence for Simkania and Waddlia spp., we propose to also use these cutoffs for these chlamydiae.

Confirmation of positive immunofluorescence results by Western blotting has been reported for sera that tested positive for Parachlamydia (48) and for Simkania (94). For Parachlamydia, proteins of approximately 60 kDa and 48 kDa were shown to exhibit the strongest antigenicity, whereas for Simkania, a strong and specific signal was observed at 64 kDa. More important, this 64-kDa signal was shown to be specific for Simkania, still being present after serum adsorption with C. pneumoniae. The degrees of cross-reactivity between the different novel chlamydia species and between every member of the Chlamydiales remain to be determined. Indeed, false-positive results may be caused mainly by serological cross-reactivities. Therefore, Western blotting and cross-adsorption, which may be of some help in confirming the specificity of any seropositive test result, should ideally be performed.

CONCLUSION

In conclusion, there is increasing evidence of the pathogenicity of Simkania negevensis (32) and Parachlamydia acanthamoebae (40), especially as agents of lower respiratory tract infection. The pathogenic potentials of additional novel chlamydiae remain to be defined (14). Consequently, there is an urgent need for efficient and standardized diagnostic approaches. Based on the published reports, a combined diagnostic strategy using both molecular approaches and serology as an initial diagnostic screening work-up seems appropriate. Positive results may then be confirmed by cell and amoebal coculture. Future diagnostic challenges include the identification of the best PCR protocols, confirmation of the serological cutoff levels that we have proposed here, and the determination of the optimal growth conditions for each novel chlamydia. We hope that the present review will help stimulate clinical diagnostic microbiology laboratory and research teams to work in this exciting emerging field.