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 ~  Abstract
 ~ Introduction
 ~  Materials and Me...
 ~ Results
 ~ Discussion
 ~ Conclusion
 ~  References
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  Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 38  |  Issue : 3  |  Page : 319-323
 

Detection of chlamydia trachomatis infection among the pregnant women attending a tertiary care hospital in Kerala - South India by polymerase chain reaction


1 Department of Microbiology, Azeezia Institute of Medical Sciences and Research, Kollam, Kerala, India
2 Department of Microbiology, SR Medical College and Research Foundation, Thiruvananthapuram, Kerala, India

Date of Submission04-Aug-2020
Date of Decision09-Mar-2020
Date of Acceptance11-Aug-2020
Date of Web Publication4-Nov-2020

Correspondence Address:
Dr. A Neena
Department of Microbiology, Azeezia Institute of Medical Sciences and Research, Kollam - 691 537, Kerala
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmm.IJMM_19_429

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 ~ Abstract 


Background: Chlamydia trachomatis infection is the most prevalent bacterial sexually transmitted infection and may influence pregnancy outcome. Aims and Objectives: This study was conducted to assess Chlamydial infection during pregnancy by PCR. Materials and Methods: Study group consists of patients who are attending the antenatal clinics. Endocervical swabs were collected from 300 patients. Results: Off the 300 samples tested, 29 were positive as per PCR which used CT F : 5' CGT GTC GGC AAT CCT GCT GAT 3' and CT R : 5' GTC GAT AAC ATA GTC ACG ATA GTC 3'as the primers. Conclusion: This suggests there is a prevalence of Chlamydia trachomatis in our population which is 10%. Hence, it should be noted as a significant public health problem especially among sexually active young women of child bearing age. Timely detection and prompt treatment of Chlamydial infection during pregnancy can eliminate its adverse outcomes.


Keywords: Chlamydia trachomatis, enzyme-linked immunosorbent assay, IgM antibodies, micro immunofluorescence, polymerase chain reaction, pregnancy, sexually transmitted infections


How to cite this article:
Neena A, Deepa R. Detection of chlamydia trachomatis infection among the pregnant women attending a tertiary care hospital in Kerala - South India by polymerase chain reaction. Indian J Med Microbiol 2020;38:319-23

How to cite this URL:
Neena A, Deepa R. Detection of chlamydia trachomatis infection among the pregnant women attending a tertiary care hospital in Kerala - South India by polymerase chain reaction. Indian J Med Microbiol [serial online] 2020 [cited 2020 Nov 28];38:319-23. Available from: https://www.ijmm.org/text.asp?2020/38/3/319/299805





 ~ Introduction Top


Chlamydia trachomatis is an obligate intracellular bacterium. It can cause mild asymptomatic as well as symptomatic infections that can lead to adverse outcomes in pregnancy. C. trachomatis is a known cause of preterm labor, premature rupture of membranes, low birth weight and still birth.[1] Mandatory screening tests should be done in patients from poor socioeconomic backgrounds and bad obstetric history to give timely treatment and to avoid adverse pregnancy outcomes. The incidence of C. trachomatis infection has dramatically increased during the past 10 years.[2]Chlamydial Pelvic inflammatory disease is the most important preventable cause of infertility and adverse pregnancy outcome.[3] The Centers for Disease Control and Prevention (CDC), the US Preventive Services Task Force and other agencies and professional organisations have guidelines recommending annual Chlamydia screening of young, sexually active women aged <25 years and at-risk older women.[4] Similar study conducted by Illan Cohen et al. suggesting the need for repeated Chlamydial testing during antenatal checkup to prevent adverse outcomes during pregnancy.[5] The greater sensitivity of nucleic acid amplification tests (NAATs) for C. trachomatis can increase the reach and decrease the costs of public health screening programs aimed at controlling C. trachomatis infection. The increased sensitivity of NAATs are due to their ability to produce a positive signal from as little as a single copy of the target DNA or RNA.[3] NAATs are having high specificity and sensitivity so that it can be used for the diagnosis of C. trachomatis genital infections.[2],[6],[7]


 ~ Materials and Methods Top


The study was carried out during the period of 2016 March to November 2018, and it was approved by the Institutional Ethical Committee of Azeezia Medical College Hospital. Written informed consent from each patient was also obtained. A total of 300 symptomatic patients aged between 18 and 39 years, visiting the gynaecology department, were enrolled for the study. Pregnant women who were reported to have signs such as severely eroded cervix with hypertrophic cervical erosions, mucopurulent endocervical discharge and symptoms such as burning micturition and abdominal pain were included under this study. Patients who were reported to have other co-morbidities were excluded from the study. Obstetric history of patients is given in [Table 1].
Table 1: Obstetric history of patients (n=300)

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Symptomatic patients were screened for the presence of C. trachomatis specific IgM antibodies by both Enzyme linked immunosorbent assay (ELISA) and Microimmunofluorescence (MIF) and the positive cases were confirmed with Polymerase chain reaction (PCR).

Sample collection

A total of 300 endocervical swabs and blood samples were collected from women who were attending antenatal clinics. The endocervical swabs for PCR were collected using sterile Dacron swabs and were transported to the laboratory in Amplicor Transport kit (Roche) maintaining the cold chain.[8] The blood samples were centrifuged at 5000 g for 7 min and then their serums were collected in 1.5 ml microtubes and used for the detection of C. trachomatis specific IgM antibodies by both MIF and ELISA.

Screening tests

Micro immuno fluorescence

The Savyon Chlamydia Sero FIA kit (Savyon Diagnostics, Israel) was used in our study. It is a micro-IF assay.[9] Purified elementary bodies of C. trachomatis (L2) were used as the antigen.

Enzyme-linked immunosorbent assay

Novatec C. trachomatis IgM kit (Germany) was used to detect IgM antibodies. The ELISA was based on a synthetic peptide from the immunodominant region of the major outer membrane protein.[10]

Procedure of polymerase chain reaction

Endocervical swabs are treated with a detergent solution to release Chlamydial DNA contained in the chlamydial reticulate bodies. A second detergent solution is then added to prepare the lysed specimen for amplification. The AMPLICOR CT/NG Test for C. trachomatis is based on four major processes specimen preparation; PCR amplification of target DNA using CT specific complementary primers; hybridisation of the amplified DNA to oligonucleotide probes specific to the target(s); and detection of the probe bound amplified DNA by colorimetric determination.[11]

DNA extraction

DNA was extracted from samples using Qiagen DNA extraction kit. Extraction was carried out according to the manufacturer's protocol. The primer sequence is given in [Table 2]. The PCR was done using 95°C initial denaturation, 94°C Final denaturation, 55°C annealing, 72° C extension with 35 cycles and 72°C final extension by agarose gel electrophoresis. The positive bands were obtained at 450 bp. In addition to chromosomal DNA, C. trachomatis contains an approximately 7,500 base pair cryptic plasmid that is common to all serovars of C. trachomatis.[12] The AMPLICOR CT/NG test for C. trachomatis uses the biotinylated primers CP24 and CP27 to define a sequence of approximately 207 nucleotides within the cryptic plasmid DNA of C. trachomatis. For performing the sequencing of the obtained C.trachomatis DNA, positive bands were taken and subjected for cycling sequencing, followed by Sanger sequencing, since cycling sequencing can enhance the sequence.[13]
Table 2: Primer sequence

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Optimisation of reaction conditions several parameters were varied for each primer set to obtain optimal reaction conditions. Annealing temperatures of 37, 42, 48, 55 and 60°C were tested. The following MgCl2, primer and oligonucleotide concentrations were analysed in all possible combinations: MgCI2, 1–10 mM; primers, 0.2, 0.5, 1.0 and 2.0 uM; and oligonucleotides, 20, 50, 100 and 200 R, M.[13]

Statistical analysis

Data were analysed using Statistical Analysis Systems software IBM Corp. Released 2011. IBM SPSS Statistics for windows, Version 20.0. Armonk, NY:IBM Corp. Chi-square was used to assess differences in proportions and P < 0.05 were considered statistically significant.

The value of the Chi-square Statistic is 7.824 and the P < 0.05 (<0.020). This clearly indicates that there is a statistically significant association between the ages group (20–25) and test results.


 ~ Results Top


Out of the 300 blood samples, 48 and 42 samples were positive for C. trachomatis specific IgM antibodies by MIF and ELISA, respectively. From the C. trachomatis specific IgM antibody-positive cases (48), 29 were positive by PCR [Figure 1] and [Table 3]. The sensitivity of PCR was found to be 95.7% and specificity was 100%.[8] Present study giving information about the prevalence of genital tract infections caused by C. trachomatis. The demographic characteristics of patients with C. trachomatis infection (PCR positive) is given in [Table 4]. The adverse outcomes noted in the current study were abortion, premature rupture of membrane, preterm birth and low birth weight.
Figure 1: Polymerase chain reaction gel picture; 1-control, 2,3,4-samples-1,2,3

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Table 3: Association between polymerase chain reaction, migration inhibitory factor, ELISA and age groups

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Table 4: Demographic characteristics of Chlamydia trachomatis (Polymerase chain reaction positive) patients

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The value of the Chi-square Statistic is 7.824 and the P < 0.05 (<0.020). This clearly indicates that there is a statistically significant association between the age groups (20–25) and test results.


 ~ Discussion Top


C. trachomatis is a highly prevalent sexually transmitted diseases in many regions, and it seriously affects public health; therefore it needs effective epidemiological control, starting with an adequate method for correct and effective diagnosis. In the current study, there was an overall prevalence of 10% of C. trachomatis infection was observed among pregnant women. The major infection was found among women in the age group of 20–25 and was found to be 59% followed by 26–30, and its prevalence was 34% [Table 3]. A study conducted by Singh et al., 2003, from New Delhi showing the highest prevalence rate (28%) of Chlamydial infection in younger women.[14] As per the CDC, women ≤ 25 years are at an increased risk for C. trachomatis infection.[15] Another report from Germany by Ohman et al., 2009, also reveals the presence of C. trachomatis infection among pregnant women.[16] Similar study conducted by Patel et al., 2010, recorded a high occurrence of 23% C. trachomatis infection in women attending antenatal clinics in New Delhi.[17] In all the age groups, there were nulliparous and multiparous patients. In contemporary study, the maximum infection rate was found among multiparous women. A similar study conducted in Iran also showing the highest incidence among multiparous women of reproductive age group.[18] Our study shows C. trachomatis infection was prevailing among patients who are living under low socioeconomic status as well as with low or intermediate education. Another study conducted by Götz et al. reveals the frequency of C. trachomatis infection among patients living under low socioeconomic status and low education.[19] Patients with foremost symptomatic conditions such as severely eroded cervix with hypertrophic cervical erosions, mucopurulent endocervical discharge, burning micturition and abdominal pain were showed positivity of 59% (20–25 age group) and 34% (26–30 age group). The adverse outcomes such as abortion, premature rupture of membrane, preterm birth and low birth weight were significantly correlated with the studies of Ahmadi et al., Mårdh and Silva et al.[20],[21],[22]

IgM antibody detection by MIF and ELISA remains the front line assays as it is preferred for large serological studies, especially in resource-poor settings.[23],[24] In the current study, out of the 300 patients, 16% and 14% were positive for C. trachomatis specific IgM by MIF and ELISA. The Microimmunofluorescence test (MIF) is usually considered as a gold standard in the serological diagnosis of C. trachomatis infection.[22],[25] According to Silva et al., the MIF test is time-consuming and labor-intensive, and the reading of the fluorescent signals is prone to subjective evaluation.[26] Since ELISA tests are easier to carry out, less expensive, and able to be read more objectively than the MIF assay, they might be good alternatives to the MIF assay for the detection of C. trachomatis antibodies.[20] Thus, the affordability, easy application and high throughput of the immunoassay, makes it a practical screening tool in regions of high C. trachomatis prevalence and a good substitution for the more sensitive and specific PCR, especially low-income countries.[27] However, Black reported that the presence of C. trachomatis IgM antibodies was an unreliable marker of acute infection in adolescents and adults.[28] The enzyme immunoassay is a commonly used front-line assay for the diagnosis of C. trachomatis infection.[30]

The conventional serological assays are not without limitations as it has been reported that serum antibody is not always consistent with active infection, especially in regions of the high prevalence of C. trachomatis.[29] The presence of antibodies cannot differentiate an acute, chronic or a resolved C. trachomatis infection and cross-reactive antibodies are sometimes produced in response to lipopolysaccharides of other chlamydia species such as C. pneumoniae and Gram-negative bacterial lipopolysaccharides leading to a high number of false-positive results.[30]

Screenings of symptomatic patients (out of 300 patients) were done with MIF and ELISA and the positive cases (48 samples) were confirmed with PCR in the current study. C. trachomatis PCR assays performed on endocervical swab provide the highest sensitivity, thereby leading to an early diagnosis which minimises the risk of disease sequel and continued transmission of infections.[31] Amplicor PCR has been relatively well evaluated for both urogenital and urine specimens, with an overall sensitivity and specificity of 90 and 99%–100%, respectively.[32]

NAATs are more superior for the diagnosis of C. trachomatis infection.[33] Similar detection rates of C. trachomatis in infertile women by PCR have been reported in previous studies from developing countries.[34],[35] The efficiency of the PCR technique is influenced by different parameters, the most important of which seems to be the composition of primers, both in relation to each other and in relation to the target DNA, and the temperature at which annealing of primers is performed. However, this is not practicable in resource-poor regions and comparable alternatives are of upmost importance. Hence, immunoassays are frequently used as a practical screening tool in regions with high prevalence C trachomatis genital infections.[36]

Studies reported from India also testify the prevalence of genital C. trachomatis in a higher range of 4, 14, 19.9 and 28.5% percent, respectively.[37],[38],[39],[40] Our study highlighted the importance of early laboratory diagnosis and specific treatment of these agents as these increase the risk of transmission.


 ~ Conclusion Top


We conclude that the PCR technique is a valuable tool for diagnosing genital C. trachomatis infections because of its high sensitivity and specificity. With these results, we can say that pregnant women under 25-year-old and low economic resources are one of the populations in which the screening programs of C. trachomatis should focus. The prevalence of C. trachomatis infection among fertile women detected by molecular methods seems to be a good alternative. By considering the high specificity and sensitivity of PCR, it could be used as a screening technique during the antenatal check-up. However, this suggests that ELISA is a practical and cheaper alternative to the molecular tests for routine screening and diagnosis, especially in resource-poor countries.

Acknowledgement

We would like to thank all patients who were voluntarily participated in this study. We also acknowledge the cooperation of the Principals, HODs and Faculties of Departments of Microbiology and Obstetrics and Gynaecology of Azeezia Institute of Medical Sciences and Research for allowing us to conduct the study. We are also thankful to all medical and paramedical staffs of concerned Departments of these Institutions for their valuable support.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
 ~ References Top

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  [Full text]  
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    Figures

  [Figure 1]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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