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 conditions

Real-time PCR cycling conditions

Culture of CT clinical isolates

Confirmation of plasmid-less clinical isolate of CT

Statistical Analysis

Results

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 ompA real-time PCRs and 19 (6.1%) samples were positive by pCT real-time PCR only (Table 1). Moreover, the number of samples detected positive for both pCT and ompA real-time PCR were significantly higher (p<0.001) than detected using either of the single test. The analytical detection limit was 0.2 copies µl ^-1 (p<0.05) and 0.5 copies µl ^-1 (p<0.05) for pCT and ompA real-time PCRs respectively. All the 77 samples detected positive for real-time PCR were also found positive in culture system.

Figure 1

OmpA CT real-time PCR (Fig 1a. Panel A1, B1) was able to detect a probable plasmid- less isolate of urogenital CT as pCT was not detected in the same run by using the pCT real-time PCR (Fig 1a. Panel A2, B2).

Figure 2

Figure 1a: Identification of plasmid-less isolate of

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 C. trachomatis specific detection channel (FAM). Sample RT115 was analyzed by using an ompA based assay (panel A1 = first run, panel B1 = rerun of this sample) and an cryptical plasmid based assay (panel A2 = first run, panel B2 = rerun). In both PCR runs inhibitions of this sample were not observed (data not shown). Patient sample RT115 was repeatedly shown to be C. trachomatis positive by using the RealArt Standard kit (ompA based, panel A1 and B1) and negative by using the modified RealArt Plus kit (cryptical plasmid based, modified by removal of ompA specific primers and probes, panel A2 and B2).

DNA sequencing of ompA real-time PCR amplicon (106bp) of the plasmid-less clinical isolate confirmed its specificity (Table 2).

Figure 3

Table 2: BLAST analysis (http://www.ncbi.nlm.nih.gov/BLAST/) and Taxonomy Report of DNA sequencing of amplified plasmid-less isolate DNA using real time PCR.

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.

Figure 4

Figure 1b: Southern blotting hybridization for confirmation of plasmid-less isolate of using 517 bp pCT PCR product and digoxigenin labeled probes

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 Plus kit assay and positive for ompA using the RealArt standard kit. Lanes 4 and 5- Samples positive for modified RealArt Plus kit. Lane M- 100 bp DNA ladder (Bangalore Genei, Bangalore, India).

Discussion

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 ompA real-time PCR and 19 samples (6.1%) were positive for pCT only. One sample was detected positive for ompA but not for pCT and was subsequently confirmed by DNA sequencing, southern blotting and cell culture as plasmid-less isolate. Due to lack of a national screening program there was no such accountability till date about the prevalence rates of such isolates of CT in the semi-urban and rural population of India. The patient harbouring this isolate was a resident of New Delhi, the capital of India and a major cosmopolitan city in India. Since tourism, business and educational exchanges between India and the world have seen a rapid increase in the last decade there is a huge exchange of global populations from different geographical regions. Hence the identification of plasmid-less CT strains in the Indian sub-continent should be pondered upon with caution and efforts should be made to stop the cross pooling of such isolates.

Reports of a genetic variant of CT in Europe are thrilling and have provided several lessons (Herrmann, 2007; Schachter, 2007). Ripa et al (Ripa and Nilsson, 2006) reported deletion of 377 base pairs in the pCT target area using Abbott and Roche commercial kits, which detected false CT negatives and found a variant that was positive using RealArt Standard kit. The high copy number (7-10 copy cell ^-1 ) pCT is universal among all strains of CT and hence serve as targets for nucleic acid amplification tests along with 16S rRNA and ompA gene. However, the identification of plasmid-less isolates of CT stress upon the fact that there is need for designing nucleic acid tests which simultaneously target the pCT for its multiple copy number and the genome for negating the probability of false negative results while detecting CT. Use of commercial assays which exclusively detect CT plasmid sequences, provides a further reason for caution - ongoing or new therapies based on these diagnostic kits will ONLY eliminate plasmid bearing strains, whereas plasmid-less strains will remain undetected and may accumulate in the population.

Presence of plasmid-less isolates in nature (Peterson et al., 1990) can help understand Chlamydia biology in several ways .The fact that pCT is positively selected during evolution confers the fact that it provides advantages to the pathogen for host infection, replication and survival by modulation of host's immune system. Hence, availability of such strains would favour further studies in pCT's role in pathogenesis of the organism.

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

Correspondence to

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: amittal_cp@rediffmail.com, amittal_iop@yahoo.com