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SHORT COMMUNICATION
Year : 2014  |  Volume : 3  |  Issue : 1  |  Page : 66-69

Simple, non-invasive and cost-effective method for detection of Chlamydia trachomatis infection (a silent, sexually transmitted pathogen that can cause infertility)


Department of Microbiology, Sri Aurobindo Institute of Medical Sciences Medical College and PG Institute, Indore, Madhya Pradesh, India

Date of Web Publication15-Apr-2014

Correspondence Address:
Trupti Bajpai
Department of Microbiology, Sri Aurobindo Institute of Medical Sciences Medical College, MR 10 Crossing, Indore Ujjain Road, Indore, Madhya Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2278-344X.130623

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  Abstract 

Background: Chlamydia trachomatis (C. trachomatis), an obligate intra-cellular bacterial pathogen has been recognized as one of the major cause of sexually transmitted infections throughout the world. Being asymptomatic, these infections have severe ramifications for the reproductive health of women leading to the long term complications like infertility. Screening women for C. trachomatis is highly desirable in developing countries. Aims: To determine the prevalence of Chlamydia trachomatis infection in infertile women attending In vitro fertility (IVF) centers. Also to highlight the importance of non-invasive serological diagnostic tool for screening infertile women to rule out Chlamydia as one the causes of infertility. Materials and Methods: The present study was carried out in the Microbiology section of Central Laboratory of a tertiary care hospital from Sep. 2012 to August 2013. Serum samples from 128 suspected women patients were diagnosed by C. trachomatis IgG ELISA (Calbiotech). Positive and borderline positive samples were retested after one month of initial testing by the same method. Results: Out of 128 serum samples tested, six (4.68%) samples were found to be positive for C.trachomatis IgG while seven (5.46%) samples were borderline positive. Paired sera-analysis finally confirmed 8.59% sero-positivity. Conclusion: IgG antibody detection is an effective and non-invasive tool for the detection of Chlamydia and more viable option than other techniques in India. Screening of women with secondary infection for C. trachomatis is strongly recommended to allow early therapeutic interventions. Since, a significant proportion of women expressed the evidence of exposure to C.trachomatis in our study; this cannot be ignored as one of the probable cause of infertility in women.

Keywords: Chlamydia trachomatis , ELISA, Infertility


How to cite this article:
Bajpai T, Bhatambare G S, Shrivastava G, Patel K B. Simple, non-invasive and cost-effective method for detection of Chlamydia trachomatis infection (a silent, sexually transmitted pathogen that can cause infertility). Int J Health Allied Sci 2014;3:66-9

How to cite this URL:
Bajpai T, Bhatambare G S, Shrivastava G, Patel K B. Simple, non-invasive and cost-effective method for detection of Chlamydia trachomatis infection (a silent, sexually transmitted pathogen that can cause infertility). Int J Health Allied Sci [serial online] 2014 [cited 2020 Feb 22];3:66-9. Available from: http://www.ijhas.in/text.asp?2014/3/1/66/130623


  Introduction Top


Chlamydia trachomatis (C. trachomatis) is a gram negative, obligate, intra-cellular, pathogenic bacterium with a bi-phasic developmental cycle. [1],[2],[3] Chlamydial infections in women have major epidemiological and clinical significance and are usually asymptomatic up to 80%. [4],[5],[6] Genital Chlamydial infections are currently, the most prevalent among the sexually transmitted infections throughout the world. Urogenital infections due to C. trachomatis are frequently in apparent. Most of the infections run an insidious and chronic course, which may persist for months to years causing irreversible tissue damage in unidentified and untreated cases. [7] Especially, in women, if left untreated, lower genital tract infections may ascend to upper genital tract and can cause potentially fatal ectopic pregnancies, pelvic inflammatory disease (PID) and serious clinical conditions like infertility. [3],[4],[5],[6],[8],[9],[10]

Many women aren't even aware that they were exposed to it, until they try to have a baby and can't. The bacteria often go undetected, but are easy to test for and treat.

Screening programs usually aim at reducing morbidity in individuals by early detection and treatment, and at decreasing to overall prevalence of infection in the population. [5] Although national screening programs are in place in developed countries, such programs are not existent in most of the developing countries even in high risk population such as sex workers. [4] Even, in the country like India, such practice has not been adopted probably due to lack of information on the magnitude of this problem. [11] However, considering the high prevalence of infection in India, there is an urgent need to design tests that are simple, inexpensive and can be used to improve diagnosis as well as specificity of the syndromic management. [4]


  Matrials and Methods Top


The present study was carried out from September 2012 to August 2013 in the Microbiology section of the Central Laboratory located in the heart of a tertiary care hospital. One hundred and twenty eight infertile female patients of reproductive age group seeking help in "In Vitro Fertility" (IVF) center of our multi-super specialty tertiary care hospital were considered for study. The study protocol was approved by the institutional ethical committee. Aseptically collected blood sample from the study subjects were kept at room temperature for 20 minutes. After clotting, the clot was retracted and centrifuged. Sera were separated and stored at -20 o C until tested. [6]

Determination of Immunoglobulin G (IgG) antibody against C. trachomatis was done using Enzyme Linked Immunosorbant Assay (ELISA) Kit (CALBIOTECH). [12] The sensitivity of the test was 93.8% and specificity was 92.2%. All assays and calculations were performed according to the manufacturer's instructions.

Diluted serum was added to wells coated with purified lipo polysaccharide antigen from Chlamydia strains. IgG specific antibodies, if present binded to the antigen. All the unbound material was washed away by using wash buffer. The micro-titer assay used peroxidase-conjugated IgG antibodies to bind to antigen-antibody complexes. Again, the excess enzyme conjugate was washed off using wash buffer. After incubation with tetra-methyl benzidine (TMB) substrate, the reaction was stopped by the addition of sulphuric acid (stop solution). The absorbance or optical density (OD) was read photometrically at 450 nm. by ELISA-reader within 15 minutes. The intensity of the color generated was proportional to the amount of IgG specific antibody in the sample.

Calculation of cut-off value for a certain set of ELISA test was done by multiplying calibrator OD with calibrator factor value mentioned on the calibrator bottle. The antibody-index for the patient's sample was calculated by dividing sample OD by cut-off value. The antibody-index value for a certain sample was considered as positive when it was greater than 1.1 and negative when it was less than 0.9. If the value ranged in between 0.9 and 1.1 then it was considered as borderline positive. [3],[4]

The positive samples were retested by the same method after 1 month of the initial test.


  Results Top


The present study was undertaken to determine the prevalence of C. trachomatis antibodies infection in females attending IVF center, because of infertility. Additional aim was to highlight the importance of non-invasive serological diagnostic tool for screening the women patients in order to rule out Chlamydia as one of the causes of infertility.

Out of the 128 serum samples considered for study, six (4.68%) samples were found to be positive and seven (5.46%) samples were borderline positive by ELISA IgG antibody detection test. On retesting after one month, out of six positive samples, five samples showed a four-fold rise in titer, while one sample showed a reduction in titer giving a borderline positive result. Out of seven borderline positive samples, five samples showed a rise in antibody titer while two samples showed negative results.


  Discussion Top


Due to their obligate intracellular nature, the detection and manipulation of Chlamydia have proved challenging. [2] The large pools of asymptomatic infected women are not only at the risk of developing serious long-term sequel but would also transmit the infection. Diagnosis of Chlamydial infection is even more difficult in asymptomatic and in chronic or persistent infections where the pathogen load would be low. [4]

Immunoglobulin G antibody detection is an effective and non-invasive tool for the detection of Chlamydia and a more viable option than other techniques in India. [5] Chlamydia antibody testing (CAT) is a simple blood test, technically simple to perform, less expensive and causes little inconvenience to patients. [7],[9] Our results were found to be comparable with the results of Dwibedi et al. (7.04%) in symptomatic women in Odisha, Moaiedmohseni et al. (5% of all patients and 10% of infertile women), WHO, Stamn, Patel et al., Imai et al. and Satterwhite et al. (ranging from 3-9%). [2],[9],[11] All the positive cases in our study can be assumed to having past exposure to genital Chlamydial infection. [7],[13] All the six IgG positive samples and seven borderline positive samples that were retested after one month of initial test provided reasonable inferences. Out of six positive samples, five samples showed a four-fold rise in titer, confirming the Chlamydial infection, while one sample showed a reduction in titer giving a borderline positive result. This may be because the patient would have been in peak of infection during the initial testing and the IgG titer would have reduced during the second testing. Out of seven borderline positive samples, five samples showed a rise in antibody titer while two samples showed negative results means, these two samples had produced false positive results during the initial testing. That means overall sero-positivity detected after paired sera analysis was 8.59% almost similar to other studies. [2],[9],[11]

Estimation of IgG titers serve useful in clinical situations like ascending upper genital tract infections with C. trachomatis. The elevated antibody titer in single serum is diagnostically suggestive of deep-seated Chlamydial infections involving severe upper genital tract complications such as PID and Infertility. Rise in antibody titer on paired sera analysis confirmed the diagnosis of C. trachomatis infection in infertile women patients. This can be further confirmed by invasive and highly sensitive diagnostic tools like Hysterosalpingography (HSG) or Laparoscopy. Serological tests are also useful in identifying Chlamydial etiology in ascending upper genital tract infections where direct and specific tests fail to identify the organism. Non-invasive serological testing reduces the risk of introducing infections to the upper genital tract by avoiding procedures that involve instrumentation such as HSG or Laparoscopy. [7]

Chlamydia can cause both adnexal adhesions as well as tubal obstruction. High Chlamydia antibody titers are commonly observed in patients with adnexal adhesions. These are also associated with inflammatory tissue damage and increased risk of tubal pregnancy. Hence, women with high IgG titer should have laparoscopy. This is because such complications are best picked up by more sensitive but invasive laparoscopy instead of less sensitive and less invasive HSG. However, HSG combined with antibody titers can reduce unavoidable false positive and false negative results significantly and therefore are best used in patients with low antibody titers. [3],[9]

Thus, studies show that the choice of the invasive diagnostic tests for tubal assessment should depend upon the values of antibody titer of the CAT, and hence, should be done after the results of CAT are known. [3],[7],[10]

Paired sera analysis is useful in confirming the positive results and avoiding false positive results. However, the test has limitation like its negative result can only rule out C. trachomatis as the cause of infertility but it cannot detect infertility due to other microorganisms. Also, the test usually, non-specifically detects antibodies produced on exposure to other species of Chlamydia.

Therefore, to conclude, IgG antibody detection is an effective and non-invasive tool for the detection of Chlamydia and a more viable option than other techniques in India. [5] C. trachomatis antibody testing should be preferred as a routine baseline investigation in infertility clinics. [9] Screening of infertile women for C. trachomatis is recommended for early therapeutic options. [5],[11]


  Acknowledgment Top


The authors wish to thank the Chairperson and Dean of the institute for providing laboratory facilities and healthy working atmosphere during the study period. The authors are also thankful to the technical staff of the institute for providing necessary helping hand during the endeavor.

 
  References Top

1.Paniker CK, Ananthanarayan R. Textbook of Microbiology. 8 th ed. Universities Press (India) Pvt. Ltd.; Hyderabad, 2013.  Back to cited text no. 1
    
2.Demetra S, Bleotu C, Miron N, Socolov R, Boiculese L, Mares M, et al. Correlation between Chlamydia trachomatis Igg and Pelvic Adherence Syndrome. In: Mihai M, editor. Chlamydia, Publisher Intech., 2012.  Back to cited text no. 2
    
3.Keyhani AH, Nazer M, Mirsalehian A. Evaluation of serum chlamydial igg antibody in women with tubal factor infertility. Res J Med and Med Sc 2006;1:150-3.  Back to cited text no. 3
    
4.Patel AL, Sachdev D, Nagpal I, Choudhry U, Sonkar SC, Mendiratta SL, et al. Prevalence of chlamydia infection among women visiting a gynecology outpatient department: Evaluation of an In-house PCR assay for detection of Chlamydia Trachomatis. Ann Clin Microbiol Antimicrobiol 2010;9:1-10.  Back to cited text no. 4
    
5.Pratibha G, Joseph P, Innocent D, Prabhu N. Prevalence of chlamydia trachomatis infection in women in Chennai, India. Ann Biol Res 2010;1:76-81.  Back to cited text no. 5
    
6.Saleem M, Innocent D, Joseph P, Prathiba G. Detection of Chlamydial Igm and igG antibodies in associated infertile population. J Adv Res Biol Sc 2013;5:96-9.  Back to cited text no. 6
    
7.Joyee AG, Thyagarajan SP, Vikram Reddy E, Rajendran P, Venkatesh C, Ganapathy M. Diagnostic utility of serological markers for genital chlamydial infection in std patients in Chennai, India. J Asso Physicians India 2007;55:777-80.  Back to cited text no. 7
    
8.Malik A, Jain S, Hakim S, Shukla I, Rizvi M. Chlamydia trachomatis infection and female infertility. Indian J Med Res 2006;123:770-5.  Back to cited text no. 8
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9.Moaiedmohseni S, Owje M. The value of chlamydia trichomatis antibody testing in prediction of tubal factor infertility. J Family and Reproductive Health 2007;2:29-32.  Back to cited text no. 9
    
10.Idahl AC. Trachomatis as a risk factor for infertility in women and men, and ovarian tumor development, 2009. Umeå University Medical Dissertation; New Series No 1255 ISSN 0346-6612 ISBN 978-91-7264-759-6.  Back to cited text no. 10
    
11.Dwibedi B, Pramanik JM, Sahu P, Kar SK, Moharana T. Prevalence of genital chlamydia infection in females attending an Obstetrics and Gynecology outpatient department in Orissa. Indian J Dermatol Venerol Leprol 2009;75:614-16.  Back to cited text no. 11
    
12.Poussin M, Fuentes V, Corbel C, Prin L, Eb F, Orfila J. Capture- ELISA: A new assay for the detection of immunoglobulin M isotype antibodies using chlamydia trachomatis antigen. J Imminol Methods 1997;204:1-12.  Back to cited text no. 12
    
13.Numazaki K. Serological tests for chlamydia trachomatis infections. Clin Microbiol Rev 1998;11:228-9.  Back to cited text no. 13
    



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Abstract
Introduction
Matrials and Methods
Results
Discussion
Acknowledgment
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