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

摘要

Reports of Chlamydia trachomatis (CT) plasmid-less isolates worldwide have caused concern. Hence, this study was aimed to determine if plasmid-less isolates of CT is present in India. Endocervical swab samples collected from 311 symptomatic women were screened for CT using real-time PCR targeting ompA gene and plasmid (pCT), culture and direct fluorescent assay (DFA). Of 311 samples, 57 (18.32%) were positive for both pCT and ompA and 19 samples (6.1%) were positive for pCT only. Samples detected positive for both pCT and ompA real-time PCR was significantly higher (p<0.001) than using either of the single test. One sample was detected positive for ompA but not for pCT and was subsequently confirmed by DNA sequencing, southern blotting and culture as plasmid-less isolate. The existence of plasmid-less isolate of CT in India demands the use of real-time PCR assays for better clinical management. Introduction Genital tract infections due to Chlamydia trachomatis (CT) are a major cause of morbidity in sexually active individuals worldwide and in developing countries including India. A high prevalence of female genital chlamydial infection has been previously reported from India (Singh et al., 2002; Joyee et al., 2004). In women untreated infections may progress to serious reproductive sequalae including ectopic pregnancy, pelvic inflammatory disease and salpingitis with tubal scarring and infertility (Low et al., 2006). Therefore, early and accurate diagnosis is necessary to prevent these complications and thereby controlling the spread of infection. 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 specific DNA in endocervical swab samples have been developed and evaluated (Chan et al., 2000; Jensen et al., 2003). Real-time PCR for detection of Chlamydiae is considered highly sensitive and has a high degree of specificity over conventional PCRs (Hardick et al., 2004; Boel et al., 2005).The evolutionary conserved, 7.5- kb CT cryptical plasmid (pCT) is present in seven to ten copies per cell and is the target of most molecular diagnostic tests. Inspite of being under positive selection pressure, several clinical isolates of Chlamydia sp. have been documented worldwide, which lack the cryptical plasmid (Peterson et al., 1990; An et al., 1994; Farencena et al., 1997). These isolates are crucial for research to gain an understanding of the functions of the plasmid and its contributions to the morphology, development and pathogenecity of this bacterium. Hence this study was aimed at screening for plasmid-less isolates of CT in India using dual target (plasmid and ompA) real-time PCR assays. Materials and methods Study population and sampling conditions311 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 conditionsChlamydial DNA was extracted from all clinical samples and confirmed for positivity by a human beta (β) - globin PCR as described previously (Singh et al., 2003). All β-globin PCR positive samples were diagnosed for CT by RealArt Standard kit and modified RealArt Plus kit (modified by removal of ompA specific primers and probes) (QIAGEN Diagnostics GmbH, Germany) targeting 106 bp of ompA gene and 111 bp fragment of pCT. To determine the analytical sensitivity of the RealArt Plus kit, plasmid dilution series was set up from 0.66 to 0.002