Distribution of Chlamydia trachomatis omp A genotypes in patients attending a sexually transmitted disease outpatient clinic in New Delhi, India.

Background & objectives: Limited data are available on the typing of Chlamydia trachomatis in India. Serovars D to K of C. trachomatis are chiefly responsible for urogenital infections. Thus, this study was conducted to determine the distribution of C. trachomatis serovars in patients with urogenital infections and to characterize omp A gene of the detected C. trachomatis isolates by sequence analysis. Presence of other co-infections was also evaluated.
Methods: Endocervical swabs were collected from 324 women and urethral swabs/urine were collected from 193 men attending the sexually transmitted diseases outpatient clinic. The samples were screened for C. trachomatis by cryptic plasmid PCR and omp A gene PCR. Genotyping was performed by PCR-restriction fragment length polymorphism (RFLP) and sequencing of the omp A gene. Samples were screened for genital mycoplasmas, Neisseria gonorrhoeae, Treponema pallidum and human immunodeficiency virus (HIV).
Results: C. trachomatis was found in 15.0 per cent men and 10.8 per cent women. Serovar D was the most prevalent followed by serovars E, F, I and G. Twenty two C. trachomatis isolates were selected for omp A gene sequencing. No mixed infection was found. Variability in omp A sequences was seen in 31.8 per cent cases. Both PCR-RFLP and omp A gene sequencing showed concordant results. The presence of Ureaplasma spp. and Mycoplasma hominis was observed in 18.7 and 9.5 per cent patients, respectively. Co-infection of C. trachomatis was significantly associated with Ureaplasma urealyticum and HIV. Interpretation & conclusions: The high occurence of C. trachomatis infections warrants its screening in addition to other sexually transmitted infections namely U. urealyticum and HIV. Genotyping of the omp A gene may provide additional information for vaccine development.

Chlamydia trachomatis urogenital infections are the most commonly reported bacterial sexually transmitted diseases (STDs)1. Genital infections with C. trachomatis (serovars D-K)are associated with urethritis, pelvic inflammatory disease and infertility2. C. trachomatis infection facilitates the transmission of human immunodeficiency virus (HIV) and is often associated with other STDs3.

The major outer membrane protein (MOMP) of C. trachomatis contains specific antigens that differentiate chlamydial strains by serovars (based on antigenic cross-reactivity on microimmunofluorescence) or genotype (based on nucleotide sequencing of the ompA gene)4. Whether different serovars of C. trachomatis demonstrate different virulence potential is unclear. Genotyping of C. trachomatis strains is important to monitor contact tracing, to enable thorough understanding of the pathogenesis and epidemiology of genital chlamydial infections5. The genital mycoplasmas include Ureaplasma spp. and Mycoplasma hominis which are potentially pathogenic species playing an aetiologic role in genital infections. Most colonized individuals remain asymptomatic, but there are considerable evidences that M. hominis and Ureaplasma spp. also cause disease6.

Limited information is available on the typing of C. trachomatis from India. The aim of the present study was to determine the presence of C. trachomatis infection in patients with urogenital infections, tocharacterize the ompA gene of the detected C. trachomatis isolates by sequence analysis of DNA, to determine the occurrence of other aetiological agents (viz. Ureaplasma spp. and M. hominis) and to evaluate the presence of STD co-infections.

Material & Methods

All consecutive sexually active adults attending the STD Outpatient Clinic at the All India Institute of Medical Sciences, New Delhi, India, from February 2009 to February 2014, were included in the study. Patients who had been treated with antibiotics within the past four weeks and those who tested positive for bacterial vaginosis and Candida were excluded, whereas patients tested positive for Niesseria gonorrhoeae, Treponema pallidum and HIV were included in the study.

A total of 517 patients (324 women and 193 men) were eligible for enrolment. Ethical Committee approval for study protocol and written informed patient consents were taken for this study.

Three Dacron-tipped endocervical swabs from women and three urethral swabs were collected from men. Twenty millilitres of the first void urine (FVU) was also collected from men. The first swab was transported to the laboratory in 0.2 M sucrose phosphate buffer chlamydial transport medium (7.5 g sucrose, 0.052 g KH2PO4, 0.122 g K2HPO4, 72 g glutamine, 10 μg/ml gentamycin, 10 μg/ml amphotericin B) for C. trachomatis PCR assays. Two swabs were placed in two screw cap test tubes containing 2 ml pleuropneumonia-like organism (PPLO) broth for detection of Ureaplasma spp. and M. hominis. The PPLO broth for Ureaplasma spp. contained 2.1 g PPLO broth (Difco, USA), yeast extract (25%), horse serum (unheated), urea solution (50% w/v), penicillin solution (104 units/ml), trimethoprim (7.5 mg/ml), and phenol red (0.2 % w/v), and for M. hominis 2.1 g PPLO broth, yeast extract (25%), horse serum (unheated), arginine (20%), penicillin solution (104 units/ml), trimethoprim (7.5 mg/ml), thallium acetate solution (1/80 w/v), and phenol red (0.2% w/v). DNA was extracted from all the three swabs and FVU using QIAamp Mini Kit (Qiagen, Hilden, Germany). The extracted DNA was stored at −20°C till further use. The reference strains from National Collection of Type Culture Ureaplasma (NCTC10177) and M. hominis (NCTC10111) were used as positive controls.

Cryptic plasmid PCR: C. trachomatis DNA was detected by PCR targeting a sequence of the cryptic plasmid using primers KL-1 and KL-2 as described by Mahony et al7. Template DNA of C. trachomatis serovar D ATCC VR-885 (also known as prototype strain D/UW3) was used as positive control in each PCR assay.

OmpA gene PCR:Samples positive for C. trachomatis by cryptic plasmid PCR were confirmed by a second PCR targeting the ompA gene using primers NLO and NRO, and nested ompA gene PCRs were run using 5 μl of ompA primary PCR products using primers NLI and NRI as described by Gao et al8. Template DNA of C. trachomatis serovar L2 was used as positive control in each PCR reaction.

Multiplex PCR for Ureaplasma spp. and Mycoplasma hominis:In addition to culture, multiplex PCR was performed for the detection of genital mycoplasmas with primers specific for urease gene of Ureaplasma and 16S rRNA gene of M. hominis9. All the isolates of Ureaplasma were further biotyped in a second PCR targeting the multiple banded antigen (MBA) gene10. PCR positive for biovar 1 was further subtyped into serovars as described earlier by De Francesco et al11.

Restriction fragment length polymorphism (RFLP) and genotyping: Ten microlitres of the nested ompA PCR products were digested with 1 unit AluI (New England Biolabs, MA, USA). Products were electrophoresed through a polyacrylamide gel (acrylamide/bisacrylamide, 29:1; 12 V/cm for 1.5 h) to enable the identification of serovars D to K (Figure A). Further, Serovars H and I were separated with 1 unit of HhaI (Figure B). The digested products were electrophoresed through a seven per cent polyacrylamide gel (acrylamide/bisacrylamide, 29:1; 1× tris-borate-EDTA; 5 V/cm, 1.5 h) to differentiate serotypes.

Figure

(A) Restriction fragment length polymorphism patterns of ompA gene after restriction with AluI for differentiating serovars D to K. (B) Restriction fragment length polymorphism patterns of ompA gene after restriction with HhaI for differentiating serovars H and I.

Sequencing of the ompA gene: The sequencing of the ompA gene was carried out on ABI PRISM 310 Genetic Analyzer (PE Biosystems, Foster City, CA, USA) using a BigDye DNA sequencing kit (PE Biosystems) using primers NLI: 5'-TTTGCCGCTTTGAGTTCTGCT-3’ and NRI: 5'-CCGCAAGATTTTCTAGATTTC-3'according to the manufacturer's instructions8.

Phylogenetic analysis: Sequences were manually aligned and adjusted to prototype sequences. Phylogenetic analysis was performed by using the maximum likelihood method implemented in MEGA6 program12.

Statistical analysis: All statistical analyses were performed using STATA version 11.2 software (STATA Corp LP, College Station, TX, USA). The difference in the clinical features and sociodemographic data was analyzed by multivariate analysis.

Results

C. trachomatis was detected in 12.3 per cent (64/517) of patients with urogenital infections, of whom 10.8 per cent (35/324) were women and 15.0 per cent (29/193) were men. Of the 38 couples enrolled in the study, 11 were found to be infected; the infection involved partners in 54.5 per cent (6/11) of couples, only the male partner in 36.4 per cent (4/11) and only the female partner in 9.1 per cent (1/11) of couples.

The presenting symptoms and demographic characteristics of patients with urogenital infections in relation to C. trachomatis status are given in Table I. The mean age of the patients was 31.59±8.64 yr. In women, C. trachomatis infection was significantly associated with vaginal discharge (P=0.007), abdominal pain (P=0.04), low back pain (P=0.02), burning micturition (P<0.05) and unprotected sex (P=0.008).

Table I

Demographic and clinical characteristics in patients with (n=64) and without (n=453) Chlamydia trachomatis infection from patients with urogenital infections

Characteristics	C. trachomatis positive (%), (n=64)	C. trachomatis negative (%), (n=453)	P	Crude OR	95% CI
Demographic characteristics
Sex
Male	30 (46.9)	163 (36.0)	0.14	0.67	0.39-1.15
Female	34 (53.1)	290 (64.0)
Age group (yr)
<30	28 (43.8)	202 (44.6)	0.7	1.21	0.62-2.35
30-34	16 (25.0)	96 (21.2)
35+	20 (31.3)	155 (34.2)
Education level
Illiterate	13 (20.3)	78 (17.2)	0.67	0.85	0.38-1.97
Primary	20 (31.3)	141 (31.1)
High school	29 (45.3)	157 (34.7)
University	2 (3.1)	77 (17.0)
Clinical characteristics
Cervicitis*
Yes	34 (53.1)	290 (64.0)	0.6	1.11	0.65-1.88
No	30 (46.9)	163 (36.0)
Urethritis†
Yes	28 (43.8)	149 (32.9)	0.08	1.58	0.93-2.69
No	36 (56.2)	304 (67.1)
Vaginal discharge*
Yes	27 (42.2)	290 (64.0)	0.007	5.76	1.37-24.09
No	37 (57.8)	163 (36.0)
Abdominal pain*
Yes	9 (14.1)	31 (6.8)	0.04	2.22	1.0-4.92
No	55 (85.94)	422 (93.2)
Low back pain*
Yes	3 (4.7)	67 (14.8)	0.02	0.28	0.05-0.91
No	61 (95.3)	386 (85.2)
Burning micturition
Yes	43 (67.2)	188 (41.5)	<0.05	2.88	1.65-5.02
No	21 (32.8)	265 (58.5)
Pruritis
Yes	29 (45.3)	172 (38.0)	0.25	1.35	0.77-2.37
No	35 (54.7)	281 (62.0)
Dyspareunia*
Yes	11 (17.2)	43 (9.5)	0.06	1.97	0.96-4.07
No	53 (82.8)	410 (90.5)
Risk factors associated
Protection
Yes	2 (3.1)	70 (15.5)	0.008	0.18	0.02-0.69
No	62 (96.9)	383 (84.5)
Sexual history
One partner	50 (78.1)	365 (80.6)	0.4	1.02	0.51-2.06
Two partners	11 (17.2)	78 (17.2)
>Two partners	3 (4.69)	10 (2.2)
Source of infection
ME	51 (79.7)	362 (79.9)	0.9	1.01	0.52-1.94
EME+PME	13 (20.3)	91 (20.1)

*Female patients only; †Male patients only. ME, marital exposure; EME, extramarital exposure; PME, premarital exposure; C. trachomatis, Chlamydia trachomatis

Of the 517 patients, 18.7 per cent (97/517) were positive for Ureaplasma spp., 9.5 per cent (49/517) for M. hominis, 1.2 per cent (6/517) for N. gonorrhoeae, 0.6 per cent (3/517) for T. pallidum and 4.6 per cent (24/517) were HIV seropositive. Co-infection with Ureaplasma and M. hominis was detected in 6.2 per cent (32/517) of patients by culture and/or PCR. U. parvum (biovar 1) was detected in 84.0 per cent (81/97) and Ureaplasma urealyticum was detected in 16.5 per cent (16/97) of patients. None of the patients were infected with both biovars. U. parvum isolates were further subtyped into different serovars. Serovar 3/14 (58.0%; 47/81) was the most frequent isolate followed by serovar 1 (27.2%; 22/81) and serovar 6 (14.8%; 12/81).

Multivariate analysis was done to find the possible association of C. trachomatis co-infection with other STDs (Table II). The rate of co-infection of C. trachomatis with HIV [18.8% (12/64); P=0.001; OR=8.48; 95% CI=3.27-21.68] and Ureaplasma spp. (17.2% (11/64); P=0.02; OR=2.27; 95% CI=0.98-4.86) was significantly associated, whereas no significant association was found between M. hominis (21.9%; 14/64), N. gonorrhoeae (3.1% ; 2/64) and T. pallidum (4.7% ; 3/64 with C. trachomatis infection.

Table II

Chlamydia trachomatis co-infection with genital mycoplasmas, Treponema pallidum, HIV and Neisseria gonorrhoeae

C. trachomatis infection
Co-infecting pathogens	Positive (%) (n=64)	Negative (%) (n=453)	Total (n=517)	P	OR	95% CI
Ureaplasma spp.
Yes	11 (22.4)	38 (77.6)	49 (100)	0.02	2.27	0.98-4.86
No	53 (11.3)	415 (88.7)	468 (100)
Mycoplasma hominis
Yes	14 (14.3)	84 (85.7)	98 (100)	0.52	1.23	0.60-2.39
No	50 (12.0)	369 (88.0)	419 (100)
Treponema pallidum
Yes	3 (100)	0	3 (100)	_	_	_
No	61 (11.9)	453 (88.1)	514 (100)
HIV
Yes	12 (50.0)	12 (50.0)	24 (100)	0.001	8.48	3.27-21.68
No	52 (10.5)	441 (89.5)	493 (100)
Neisseria gonorrhoeae
Yes	2 (100)	0	2 (100)	_	_	_
No	62 (12.0)	453 (88.0)	515 (100)

Distribution of C. trachomatis serovars by PCR-RFLP: Of the 64 ompA C. trachomatis-positive samples, serovars D (48.4%; 31/64), E (32.8%; 21/64), F (7.8%; 5/64), G (1.6%; 1/64) and I (9.4%; 6/64) were the most common C. trachomatis serovars accounting for 53 and 47 per cent of the infections in women and men, respectively. Serovars in women and men, respectively, included serovars D (26.6%; 17/64 vs. 21.9%; 14/64), E (15.6%; 10/64 vs. 17.2%; 11/64), F (4.7%; 3/64 vs. 3.1%; 2/64), I (6.3%; 4/64 vs. 3.1%; 2/64) and G (0.0% ; 0/64 vs. 1.6% ;1/64) (Figure A and B).

An analysis was done to identify the possible relationship between C. trachomatis serovars with demographic and clinical manifestations. However, no significant association was found except for the serovar E with lower abdominal pain (P=0.01) in women and serovar D with smoking (P=0.03) in men.

Sequencing of ompA gene: Optimization of the DNA sequence analysis was performed using reference DNA for C. trachomatis serovars D, E, F, G, H, I, J and K. Of the 64 C. trachomatis-positive cases, 31 (48.4%) isolates were randomly selected for sequencing. Of which, 22 (71.1%) C. trachomatis isolates were successfully sequenced and analyzed. The sequence data showed no sign of mixed infections. Variability in ompA sequences was seen in seven isolates (31.8%). Of these seven C. trachomatis isolates, five (71.4%) resulted in point mutations (one sample of serovar D and serovar I and two samples of serovar E). Insertion was observed in one (12.5%) of serovar I and silent mutation was observed in two isolates (25%) of serovar D (Table III).

Table III

Mutation in seven clinical samples from patients with urogenital infections at baseline compared with ATCC reference strains

Serial number	Serovar	Number of nucleotide changes	Nucleotide change	Position (bp)	Accession number
1	D	2	C---T	841	KP015820
			C---T	961
2	D	2	T---A	681	KP015821
			A---G	729
3	D	3	A---T	678	KP015822
			T---A	704
			T---G	738
4	E	1	A---G	622	KP015823
5	E	2	A---T	622	KP015819
			T---A	740
6	I	2	T---A	750	KP015818
			A---G	941
7	I	2	G---A	1006	KP015824
			T---A	1031

On the basis of sequence similarity of ompA gene, phylogenetic analysis was performed to find out the evolutionary relationship among 22 C. trachomatis isolates. In B-complex, among 12 C. trachomatis isolates of serovar D, eight clinical samples were identical to the reference strain D/UW-3/CX, whereas the other four were closely related, showing 98 per cent of identity. Among the three isolates of serovar E, two were identical to reference strain E/UW-5/CX, showing 98 per cent of identities, whereas the other isolate showed 94 per cent of identity which was closely related. In F/G group, one isolate of serovar F and one strain of serovar G were identical to the reference strain F/IC-CAL3 and G-UW57, showing 97 per cent of identity. In C-complex, only four clinical isolates of serovar I were identified and all were closely related to the reference strain I/UW-12, showing 95 per cent of identity.

Discussion

Genitourinary tract infections due to C. trachomatis are a major cause of morbidity in sexually active individuals13, and women carry the major burden of the disease14. In the present study, C. trachomatis was found in 12.3 per cent samples by PCR assays; similar detection rates of C. trachomatis in patients with urogenital infections have been reported in previous studies from developing countries15. In our study, the highest rate of chlamydial infections was found in ≤30 yr of age group. This is the sexually active group and more vulnerable to sexually transmitted infection (STI) acquisition1617. A multivariate analysis showed that the most common presenting symptoms which were significantly associated with C. trachomatis-infected women were vaginal discharge, low back pain and dysuria. The clinical presentation was similar to those described in previous studies1819.

Ureaplasma spp. and M. hominis have been implicated in a variety of clinical conditions primarily related to lower genital tract colonization and infection. In the present study, 28.2 per cent of patients with urogenital infections were infected by genital mycoplasmas. Similar rates of infection have been reported by others2021. Among the Ureaplasma isolates, U. parvum (Biovar 1) was the most prevalent and serovar 3/14 was the most frequent serovar detected, suggesting a possible pathogenic role of U. parvum serovar 3/1411.

The non-ulcerative STIs caused by C. trachomatis and genital mycoplasmas, namely Ureaplasma spp., and M. hominis, potentially increase the susceptibility of acquiring and transmitting HIV22. Our study highlighted the importance of early laboratory diagnosis and specific treatment of these agents as these increase the risk of transmission many folds when exist together.

To develop epidemiological data and to detect multiple serovar infections, accurate and specific typing of C. trachomatis isolates is required23. Sequencing of the amplified ompA gene, which encodes the MOMP, is currently considered to be more sensitive, and more specific methods are available for identifying C. trachomatis serovars24.

In our study, serovar D was found to be the predominant serovar followed by serovars E, F, I and G. A similar distribution of C. trachomatis serovars has been reported2325, whereas others have shown serovar E as the most prevalent one worldwide26. In a study by Gita et al27, serovar D was found to be the most predominant serovar amongst patients with urogenital infections, followed by E, F and I. However, in another study, serovar E was found to be the most predominant followed by genovars D and F in patients with infertility28. In our study other serovars H, J and K of C. trachomatis were not found which have been reported by other workers2629.

Clinical symptoms were analyzed for possible associations with particular C. trachomatis serovars. Serovar E was significantly associated with lower abdominal pain in women, which was in contrast with previous studies which reported that women infected with intermediate group F/G genotypes more often complained of lower abdominal pain425. Most studies investigating the association between C. trachomatis serovars and clinical symptoms of infection showed contradictory results30.

Sequencing for a subset of 22 C. trachomatis isolates was performed and mutations were observed. However, no visible trace of recombination was found in ompA in our genetic variants compared to the respective prototype strain, suggesting that the genetic variability observed in our study was strictly a consequence of the occurrence of point mutations. A minor sequence variation was observed within the genotypes in our clinical isolates. Single-nucleotide variation was observed in one isolate of serovar E, which made it difficult to exclude the possibility of sequencing artefacts. However, two to three nucleotide substitutions were observed in five isolates, which indicated that these were accurate. Two (28.6%) of the seven C. trachomatis isolates were synonymous (i.e. silent), which suggested that these were evolutionary neutral.

In conclusion, patients infected with C. trachomatis have a significant risk of being infected with other STIs, namely U. urealyticum and HIV, suggesting screening of these agents along with C. trachomatis. Genotyping of the ompA gene of C. trachomatis isolates could be useful for epidemiological characterization of circulating C. trachomatis strains in the community and could provide additional information for vaccine development.