Serological findings of Chlamydial pneumoniae in the Czech Republic – control group of patients examined in the study: Chlamydia pneumoniae in the aetiology of keratoconjunctivitis sicca

Summary

One of the aims of IGA NS-10016 Project “Chlamydia pneumoniae in the etiology of keratoconjunctivitis sicca“ was to evaluate the serologic results regarding chlamydia infections in patients affected by keratoconjunctivitis sicca (KCS), and to compare these findings with serology tests of the control group. Serologic examination comprised anti-LPS (family specific) antibodies detection, Chlamydia pneumoniae and Chlamydia trachomatis species specific antibodies and antibodies against chlamydia heat-shock protein cHSP60 IgG.

The control group was formed by FNKV Ophthalmology Clinic patients who were coming for pre-operative investigation. At the time of laboratory sample acquisition or clinical investigation no acute ocular or general disease was found in these persons. During the last six moths, they had neither antibiotic therapy, nor therapy of respiratory tract chronic inflammation, asthma, joint or cardiovascular disease in personal history. The control group consisted of 103 subjects.

We detected anamnestical antibodies against CP in 69% of subjects in the control group, in 31% of them IgA and/or IgM were also detected in our study. In 45% of them anti-cHSP60 IgG was positive even in subjects without symptoms of acute infection or inflammatory laboratory markers. It is obvious, that this finding can be misleading during serologic investigation and its interpretation, and always it is necessary to evaluate the results in relation to the clinical condition.

Key words:
Chlamydia pneumoniae, antibody, serologic investigation, chlamydia heat-shock protein, conjunctivitis, keratoconjunctivitis sicca.

INTRODUCTION

Chlamydia ranks among the most widespread microorganisms in the human and also in the animal world. These pathogens are categorized among gram-negative obligatory intracellular bacteria. They are called energy parasites of eucaryotic (nuclear) cells, for they use host cell ATP for metabolic processes, not being equipped with an own ATP production system. Their presence is cosmopolitan (1).

Present days used taxonomy describes 1 family Chlamydiaceae, 1 genus Chlamydia a 4 species: Ch.trachomatis, Ch.pneumoniae, Ch.psittaci, Ch.pecorum. Everett, Bush and Andersen published in 1999 a new proposal of taxonomy classification based on phylogenetic analyses of rRNA genes 16S and 23S (2). According to this proposal we should distinguish 2 genus Chlamydia and Chlamydophila and nine species. This new taxonomy appeared soon in publications and it was frequently used, however the proposal was not generally accepted and the debate about its necessity and correctness have been under way in professional spheres so far (3).

The most frequent from all recognized chlamydia species in human is Chlamydia pneumoniae and Chlamydia trachomatis (4,5). The interpersonal transfer of these two species is very different and there are also differences in the clinical symptomatology, diagnostics and therapy.

Ch. pneumoniae is a respiratory agent and the clinical signs of infection are connected predominantly with upper and lower respiratory airways, however, one of the first isolations of this agent came from the ocular conjunctiva smear culture. This species was isolated in Taiwan in 1965 from child ocular conjunctiva during trachoma vaccination study (6). Chlamydia pneumoniae was definitively identified as a separate species in the end of the 20^th century last decade (7) and since that time the knowledge of this chlamydia species have been still growing. Many studies investigated the relation of Ch. pneumoniae to the lower respiratory tract infections (bronchitis, pneumonia)(8), but their results sometimes significantly varied. The role of Ch. pneumoniae in diseases of the upper respiratory ways is not exactly defined yet. Asymptomatic presence without serologic proof of infection is possible (9,10). The involvement of Ch. pneumoniae in development or in progression of some non-respiratory diseases, as cardiovascular or central nervous system illnesses are, is also unclear yet (11, 12).

Ch.trachomatis (serotypes D-K) infects in adults most frequently urogenital tract, in neonates it infects ocular conjunctiva and respiratory tract (because of the infected labour passages nearly two thirds of neonates can be infected). Most of the urogenital infections pass asymptomatically or with minimal clinical signs. When symptoms are present, they may even resemble gonorrhoea. Mucous membrane of urethra, uterus cervix or rectum are primarily afflicted, during oral sex pharynx can be infected. Ocular conjunctiva can be also infected (in adults usually self-infection from urogenital tract) (1, 13, 14).

The results of Czech Republic Ministry of Health IGA investigational project, NI–66811 from 1999 – 2001 called ”The importance of Chlamydia pneumoniae in pathogenesis of chronic human diseases” showed a possible link between chlamydia caused chronic follicular conjunctivitis and the development of keratoconjunctivitis sicca (KCS). Another IGA project NS-10016 called ”Chlamydia pneumoniae in the etiology of keratoconjunctivitis sicca” was initiated in 2008 and it directly concentrated on this issue and on its relation with possible adult population infestation with this infection.

MATERIAL AND METHODS

One of the aims of this project was to evaluate serologic findings related to chlamydia infections in patients with chronic follicular conjunctivitis and coincident symptoms of KCS, and to compare these findings with the serologic investigations of control group patients. Another aim was to evaluate also the importance of various diagnostic methods (serology, chlamydia antigen detection and chlamydia DNA detection) for the diagnostic proof of this infection in KCS patients.

Serological investigation comprised anti LPS IgG, IgA, IgM antibodies (genus specific) (medac, Germany), species specific anti-Chlamydia pneumoniae IgG, IgA, IgM antibodies (Savyon Diagnostics, Israel), anti-Chlamydia trachomatis IgG, IgA antibodies (medac, Germany) and antibodies against chlamydia heat-shock protein cHSP60 IgG (medac, Germany).

Into the control group were assigned the patients from FNKV Ophthalmology Clinic who were coming for pre-operational investigation before future intra-ocular procedures during the period from May 2009 to May 2010. Not any laboratory or clinical acute or general disease was diagnosed in these subjects during the time of biological samples acquisition. They had not used antibiotics during the last six months, they denied the therapy of chronic respiratory tract inflammation, asthma, joint or cardiovascular disease. One hundred and three subjects were assigned to the control group (49 men and 54 women in the age from 21 to 90 years, median 52 years).

During this period 37 patients with follicular conjunctivitis and contemporary KCS symptoms were also investigated.

RESULTS

Control group

During the serologic results evaluation the control group containing 103 subjects was divided into three parts according to the anti-cHSP60 IgG positivity. These antibodies were chosen to be the main marker in order to compare the process activity in relation to species antibodies (anti-Ch. pneumoniae) and genus antibodies (anti-chlamydia). Microbial heat-shock proteins (HSP) are strong immunogens and during any bacterial infection their synthesis significantly increases, infectious agent struggles to protect against host immunity mechanisms. Human HSP are discharged at the same time in order to protect human cells. Cross-reactions may occur because of significant resemblance of the proteins and autoimmune reactions may be the consequence.

Anti-cHSP60 IgG positivity and coincident chlamydia genus and species antibody negativity in all immunoglobulin groups was found in three serum samples. It cannot be excluded, that the anti-cHSP60 IgG positivity in these cases is a result of cellular stress caused by another reason but chlamydia infection. Therefore, these three plasma samples were excluded from the evaluation. Serological results of the control group are summarized in Table 1.

Negative anti-cHSP60 IgG were found in 55 (55%) serum samples

Antibodies against Ch. pneumoniae were negative in 20 serum samples in this group – 18 serum samples showed no species antibodies positivity (anti-Ch. pneumoniae, anti-Ch. trachomatis), but in 13 of them positivity was found in genus antibodies (anti-chlamydia IgA and/or IgM) and 2 serum samples showed positivity for species antibodies only against Ch. trachomatis (IgG).

Another 25 serum samples contained only anamnestical antibody levels against CP (i.e. IgG), in six of them was also coincident positivity for genus anti-chlamydia antibodies (anti-chlamydia IgA and/or IgM), in 3 serum samples positivity for anti-Ch. trachomatis antibodies was found.

The last 10 serum samples showed positivity for antibodies against Ch. pneumoniae not only in IgG class, but also in IgA and/or IgM classes (in six cases together with positivity for anti-chlamydia IgA and/or IgM). This group of patients had no clinical signs of acute infection and antibody positivity was considered more as an anamnestic consequence of recently passed infection (also in relation to coincident negativity for anti-cHSP60 IgG).

Positive anti-cHSP60 IgG were found in 45 (45%) serum samples

• a) Marginal or slightly positive (positivity index up to 1.8) anti-cHSP60 IgG were found in 22 serum samples. Three samples in this group showed no positivity for species antibodies (anti-Ch. pneumoniae), in two of them only genus antibody positivity was found (anti-chlamydia IgG) and in 1 serum sample only positivite anti-Ch. trachomatis species antibodies (IgG) were found.
  Another 11 plasma samples showed only anamnestical antibody levels (i.e. IgG) against CP, in 3 of them also coincident positivity for genus anti- chlamydia IgM and in one sample positivity for anti-Ch. trachomatis antibodies was found.
  Eight serum samples altogether showed positivity for IgA or IgM genus (anti-chlamydia) or species (anti-Ch.pneumoniae or also anti-Ch.trachomatis) antibodies. These findings we evaluated as a suspect clinically insignificant active infection, because the patients evinced no inflammatory process symptoms related to studied agent. This conclusion was also supported by low cHSP60 positivity index. Coincident positivity for species anti-Ch. pneumoniae and genus anti-chlamydia IgG and IgM or IgA antibodies were found in two patients of this group. This finding we evaluated as a suspect active infection and therefore we organized follow-up serologic investigation after six moths in these subjects.
• b) Moderately or significantly positive (positivity index over 1.8) anti-cHSP60 IgG were detected in 23 serum samples. Eight samples in this group were negative for anti-Ch. pneumoniae antibodies – 3 samples showed no positivity for species antibodies (anti-Ch.pneumoniae, anti-Ch.trachomatis) and only genus antibody positivity was found (anti-chlamydia IgG and/or IgA, IgM). Positivity for species antibodies only against Ch.trachomatis (IgG or also IgA) was found in 5 serum samples.

Anamnestical antibody (i.e. IgG) levels against CP (in 1 sample also against CT) and anti-chlamydia IgG were found in 2 serum samples.

Altogether 13 serum samples showed positivity for IgA and/or IgM in species (anti-Ch.pneumoniae) and/or genus (anti-chlamydia) antibodies. We evaluated this finding as a suspect active infection also because of significant or moderate anti-cHSP60 IgG positivity. However, white blood count and FW tests done during regular pre-operative investigation were within physiological limits. After six months we invited these 13 subjects together with 2 subjects with the most significant serologic findings from the previous group to a follow-up investigation. After this period the antibody levels were more or less without change in all these patients, except for 2 absent subjects.

Patients with KSC

There were altogether 37 adult patients with follicular conjunctivitis and KCS symptoms investigated up to now. Laboratory results (serology, antigen detection and DNA) supported ATB therapy initiation in eleven of them (6 women and 5 men, age 28-81 years, median 50 years). Anti-Ch. pneumoniae IgA and/or IgM positivity was found in 10 patients and coincidently anti-chlamydia IgA and/or IgM positivity was found in 8 patients. Positive smear cultures were recorded in 6 patients. Serologic findings were insignificant in 2 treated patients, but Ch. pneumoniae DNA positivity in peripheral leukocytes indicated Ch. pneumoniae infection. History of symptoms in these 2 patients had lasted six months (in comparison with other patients they were relatively short). Detailed analysis of clinical and laboratory findings of these patients is subject of another article (15).

DISCUSSION AND CONCLUSION

Evaluation of serologic findings in chlamydia infections related to clinical diseases must be done with some caution. Serologic findings in control group subjects, i.e. in patients free from laboratory and clinically confirmed acute disease at the moment of samples acquisition (who anamnesticaly had not reported therapy of chronic inflammatory disease) indicate importance of this issue. Two thirds (69%) of subjects of our cohort had anamnestic (i.e.IgG) antibodies against Ch. pneumoniae. This result corresponds with serologic survey reports published in 1998 (3). However, 31 % (nearly one half of the patients) showed also serologic signs of active infection with Ch. pneumoniae (i.e. positivity for IgA and/or IgM in species and/or genus antibodies).

According to the division of findings with respect to anti-cHSP60 IgG positivity, only 55% of subjects were negative, 22% had marginal or weak positivity (positivity index up to 1.8) and 25% moderate or significant positivity (positivity index over 1.8). Consequently, in the group with significant anti-cHSP60 IgG positivity there was a majority of antibodies against Ch. trachomatis (in 15 serum samples) found. Again, nearly one half out of 45 serum samples (21 samples) with antibody positivity against chlamydia heat-shock protein showed serologic signs of active infection by Ch. pneumoniae (i.e. IgA and/or IgM positivity for species and/or genus antibodies).

We assume that the detection of IgA or IgM positivity (species or genus) in one third and the detection of anti-chlamydia HSP antibody positivity in nearly one half of relatively healthy adult population significantly preclude to interpret serologic findings. Present days Ch. pneumoniae infection diagnostics is based mainly on the proof of antibodies. The production of antibodies in chlamydia infections is delayed and starts approximately on the 10th day from the onset of the disease (species antibodies even more lately – in 2 – 3 weeks, and probably more lately). As a rule, IgM antibodies are produced during the prime infections, while during recurrent infections or reactivations usually IgG and IgA antibodies increase. Isolated IgG finding is a sign of passed disease or of a contact with infection. However, in order to interpret optimally the serologic finding it is necessary to follow the dynamic of antibody production. Unequivocal evidence of acute infection is a record of seroconversion or quadruple antibody increase in paired serum samples (the1st sample at the beginning of the infection, the 2nd sample after 2-3 weeks) (16). Diagnostics of “chronic” Ch. pneumoniae infections is present days probably the most difficult. Long lasting IgA and IgM positivity is usually considered to be a proof of chronic infection in literature and medical practice, but we have not any certified serologic marker of chronic or persistent chlamydia infection yet (17). When evaluating the serologic findings, it is necessary to consider also the possibility of polyclonal antibody activation (e.g. during the EBV or Mycoplasma pneumoniae infection).

Heat-shock proteins (HSP) are highly conserved cell stress proteins, excreted in cells during cell stress, caused for example by increased temperature, inflammation, infection, toxins, radiation or lack of nourishment. These proteins are found in all prokaryotic and eukaryotic cells and they are remarkably highly homologous. HSP homology of man and mouse is 99.9% and homology of man and bacteria approximately 60% (18). Cross reactions may occur due to high homology of proteins and auto-immune reaction may be the consequence. cHSP60 IgG Elisa (medac) test uses recombinant heat-shock protein 60 (cHSP60) from CT as an antigen. Anti-HSP60 CT antibodies are mainly detected by this test, however, because of very high homology of chlamydia species proteins (95%), the detection of anti-CP antibodies may be possible (19). It is impossible to exclude that positivity of these antibodies is also a sign of cell stress, caused by other reasons than chlamydia infection. This alternative is probable in three anti-cHSP60 IgG positive serum samples, which were excluded from the cohort because of chlamydia antibodies negativity.

The detection of antibodies is an indirect diagnostic method and one must have it in mind during the interpretation. Adult population infestation with Ch. pneumoniae is high and therefore anamnestic antibodies against this agent can be detected very often. In our control group we have also detected “acute infection markers” (positivity for IgA and/or IgM and anti-cHSP60 IgG positivity) in one third or even in one half of the subjects. Follow-up serologic investigation six months later was practically without change in 13 patients with the most significant initial findings. There was no reason to initiate antibiotic therapy because these patients were clinically asymptomatic.

Serologic findings should be assessed always with respect to the clinical condition and together with other laboratory results. It is necessary to examine paired sample in order to prove acute infection. When possible, serologic investigation should be supported by direct diagnostics (DNA screening). An isolated IgA, IgM or anti-cHSP60 IgG positivity is not a reason to initiate antibiotic therapy. 

Supported by grant IGA – NS 10064

MUDr. Dana Hrubá
VIDIA-DIAGNOSTIKA s.r.o.
Gen.Janouška 902
198 00 Praha 9
Tel. 281 012 033
d.hruba@seznam.cz