|Year : 1999 | Volume
| Issue : 2 | Page : 111-116
Evaluation of PCR assay for common endogenous plasmid and major outer membrane protein gene of C. trachomatis in diagnosis of follicular conjunctivitis
Gita Satpathy1, Sujata Mohanty2, SK Panda2
1 Dr. R.P. Centre for Ophthalmic Sciences, All India Institute of Medical Sciences New Delhi, India
2 Department of Pathology, All India Institute of Medical Sciences New Delhi, India
Dr. R.P. Centre for Ophthalmic Sciences, AIIMS, New Delhi - 110 029
Source of Support: None, Conflict of Interest: None
Purpose: To evaluate polymerase chain reaction (PCR) in diagnosis of Chlamydia trachomatis. Methods: In this study PCR assay was used to amplify the 517bp region of common endogenous plasmid in conjunctival specimens from 178 patients with follicular conjuntivitis, and to amplify the 1000bp region of major outer membrane protein (MOMP) gene of C.trachomatis from 71 of these 178 patients. The PCR-amplified products were visualised by agarose gel electrophoresis and ethidium bromide staining and Southern hybridisation with radio-labelled internal probes. The test was compared with a direct immunofluorescence assay using monoclonal antibody for Chlamydia antigen detection. Results: The plasmid PCR assay was positive in 95 (53.37%) of the 178 specimens processed whereas the Chlamydia antigen was detected in 69 (38.76%) of the 178 specimens by direct immunofluorescence assay (p= 0.005). In the 71 specimens processed for both the PCR assays, plasmid PCR was positive in 52 (73.23%) and MOMP PCR was positive in 43 (60.56%) of the specimens (p=0.10). Thirty seven of these 71 specimens which were positive in both PCR assays were also positive in direct immunofluorescence assay. Conclusion: The PCR assays could detect Chlamydia in a significantly larger number of specimens than conventional antigen detection assay, and being marginally more sensitive, the plasmid PCR assay has the potential for wider use in the diagnosis of trachoma.
Keywords: Polymerase chain reaction assay, direct immunofluorescence assay, Chlamydia trachomatis, endogenous plasmid, major outer membrane protein, follicular conjunctivitis
|How to cite this article:|
Satpathy G, Mohanty S, Panda S K. Evaluation of PCR assay for common endogenous plasmid and major outer membrane protein gene of C. trachomatis in diagnosis of follicular conjunctivitis. Indian J Ophthalmol 1999;47:111-6
Trachoma, a follicular conjunctivitis characterised by the presence of papillae and follicles in upper tarsal conjunctiva, is caused by A to C serovars of C.trachomatis, an obligate intracellular bacteria. Untreated, an inflammatory state is maintained due to repeated infections leading to pannus, scarring, trichiasis, and eventually blindness., Trachoma is a leading cause of preventable blindness in the world. Moreover, with the sharp worldwide increase of genital Chlamydial infections, follicular conjunctivitis caused by genital serovars of C.trachcomatis is also becoming important. Precise and timely laboratory diagnosis is essential to measure the prevalence of the disease, to study the pathogenesis and to institute appropriate therapy. However, in a large percentage of cases, the organism cannot be demonstrated either by cytoimmunological techniques or by tissue culture. Tissue culture isolation of Chlamydia is laborious, time consuming and the results are affected by improper transport, storage, and handling of specimens.
Currently, polymerase chain reaction (PCR) assay, a highly sensitive and specific technique, is being used to detect micro-organisms in clinical specimens., In the present study we used PCR assay for amplification and detection of a specific 517 bp sequence of the common endogenous plasmid from conjunctival specimens in 178 patients with follicular conjunctivitis, and the gene encoding the major outer membrane protein (MOMP) of C.trachomatis from 71 of these specimens. The results of the PCR assay were compared with a monoclonal-based direct antigen detection assay. The results obtained in some of the specimens using plasmid primer-directed PCR assay were confirmed using MOMP outer membrane protein primer-directed PCR assay.
| Materials and Methods|| |
| Clinical Specimens|| |
Conjunctival specimens were collected, using sterile cotton-tipped swabs from 178 patients with follicular conjunctivitis attending the outpatient department of the Dr R P Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi, from January 1994 to September 1995. The patients ranged in age from 10 to 52 years. Ninety eight of these patients were males and 80 were females and presented with conjunctival hyperaemia, discharge, or both, of more than 2 weeks duration. They had follicles in the upper tarsal conjunctiva. The specimens were collected prior to medication.
A smear from each of the specimens was made on a Teflon-coated slide for immunofluorescence assay for antigen detection. The other swab from each patient was collected in 0.2M sucrose phosphate (1 mL) buffer pH 7.2 for PCR assay. The specimens were brought to the laboratory as soon as possible. The slides for the immunofluoresence assay were fixed in cold methanol and preserved at -20° C until they were tested. The specimens for the PCR assay were preserved at -70°C till processed.
| Direct immunofluorescence assay|| |
The conjunctival smears on glass slides were stained with fluorescent tagged anti-Chlamydia monoclonal antibodies (Syva Microtrak, USA) according to the manufacturer's instructions and observed under the 100x objective of a fluorescent microscope. (Nikon, Japan)
| The Polymerase Chain Reaction Assay|| |
| DNA isolation|| |
DNA was isolated from 500 μl of thoroughly mixed clinical specimens. The extraction was first tried with proteinase K 100 g/ml (Life technology, Inc., USA) and SDS (Sigma, USA) treatment. Consistent results could not be obtained. Consequently, the specimens were treated with proteinase K 100 mg/ml, 0.45% NP40 (Sigma, USA), 0.45% Tween 20 (Sigma, USA), 2.5 mM Magnesium chloride (Sigma, USA), 50 mM Potassium chloride (Sigma, USA), in 10 mM Tris HC1 buffer pH 8.3. The mixture was incubated at 56°C for 3 hours. This was followed by phenol chloroform isoamyl alcohol extraction and ethanol precipitation.
DNA was also extracted from C.trachomatis L2 (434 Bu strain) grown in the yolk sac of embryonated hens' eggs and purified by renograffin gradient centrifugation 10 to serve as positive control
| Primers and the PCR procedure|| |
Two sets of primers were synthesised on a DNA synthesiser 392 (Applied Biosystems, USA) by the methoxy phospharamidite method. A set of 22 nucleotides (nt) sense and 26 nt antisense primers were synthesised for the amplification of 1 kilo base pairs (kbp) sequences spanning the entire length of ompl gene coding for the major outer membrane protein as per published sequences by Sayada et al. A 21 nt internal probe complimentary to the conserved region I of the gene was also synthesised as per published sequences by Ossewarde et al. The primer and probe sequences were as follows:
5' ATG AAA AAA CTC TTG AAA TCG G 3' Sense
5' CAA GAT TTT CTA GAT TTC ATT TTG TT
5' GAT CCT TGC GAT CCT TGC ACC 3' Probe
Another set of 18 nt sense and 18 nt antisense primers and a 30 nt internal probes were synthesised as above to amplify a 517 bp region of 7.2 kb common endogenous plasmid of C.trachomatis as per published sequences by Class et al. The primer and probe sequences were as follows:
5' GGA CAA ATC GTA TCT CGG 3' Sense
5' GAA ACC AAC TCT ACG CTG 3' Antisense
5' CGC AGC GCT AGA GGC CGG TCT ATT TAT GAT
The different parameters of the PCR assay, that is, the required concentrations of Magnesium chloride, primer, and dNTP; the reaction temperature profile, and the number of cycles to be performed were standardised. The PCR was performed in 50 μl of solution containing 10 mM Tris HC1 pH 8.3, 50 mM KC1 (Sigma,USA), 1.5 mM Mgcl2, 200 mM each dATP, dCTP, dGTP and dTTP (Stratagene, USA), 100 pmol of each primer, 1U of Ampli Taq DNA polymerase (Perkin Elmer Cetus, USA) and 10 μl of denatured ( at 95°C for 10 minutes) sample DNA. The mixture was overlaid with 3 drops of mineral oil. The PCR was carried out in a thermocycler (Techni, UK).
Each PCR cycle for the amplification of MOMP gene sequences consisted of denaturation at 94°C for 1 minute, primer annealing at 60°C for 2 minutes and extension at 73°C for 3 minutes. After 32 cycles of amplification a final extension for 5 minutes was done at 73°C.
Each PCR cycle for the amplification of plasmid sequences consisted of denaturation at 95°C for 1 minute, primer annealing at 50°C for 1 minute, and extension at 73°C for 1.5 minutes. After 40 cycles of amplification a final extension was done for 5 minutes at 73° C.
At the end of the PCR assay, 10 μl of each reaction mixture was analysed by electrophoresis on a 1% agarose gel. The gel was stained with ethidium bromide (0.5 μg/ml) and visualised under a UV trans-illuminator (UVP, USA) for specific amplified bands of DNA.
| Southern Blotting|| |
The amplified products were transferred from the agarose gel to nylon membrane (Hybond, Amersham, UK) by diffusion blotting in 10 mM NaOH with 1 Mm EDTA (Sigma,USA). The membranes were washed, air dried and ultraviolet fixed for 5 minutes using cross linker (UV Products, UK). The membranes were prehybridised at 50°C in 5X SSC (75 mM sodium citrate (Sigma,USA) and 750 mM sodium chloride(Sigma,USA), 10X Denhardt's solution (0.2% Bovine serum albumin (Sigma,USA), 0.2% Ficoll 400 (Sigma,USA), 0.2% polyvinyl pyrolidone (Sigma,USA)), 7% SDS, 20 mM sodium phosphate(Sigma,USA) pH 7.0 and 100 mg/ml of heat denatured sonicated salmon sperm DNA (Sigma,USA) per mL for 30 minutes. Hybridisation was performed oversight in the same solution at 50°C for over night with p-labelled oligonucleotide probe specific for MOMP gene sequences or plasmid sequences. The blots were washed for 30 minutes each in wash solution I (3X SSC. 5% SDS, l0x Denhardt's solution, 25 mM sodium phosphate pH 7.5), wash solution II (2X SSC and 2% SDS) and wash solution III (1X SSC and 1% SDS) successively. Autoradiography was performed at -80°C on Kodak X-Omat (Kodak, USA) film by using intensifying screens.
The Chi Square test was used for statistical analysis of the results.
| Results|| |
| Standardisation of the PCR assay|| |
The PCR assay was standardised step by step. Several combinations of reagents in lysis buffer were tested for DNA extraction from clinical specimens. The best results were obtained with 10 mM Tris HCl buffer pH 8.3 with addition of non-ionic detergents NP40 and Tween 20 at 0.45% concentration, 2.5 mM magnesium chloride, 50 mM potassium chloride, and 100 mg/ml proteinase K followed by standard phenol chloroform extraction. It was observed that 1.5 mM magnesium chloride, 200 mM of each dNTP and 100 pmol of each primer gave best results in the PCR assay.
| Detection of C trachomatis in clinical specimens|| |
In the direct immunofluorescence assay (DFA) the Chlamydia elementary bodies in the smear were seen as bright, apple green, spherical and regular particles [Figure - 1]. Any smear showing more than 10 such particles was considered positive. In plasmid primer-directed PCR assay, C. trachomatis plasmid DNA could be detected in 95 of the 178 (53.37%) of the conjunctival specimens. Sixty-nine of these (72.63%) positive specimens were positive for Chlamydia antigen in the DFA (p = 0.005 ). [Figure - 2] shows the 517 bp amplified plasmid DNA sequences in ethidium bromide-stained agarose gel and [Figure - 3] shows the autoradiograph of Southern hybridisation of the same product.
Seventy-one of the conjunctival specimens were processed for amplification of 1 kbp region of the major outer membrane protein gene in addition to plasmid-directed PCR assay. Fifty-two of the 71 (73.23%) conjunctival specimens were positive in plasmid-primer PCR assay, and 43 of the 52 (82.69%) positive specimens were also positive in major outer membrane protein primer-directed PCR assay (p = 0.10). Thirty seven (52.11%) of these 71 specimens, which were positive in both the PCR assays were also positive in DFA (Table). [Figure - 4] shows the amplified 1000 bp sequences of the major outer membrane protein gene in 1% agarose gel stained with ethidium bromide. [Figure - 5] shows the autoradiograph of Southern hybridisation of the same product.
| Discussion|| |
Current methods of detection of C. trachomatis in clinical specimens include cell culture, direct immunofluorescence assay (DFA), enzyme immuno assay, and hybridisation., The PCR assay promises to be more sensitive and can overcome the shortcomings of these assays. This test has been found useful in follow-up studies after antibiotic therapy in genital and ocular infections, and particularly useful in chronic infections where organism load is low.
For the detection of C. trachomatis, PCR assay, various DNA extraction procedures, namely, boiling pronase with SDS treatment, non-ionic detergents, NP40 and Tween 20 treatment,, proteinase K with SDS,sarkosyl, SDS, and EDTA with proteinase K treatment have been used. In the present study proteinase K with SDS treatment followed by phenol chloroform extraction did not give consistent results. Therefore we used non-ionic detergents NP40, Tween 20 along with proteinase K for sample preparation which gave consistent results.
Although PCR assay products could be detected in all the positive specimens by simple ethidium bromide staining of the agarose gels, Southern blotting with internal oligo probes was done for confirmation. As these were single-stage PCR assay, Southern blotting provided information on both size and specificity of amplified product; moreover, it has been claimed to be more specific., To avoid false positivity, DNA extraction, preparation of amplification mixture and detection were carried out in separate physical areas, which is a commonly practised procedure.
The present study demonstrated that PCR assay could detect C.trachomatis in a significantly greater number of conjunctival (53% Vs 39%, p= 0. 005) specimens than the DFA. DFA was used for comparison with PCR assay as this test has been widely evaluated against the tissue-culture isolation method and has been used as reference method by reputed laboratories. The detection of C.trachomatis by conventional laboratory diagnostic methods in conjunctival specimens often correlates poorly with clinical follicular and inflammatory trachoma., The PCR assay has been used successfully in diagnosis of genital Chlamydial infections, and Chlamydial DNA has been found in culture-negative, DFA-negative genital specimens by PCR., In ocular infections also the PCR assay has been able to detect Chlamydial DNA in DFA-negative specimens. The presence of Chlamydial DNA in DFA-negative or culture-negative specimens is significant as it has been postulated for some time that serious sequelae and chronic trachoma are due to repeated infection with C.trachomatis and possible incomplete clearance of the organism. [3,20] In the present study 14% of the conjunctival specimens which were Chlamydial antigen negative in DFA assay but were positive for Chlamydial DNA in PCR assay, might have been infected with C.trachomatis at levels below the detection abilities of immunofluorescence assay. Since the specificity of the reactions were always checked with controls and extreme care was taken while setting up the reactions, cross contamination of the specimens was unlikely and the results were specific. The follicular conjunctivitis cases which were clinically diagnosed as trachoma and were positive for C.trachomatis DNA by PCR assay varied in different studies. In the present study the test could detect Chlamydial DNA in 53% of the patients compared to 49% in a Tanzanian study and 51% of clinical trachoma cases in a Gambian study. Even though the results of the present study in terms of sensitivity for PCR assay for C.trachomatis detection are comparable to these two studies, 49% of those having follicular conjunctivitis were negative for C.trachomatis even in the PCR assay. Therefore it appears that all the follicular conjuctivitis cases in this study group are not due to Chlamydial infection. These might be non-Chlamydial follicular conjunctivitis cases, possibly of allergic origin, and could be due to high environmental pollution in the community. This aspect needs further study.
In the present study, the primer-directed PCR assay detected Chlamydia in a larger number of conjunctival (73% vs 61%) specimens than MOMP gene primer-directed PCR assay. The difference however was not significant. Plasmid PCR assay has earlier been reported to be more sensitive., In conjunctival specimens, 88% of those labelled positive by plasmid PCR assay were found positive by major outer membrane protein gene PCR assay in a previous report, compared to 83% in the present study. Under laboratory conditions, C.trachomatis L2 DNA could be detected up to a higher dilution by plasmid PCR assay compared to major outer membrane protein gene PCR assay. The higher sensitivity may be due to presence of multiple copies (up to 10) of the plasmid. Only one plasmid-less strain of C.trachomatis has been described so far. So the possibility of a false negative result is low. Therefore plasmid-directed PCR assay is usually chosen as the standard PCR assay for C.trachomatis,, and has been recommended for Chlamydia-screening programmes and follow-up studies. Usually some of the specimens are confirmed with major outer membrane protein gene PCR assay.,
The PCR assay is expensive, but so is the DFA using monoclonal antibodies or tissue-culture isolation of the organism. Therefore if resources permit, PCR assay may be preferable as it has a greater potential in the diagnosis of Chlamydial conjunctivitis.
| Acknowledgements|| |
We thank Prof. SK Angra, Dr Anita Panda, Dr RB Vajpayee, the resident doctors and the ophthalmologists of Dr RP Centre for the clinical specimens. This study was supported by a grant from Indian Council of Medical Research.
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[Figure - 1], [Figure - 2], [Figure - 3], [Figure - 4], [Figure - 5]
[Table - 1]