Existence of plasmid-less clinical isolate of Chlamydia trachomatis in India is a cause for concern and demands the use of real-time PCR assays
R Gupta, R Jha, S Salhan, M Eickhoff, G Krupp, A Mittal
detection, plasmid-less isolate, real-time pcr, sexually transmitted infections
R Gupta, R Jha, S Salhan, M Eickhoff, G Krupp, A Mittal. Existence of plasmid-less clinical isolate of Chlamydia trachomatis in India is a cause for concern and demands the use of real-time PCR assays. The Internet Journal of Microbiology. 2007 Volume 5 Number 2.
Genital tract infections due to
Several studies have shown that nucleic acid amplification tests are far superior to conventional tests for diagnosis of CT infection. Polymerase chain reaction (PCR) based tests that detect CT
The evolutionary conserved, 7.5- kb CT
Materials and methods
Study population and sampling conditions
311 symptomatic women (age range 20 to 40 years ) attending Gynecology outpatient department of Safdarjung Hospital, New Delhi, India were enrolled for the study. Prior written consent was obtained from each patient and the study was approved by the hospital Ethical Committee. The cervix was inspected for ulcers, warts, ectopy, erythema, discharge, and lesion if any. After cleaning the exocervix with cotton swab (Hi Media, India), endocervical swabs were collected in sterile vials containing phosphate buffered saline for diagnosis of CT infection by real-time PCRs and cell culture .
Real-time PCR cycling conditions
Chlamydial DNA was extracted from all clinical samples and confirmed for positivity by a human beta (β) - globin PCR as described previously (Singh
Culture of CT clinical isolates
Real-time PCR positive samples were cultured in McCoy cell line (National Centre for Cell Sciences, Pune, India), harvested at 66 hours post infection and subsequently examined for positivity by culture confirmation test DFA as described previously (Mittal
Confirmation of plasmid-less clinical isolate of CT
Along with culture confirmation, the specificity of
PriProbit TM version 1.63 (Kyoto University, Japan) was used to determine the analytical detection limit of real time PCRs. The statistical significance for performance of real-time assays was calculated using McNemar test.
All 311 samples were found to be positive for β-globin PCR. Out of 311 samples, 57 (18.32%) samples were positive for both pCT and
The results of two real-time PCR runs (panel A and B) of sample RT115 on the Rotor-Gene 3000 instrument are shown. Depicted in the figure is the
DNA sequencing of
The specificity of plasmid-less clinical isolate was further confirmed by non-radioactive southern blotting analysis using digoxigenin labeled probes against 517 bp plasmid PCR amplicons. The results of southern blotting analysis are shown in fig. 1b.
Lane 1- Negative control (uninfected McCoy cells). Lane 2- Positive Control (pCT positive sample cultured in McCoy cells). Lane 3 - Sample negative for pCT for modified RealArt
In the present study a highly specific and sensitive real-time PCR was used to detect the presence of plasmid-less urogenital isolate of CT in symptomatic female patients. Out of 311 samples screened in the study, 57 (18.32%) were positive for both pCT and
Reports of a genetic variant of CT
Presence of plasmid-less isolates in nature (Peterson
In conclusion, this study suggests that the national impact of existence of a plasmid-less isolate of CT should be looked at for better clinical management of this infection. The use of this real-time PCR kit will help in reviewing the prevalence rates of endocervical CT in the Indian population and the rest of the developing world. The spread of new variant of CT differs between countries but the reason for this remains unexplained and should be explored in the future. Further data is still needed to determine the size of the problem of emerging CT variant in India since only symptomatic patients were screened in our study.
Acknowledgements, Competing Interests, Funding
University Grants Commission and Indian Council of Medical Research are acknowledged for providing fellowship to Rishein Gupta and Rajneesh Jha respectively. There are no competing interests. Department of Biotechnology, New Delhi, India provided funds for carrying out the study.
Dr. Aruna Mittal, Institute of Pathology (I.C.M.R), Safdarjung Hospital Campus, New Delhi-110029, India. Tel No:91-011-26175616, Fax No:91-011-26198401; email ID: email@example.com, firstname.lastname@example.org