Use of polymerase chain reaction (PCR) for detection of Chlamydia trachomatis infection in cervical swab samples

Introduction

Chlamydia trachomatis (C. trachomatis) infection of the lower genital tract is one of the most prevalent sexually transmitted diseases in the world. Detecting chlamydial genital infection and preventing transmission and spread of this infection to the upper reproductive tract are a challenge for both clinicians and laboratory workers.[1] Genital infections caused by C. trachomatis are often asymptomaticz and early detection to avoid serious complications is of real value since effective treatment exists. Polymerase chain reaction (PCR) has recently been introduced for detection of C. trachomatis and studies have reported its superior sensitivity in comparison with culture,[3],[4],[5],[6] enzyme - linked immunosorbent assay[3],[5] or direct flurescein-conjugated antibody (DFA) staining.[7] Information regarding incidence and prevalence of laboratory confirmed C. trachomatis infection of the genital tract in India is limited to a few studies because of lack of proper laboratory facilities.[8],[9] In India reports on the use of PCR to detect C. trachomatis infection is restricted,[9] though this technique has been successfully used in clinical specimens elsewhere. In view of the sensitivity and broad applicability, we attempted to set up, a PCR based method to detect the C. trachomatis infection among the women population using individual single cervical swab samples. Subjects included were women attending the Gynaecology Department of two different hospitals in the city viz, the King Edward Memorial Hospital (KEM) and the Sir Hurkisondas Nurrotumdas Hospital (H.N. Hospital).

Materials and Methods

Specimens: Endocervical swab samples were obtained from patients attending the Gynaecology department of KEM Hospital, Parel and Sir H.N. Hospital, Prathana Samaj, Mumbai. Each swab was put in the sample collection vial containing the buffer (1 ml) supplied with the commercially available ELISA kit (Abbott′s Diagnostics, U.S.A.). Sample of each individual was screened in the hospital laboratory (Microbiology Department of KEM & Medical Research Society of Sir H.N. Hospital) using the ELISA kit for C. trachomatis. Some of these screened samples (n=30) were collected in coded form for PCR analysis to the author′s laboratory. Five of these 30 samples had a sample volume of 700µI each whereas in others (n=25) the volume of each sample was approximately 400 µl. Decoding of the supplied samples for infection by ELISA - based method was carried out after the PCR - based detection.

Preparation of samples for PCR: Several methods are available for DNA extraction. In the present study, we used two methods i.e., alkali lysis[10] and proteinase K treatment[11] in 5 of the samples where each sample volume was 700 µl. In other 25 samples, volume of each was 400 µl and DNA was extracted in these samples using the proteinase K treatment.

Primers: One pair of oligonucleotide primers specific for a 144 by region of the C. trachomatis gene coding for the major outer membrane protein (MOMP) was selected.[12] This primer pair was procured from Bangalore Genei, Bangalore. The sequences from 5′ to 3′ of these oligonucleotide primers are as follows:

Sense: 5′ CCT GTG GGG AAT CCT GCT GAA 3′

Antisense: 5′ GTC GAA AAC AAA GTC ACC ATA GTA 3′

PCR mixture and DNA amplification:

For PCR, Gene Amp(R) PCR reagents from Perkin Elmer, U.S.A. with AmpliTaq(R) DNA polymerase were used. A master mixture of these reagents was made for the samples along with positive and negative controls. The final reaction mixture of 50µI for each sample contained 0.5µM each primer; 100µM each of dAP, dCTP, dGTP and dTTP; 50 mM KCI; 10 mM Tris-HCI, pH 8.3; 1.5 mM MgCI2; 0.01% gelatin and 1.25 units of DNA polymerase (Thermus aquaticus) enzyme and 9ul of sample DNA. For positive PCR control whole bacteriophage Lambda DNA template along with primers amplifying a 500 by region were used. In 50 µl of PCR mixture, 0.5 ng of positive control was used, whereas for negative control 5 µl of autoclaved distilled water was used. Each microfuge tube containing the PCR mix of 50µI was mixed and subjected to 40 cycles of amplification. Each cycle was composed of sequential incubations of 94°C for 1 minute for DNA denaturation, 52°C for 1 minute for annealing primer to these templates, and 72°C for 2 minutes for DNA chain extension. At the end of 40 cycles, samples were kept for another 7 minutes at 72°C for completion of extension of DNA chain. The PCR product samples were immediately frozen for further analysis.

Amplified product detection: Visualisation of amplified product was carried out by agarose gel electrophoresis. A 10µI of post-PCR mixture was subjected to electrophoresis on 2% agarose gel in presence of ethidium bromide. A DNA ladder was also run simultaneously to confirm size of the amplified product.

Specificity of the amplified product

To confirm the specificity, the amplified DNA product was extracted directly from 30 µl of the post-PCR solution. Extraction was carried out with a DNA binding resin, using a kit supplied by Boehringer Mannheim, USA. The isolated DNA pellet was resuspended in 20 µl of TE buffer. A portion of the suspension (15 µl) was incubated with the endonuclease EcoRl. For this purpose, a mixture of 25 µI was prepared containing 15 µl of the isolated DNA product, 2 µl of EcoR1 endonuclease (80 units), 2.5 pl of enzyme specific buffer with 5.5 µl of autoclaved distilled water. The mixture for each sample was incubated for 3 hours at 37° C and then frozen till used for detection. This digested mixture (10 µl) along with the 5 µl of the undigested isolated DNA from each sample was then subjected to 2% agarose gel electrophoresis. An appropriate DNA ladder was also run simultaneously to determine the size of the bands obtained.

Detection level of the PCR: A standard of C. trachomatis (L2 strain, Elementary bodies from Scripps Organisation, USA) was used for determination of the minimum detection level of DNA for PCR amplification. For the purpose, various quantities of DNA (range: 0.001 µg to 5 µg) were taken for amplification. The amplification product of 144 by was detected as described earlier.

Quantitation of DNA with respect to epithelial cells for calculation of cell number in analysed samples and an expected yield of DNA, each sample after collection was observed under microscope to estimate the epithelial cells present. The samples were then processed for DNA extraction. Further quantification of each DNA was done by the optical density measurement obtained at 260 nm.

Discrepant - result analysis

If a specimen produced a PCR result discrepant with the comparison test Chlamydiazyme′ following decoding, PCR was repeated along with the confirmatory test with EcoR1 digestion. If the second amplification produced a result in agreement with the comparison test, the result from the initial amplification was not taken into consideration (not encountered during this study). A repeat positive PCR result indicated a ELISA (Ch\amy6azyme) false- negative result.

Results

Similar results were obtained when 5 of the 30 samples were processed both by alkali lysis and proteinase K methods for DNA extraction. PCR amplification using these DNA samples showed that, of the 30 samples, 23 produced the specific 144 by DNA fragment. This 144 by DNA after digestion with EcoR1, produced the 103 by fragment. The minimum amount of DNA that could be amplified for detection of the required 144 by was observed to be 1 µg. [Figure - 1] shows the PCR amplification of 144 by C. trachomatis MOMP gene along with digested product of 103 bp. A serial amplification of DNA corresponding to 16 µg to 1 µg is also presented in the [Figure - 1]. A linearity in the amplification of the expected fragment from increasing concentrations of DNA was clearly evident from the [figure]. DNA quantification and microscopic examination of samples showed that using our method, approximately 90 epithelial cells correspond to 1 µg of DNA.

Correlation with ELISA assay

The results of PCR analysis were correlated with the ELISA assay carried out in the hospitals using the ELISA "Chlamydiazyme" (Abbott′s Diagnostics, U.S.A.) and presented in [Table - 1]. By ELISA, out of 30 samples tested, 20 were found to be positive for C. trachomatxs antigen. The PCR based technique detected 3 additional samples with positive C. trachomatis infection. All these 3 PCR - positive, Chlamydiazyme negative specimens were confirmed by repeat PCR assay along with the confirmatory test of EcoR, digestion. After resolution of 3 Chlamydiazyme negative specimens that were PCR positive, the Chlamydiazyme and PCR sensitivities were 86.96% and 100% respectively, compared with the total resolved positive specimens, whereas the specificities of both Ch\amy6azyme and PCR were 100%.

Discussion

Detection of Chlamydia in patient specimens by DNA amplification has expanded the spectrum of pathogens amenable to PCR. The molecular biology of the Chlamydiae is incomplete. Few genes have been sequenced and little is known of the molecular aspects of its pathogenesis. Because of its relative abundance and antigenic importance, the major outer membrane proteins (MOMPs) of several isolates of C. trachomatis have been cloned and sequenced. The MOMPs have several regions of highly conserved nucleotide sequences, which have been used for primer selection.[12] These primers allow for generation of PCR fragments spanning several regions of the MOMP gene, with an internal restriction enzyme site. The primer pair which has been chosen in this study, has been able to identify the fermalinized serotypes of C. trachomatis culture products.[13] In the present study, these primer pairs also identified the 144 by sequence of MOMP gene of C. trachomatis infection present in individual cervical swab sample. Mumbai being a metropolitan city, patients coming to different hospitals (KEM Hospital, Parel and Sir H.N, Prathana Samaj, Mumbai) belong to different ethnic and socio-economic groups. These patients originated from different States. Thus the pair of primers used could successfully identify the infection present in single cervical swab samples of these individuals with mixed socio-economic backgrounds. The three cases which were positive by PCR but negative by ELISA, were the true positive as confirmed by repeat PCR and as is also reported PCR is superior in its sensitivity to ELISA.[3],[4],[5] In the first study, it was reported that the sensitivity of ELISA (Chlamydiazyme) when compared with PCR was 60%.[3] According to the other study, when PCR was compared with a direct specimen enzyme immuno assay (Chlamydiazyme) using 375 cervical specimens, PCR had a sensitivity and specificity of 100%, while the ELISA had a sensitivity of 58.8% and a specificity of 100%.[5] In the observation, both the sensitivity and specificity of the PCR is 100%, while the ELISA has a sensitivity of 86.96% and specificity of 100%.The choice of a reference method, when the true disease status is unknown is difficult. Since gene amplification methods are usually more sensitive than traditional methods like culture, ELISA, DFA, the use of these traditional methods as reference methods is not appropriate.[14] In the present study, the PCR results have been compared here with ELISA, as this was readily available and facility for culture or DFA were not available.

To achieve optimum sensitivity of the PCR assay, one has to emphasize the cycle number and quality of sample collected. In the present study, PCR was carried out for 40 cycles, so that any sample with low number of C. trachomatis genome could be optimally amplified for visualisation. Attention had been given on the quality of specimens collected.

This is an equally important aspect which has been given little attention compared to the tremendous amount of energy, time and money that has been spent on improving Chlamydia detection test.[15] In the present attempt, it was observed that sufficient number of epithelial cells should be present in the sample collected for DNA extraction and amplification. Sample dilution curve using different concentration of DNA showed that 1 µg is the minimum amount of DNA that could be used for amplification of the desired 144 by fragment. This corresponds to an approximately 90 epithelial cells present in the sample.

Direct and indirect costs due to Chlamydial infections are substantial and justify the screening with the use of a high performance diagnostic test like PCR.[16] Present observation demonstrates that C. trachomatis can be detected by this PCR-based method in Indian population with a greater sensitivity than ELISA, from cervical swabs in properly collected samples using an appropriate cycle number. It offers hope for more accurate diagnosis, which would lead to a better control of this infection and to a better prevention of pelvic inflammatory disease, ectopic pregnancies and tubal infertility.

Acknowledgement

The authors are grateful to Microbiology Department of KEM Hos-pital and Medical Research Society of Sir Hurkisondas Nurrotumdas Hospital, Prathana Samaj, Mumbai for providing the selected samples to carry out the assay. We acknowledge the constant support of Dr. H.S. Juneja, Director of this Institute for developing this.