IgM ELISA: A Better Choice for the Detection of Active Chlamydia trachomatis Infection among HIV PatientsCorrespondence Address :
Dr. Padma Krishnan
Asst Professor, Dept of Microbiology,
Dr. ALM Post Graduate Institute of Basic Medical Sciences,
University of Madras, Taramani campus, Chennai- 600113.
Phone: +91 9840742105; Fax: +91 44 24540709
E-mail address email@example.com
Context: Co-morbid STIs are documented to serve as risk factors that facilitate the acquisition and transmission of HIV. The cryptic plasmid has been linked to Chlamydial virulence and serves as a key target of diagnostic PCR assays. Reports on the higher incidence of Chlamydia trachomatis infection and the emergence of plasmid-less isolates in developing countries have raised concern.
Aim: The study was aimed to determine the prevalence of active C. trachomatis infection among high risk subjects in South India.
Materials and Methods: Serum and first-void urine samples were screened for C. trachomatis using IgM ELISA and COBAS AMPLICOR PCR respectively.
Results: The study participants include, 100 HIV seropositive patients, 51 Non-HIV-subjects attending STI clinic and 25 healthy adults. The incidence of C. trachomatis infection among HIV patients (n= 100) as measured by positive IgM ELISA result was 3%, while 0% positivity was observed among the cohort II and III. However, PCR analysis revealed that the cryptic plasmid was not detected in the three EIA positive patient samples.
Conclusions: We report here of low incidence of C. trachomatis infection among HIV patients in south India. Discrepancies were noted between the results of IgM ELISA and plasmid- PCR. Our results suggested the existence of plasmid-less/natural variant strains of C. trachomatis in South India. Thus, IgM ELISA still remains the method of choice in the laboratory diagnosis and obviates the use of plasmid based NAATs which could only under estimate the incidence of C. trachomatis infection among the HIV patients.
Chlamydia trachomatis, ELISA, PCR, Serology
Chlamydia trachomatis is the most common cause of treatable sexually transmitted infection (STI) worldwide. C. trachomatis infections are clinically silent and hence are of major public health concern (1). A previous South Indian study has reported a high prevalence of female genital chlamydial infection (2). Untreated comorbid STIs serve as risk factors that facilitate the acquisition of HIV (3). Also, early detection and treatment of STIs could reduce the burden of HIV (4). Condoms confer better protection against genital infections that are transmitted via secretions (5). Diagnosis of STIâ€™s in developing countries is essentially based on clinical criteria, symptomatology and physical examination rather than laboratory diagnosis. Hence, we intended to assess the actual prevalence of C. trachomatis infection among HIV patients.
Study Population and Specimens
Sexually active asymptomatic subjects were recruited for the study following ethical clearance by the Institutional Review Board on Human Ethics and oral informed consent of the subjects to participate after explaining him or her the study protocol. Cohort I included HIV seropositive patients with CD4 count < 500 cells/ cu. mm. Patients on highly active retero-viral therapy were excluded. Cohort II included a high risk population attending a Microbiology Section STD clinic that fulfilled the criteria of HIV sero-negativity and not on anti-microbial therapy for the past one month. Cohort III included sexually active, healthy volunteers of the reproductive age group without any symptoms of genital infections. Blood and first void urine samples were collected from the study participants. The samples were transported in ice to Microbiology laboratory. The serum was separated and stored at â€“70Âş C until use. On arrival at the laboratory, well mixed urine samples were centrifuged at 3,000 x g for 30 mins. The deposit was re-suspended in 1 ml of PBS and stored at â€“70Â° C until PCR assay.
IgM antibodies to C. trachomatis was detected in the serum samples by using NovaTec C.trachomatis IgM ELISA in accordance with the manufacturerâ€™s instructions. Samples were considered positive if the absorbance value was 10% greater than the cut off value and the results were expressed as NovaTec units (NTU).
Urine samples of patients who were positive (n=3) for CT IgM ELISA were subjected to plasmid PCR using COBAS AMPLICORTM CT/NG kit. Specimens were processed and tested according to the manufacturerâ€™s instructions. Specimens that yielded CT or NG signal above the test cut off were interpreted positive, regardless of the IC results. Specimens yielding CT or NG signal below the test cut off were interpreted as negative, only if IC signal was above the required cut off.
Cohort I comprised of 100 seropositive HIV patients hospitalised at Government Hospital for Thoracic Medicine, Chennai. Cohort II comprised 51 Non -HIV patients attending the Sexually Transmitted Infection (STI) clinic of the Institute of sexually transmitted infections, Government General Hospital, Chennai. Cohort III included 25 sexually active apparently healthy subjects without any symptoms of genital infections. Among 100 HIV patients (male-50, female-48, transgender-2) enrolled in cohort I, the mean age was 36.5 years. Cohort II included 41 males, 10 females with a mean age of 38.5 years and cohort III included 25 healthy adults (13 males and 12 females) and the mean age was 27 years. Out of 176 serum samples screened, 3 samples showed the presence of anti C. trachomatis IgM antibodies by NovaTec ELISA. The overall incidence of C. trachomatis infection among HIV patients (n = 100) included in the study as measured by positive CT IgM ELISA result was 3% (females -2, Males -1), while the positive rate was 0% among the cohort II and III. None of the urine samples of the HIV patients (n=3) with active C. trachomatis infection (positive by CT IgM ELISA) when subjected to COBAS AMPLICORTM CT / NG PCR assay, yielded a positive CT amplification signal. Review of the patientâ€™s hospital records revealed that, of the 3 HIV subjects with active C. trachomatis infection, 2 subjects (male-1, female-1) had CD4 count < 200 cells/cu. mm. Also, two of the three HIV subjects who were positive for C. trachomatis IgM ELISA, were also found to be VDRL reactive, but all the 3 samples were negative for Neisseria gonorrheae.
Progressive destruction of the immune system by HIV leads to depletion of CD4 cells making the patient highly vulnerable to a variety of opportunistic infections. HIV patients are at a high risk of acquiring other STIâ€™s (especially genital C. trachomatis infection and syphilis) owing to their promiscuous nature and common route of transmission of these etiological agents. In sexually active men and women, unrecognized and untreated C.trachomatis infection can cause excessive inflammation of the genital tract which consecutively appears to increase both the HIV shedding and viral load in genital secretions (6),(7),(8),(9) thereby increasing HIV infectiousness (10), (11). Also, CD4+ T cells are attracted to either the surface of the ulcer (11) or the endocervix (12) in both ulcerative and non-ulcerative STIs, which sequentially disrupts the innate mucosal immunity and potentially enhances an individualâ€™s susceptibility to HIV infection. Recently there has been a gradual increase in the global burden of C. trachomatis infection. This in part, may be ascribed to the implementation of newer screening protocols, reporting of new chlamydia cases, improved diagnostic tools with a high degree of sensitivity, changes in sexual behaviour. Previous reports have documented a high prevalence of C. trachomatis infection in the Indian population and an urgent need for the development of costeffective, sensitive screening methods (13)(14),(15),(16),(17). The recent Centers for Disease Control (CDC) report shows that among females attending STI clinics aged 15â€“24 years, Chlamydia prevalence ranged from 14.8% to 16.3% from 2004â€“2008 and in men the incidence ranged from 20.5% to 23.7%. The HP2010 target of CDC was to reduce the overall Chlamydia incidence to 3% (18).
Conventional laboratory methods are insensitive, labor intensive and expensive. Accurate laboratory diagnosis remains a challenge; culture has long been considered the gold standard. Nucleic acid amplification methods are currently being adopted as an accurate alternative. However, antibody detection by ELISA is a convenient diagnostic tool in developing countries. Based on our EIA results, the incidence of C. trachomatis infection is found to be relatively low (3%) among the HIV seropositive subjects. This is contrary to a previous report from our region, wherein the overall C. trachomatis prevalence rate was 30.8% and C. trachomatis positivity rate among the HIV positive and HIV negative cases were 54.2%, 26.1% respectively (19). It could be speculated that there is currently a significant reduction in the CT positivity rate among the HIV cases which could possibly be attributed to increased awareness towards STI among these high risk individuals. Our results indicate that the continuing HIV awareness and control strategies adopted in our region has met the HP2010 target of CDC.
CD4 count is an important surrogate marker for the assessment of the stage of the disease and the initiation of anti-retroviral therapy, the incidence of C. trachomatis was relatively common (2/13; 15.4%) in patients with CD4 count <200 cells/cu.mm as opposed to the HIV subjects (1/ 87; 1.1%) with CD4 count > 200 cells/cu. mm.
Molecular detection of C. trachomatis by PCR is more sensitive when performed on urine samples though the numbers copy of organism are less compared to that in present urogenital swab specimens. Nevertheless, testing urine has facilitated the use of a non-invasive method of sample collection and has increased the effectiveness, reduced the cost of screening programmes especially among the asymptomatic individuals in whom compliance is a major problem.
C. trachomatis contains in addition to the chromosomal DNA, a cryptic plasmid (7.5 kb) that is common among all serovars of C. trachomatis, also viable plasmid-less isolates and variants strains with deletion mutation (377bp) within the plasmids are currently being reported (20)(21),(22). The primers CP 24 and CP 27 that were used in AMPLICOR PCR assay are specific to the DNA sequence approximately 208 bp within the cryptic plasmid of C. trachomatis. The plasmid PCR assay could not detect positivity among all the 3 IgM ELISA positive subjects.
George et al., (2003) have documented a decreased positivity by urine PCR (25.2%) as opposed to 32.2% positivity by genital swab PCR. Also, PCR inhibitors were reported in 8.6% of the urine of female patients while, 3.2% among the samples of male subjects (14). However, the positive amplification signal of the internal control in all the 3 CT EIA positive samples in our study further nullifies the possibility of the presence of PCR inhibitors in the amplification process. Hence, the negativity of AMPLICOR PCR could not be attributed to the presence of PCR inhibitory substances in the urine.
The discrepancies between the two assay results observed in our study i.e. positive IgM ELISA and negative Amplicor PCR results could possibly be due to the occurrence of variant strains of C. trachomatis that circulate in our region, which lack the specific cryptic plasmid or possesss deletion mutants. The positive IgM ELISA assay results on plasmid-PCR-negative samples did not appear to be false positives because of the reproducibility of the results when performed in triplicates. These samples appeared to be unique as the presence of anti-C.trachomatis specific IgM antibodies suggestive of active infection were detected though the infecting C. trachomatis stains were either plasmid-free or with plasmids having deletion mutations within the target region.
Variant strains of C. trachomatis lacking the evolutionarily conserved cryptic plasmid have been previously documented among patients with symptomatic STI which were not detected by AMPLICOR PCR assay, but were detected by rRNA - PCR and RNAse Protection (RNP) assay (23). Previous studies have documented the incidence of cryptic plasmid-less variants of C. trachomatis worldwide (24),(25),(26),(27),(28). Recent Swedish reports have documented the incidence and impact of the new variant of C. trachomatis which was described in October 2006. A 377bp deletion mutation in the plasmid target region lead to the negative diagnosis by commercial plasmid based NAATs that eventually resulted in the underestimation of genital Chlamydia infections in the Swedish population (29), (30). A previous study had reported the incidence of a FVU specimen that was strongly reactive in CRT, but negative with the plasmid based Amplicor PCR (Roche) and ProbeTec ET (BD) assays (31). Gupta et al., (2008) have raised concern on the existence of a plasmidless clinical isolate of C. trachomatis in a patient from New Delhi, India and has suggested the use of real-time PCR in reviewing the C. trachomatis prevalence rates in the Indian population (32). Also, Sachdeva et al., (2009) has suggested the possible occurrence of plasmid free variants of C. trachomatis in the Indian population. In their study, four samples tested positive by Roche AMPLICOR MWP kit were found to be negative by the inâ€“house PCR assay targeting the Phospholipase D endonuclease superfamily gene (13). A recent Indian study has reported 4 samples that were negative by Plasmid PCR but positive by DFA and FISH assay (33). Our results suggest that in resource limited settings like India, the use of NAATS (commercial PCR assays, real-time PCR) is questionable and IgM ELISA still stands adequate in the diagnosis of this common STI in the HIV population.
The Advisory Committee for HIV and STD Prevention (ACHSP) (34) and the World Health Organisation (WHO, 2001) (3) have recommended the early detection and prompt treatment of STIâ€™s as an effective HIV prevention strategy. Routine, improved screening for asymptomatic STIâ€™s especially C.trachomatis infection has major public health implications for STI and consequently, would reduce the spread of HIV in this high risk population. In conclusion, we report here the possible occurrence of cryptic plasmid-less or naturally occurring mutant strains of C. trachomatis in South India. Failure to detect these variant strains of C. trachomatis and hence the infected going untreated would have a substantial impact on the incidence, spread, epidemiology and disease management among the high risk patients (with multiple sexual partners) in India wherein, plasmid based COBAS AMPLICOR PCR has been routinely used for Chlamydia screening by the research laboratories. Consistent with other studies, our observations further signify the need for cost effective periodic screening programmes for the detection of active C. trachomatis infection and access to treatment among the high risk patients which would substantially reduce the risk of HIV transmission and disease progression (4), (7), (8), (10), (18), (19), (34). CT specific IgM antibody detection obviates the need for performing plasmid PCR assay and further confirmation by MOMP PCR assay and could also reduce the health care cost. Nevertheless, further studies with a larger sample size and additional molecular methods are still needed to determine the actual magnitude of the problem and would be critically informative in determining whether plasmid-less / deletion mutant variants of C. trachomatis circulate in our geographical setting.
FINANCIAL OR OTHER COMPETING INTERESTS: NONE.
Date of Submission: Nov 30, 2011
Date of Peer Review: Dec 26, 2011
Date of Acceptance: Jan 12, 2012
Date of Publishing: Feb 15, 2012
- Emerging Sources Citation Index (Web of Science, thomsonreuters)
- Index Copernicus ICV 2017: 134.54
- Academic Search Complete Database
- Directory of Open Access Journals (DOAJ)
- Google Scholar
- HINARI Access to Research in Health Programme
- Indian Science Abstracts (ISA)
- Journal seek Database
- Popline (reproductive health literature)