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 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 3  |  Issue : 2  |  Page : 67-70

Comparative evaluation of two diagnostic methods for the detection of chlamydia trachomatis in endocervical specimens


Department of Medical Microbiology, Faculty of Clinical Sciences, College of Medicine, Kaduna State University, Kaduna, Nigeria

Date of Web Publication19-Mar-2019

Correspondence Address:
Dr. Oluwafemi Temidayo Ige
Department of Medical Microbiology, Faculty of Clinical Sciences, College of Medicine, Kaduna State University, Kaduna
Nigeria
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/archms.archms_11_18

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  Abstract 

Context: Genital infections caused by Chlamydia trachomatis have been a significant healthcare problem recognized throughout the world in recent years. The World Health Organization estimates that approximately two million new cases of genital chlamydial infection occur annually in females. Objectives: The objective was to compare the performance of the glycogen assay test to the polymerase chain reaction (PCR) in the diagnosis of genital chlamydial infection. Materials and Methods: Consenting female patients were consecutively recruited for the study until the sample size of 365 was achieved. Two endocervical specimens were collected from each of the patients using sterile plastic-shaft Dacron swabs. C. trachomatis test kit (Autobio Company China) was used according to the manufacturer's instructions. The test is a biochemical assay for the detection of C. trachomatis in clinical samples taken from the genital tract. PCR was also done for each sample. Data management was done using the Statistical Package for the Social Sciences version 20.0. Results: Of the women studied, 95 (26%) were positive and 270 (74%) negative for genital chlamydial infection using the PCR, while 244 (66.8%) tested positive and 121 (31.2%) tested negative using the glycogen assay. Conclusions: With the results generated above and using the PCR as the standard, sensitivity for the glycogen assay was 86.3%, specificity was 40%, and the predictive value for a positive result was 33.6%, while that for a negative result was 89.3%. The glycogen assay kit is locally available, not expensive (compared to the very high cost of PCR) and often used in the diagnosis of genital chlamydial infection. However, with a sensitivity of 86.3% and specificity of 40% for the glycogen assay, found in this study, the test kit is far from ideal as a tool for the rapid diagnosis of genital C. trachomatis infection, as it will yield high numbers of false-positive results.

Keywords: Chlamydia trachomatis, endocervical specimen, glycogen assay, polymerase chain reaction


How to cite this article:
Ige OT. Comparative evaluation of two diagnostic methods for the detection of chlamydia trachomatis in endocervical specimens. Arch Med Surg 2018;3:67-70

How to cite this URL:
Ige OT. Comparative evaluation of two diagnostic methods for the detection of chlamydia trachomatis in endocervical specimens. Arch Med Surg [serial online] 2018 [cited 2019 Nov 15];3:67-70. Available from: http://www.archms.org/text.asp?2018/3/2/67/254571


  Introduction Top


Chlamydia trachomatis is a small obligate intracellular bacterium transmitted by sexual intercourse.[1] Genital chlamydial infection is manifested in the form of urethritis, mucopurulent cervicitis, endometritis, salpingitis, and pelvic inflammatory disorder. C. trachomatis may also cause infertility, ectopic pregnancy, premature deliveries, and perinatal morbidity; it is associated with cervical metaplasia and atypia and may contribute to the development of cervical carcinoma.[2],[3]

Testing for Chlamydia is indicated in patients with urogenital, anorectal, and ocular symptoms. Sexually transmitted infections (STIs) other than Chlamydia, sexual contacts of persons with STI.[4] At present, there is no specific routine screening or diagnostic test for C. trachomatis infection, which compares favorably with the polymerase chain reaction (PCR) analysis, with regard to sensitivity and specificity. The aim of this study was to compare the performance of an available and affordable C. trachomatis rapid diagnostic test with that of the PCR.


  Materials and Methods Top


Participants were female volunteers aged 18–44 years, attending the General Outpatient Clinic. Women who had taken antibiotics within the preceding 6 weeks were excluded from the study. Ethical approval was obtained from the hospital's research and ethics committee.

Consenting female patients were consecutively recruited for the study, until the sample size of 365 was achieved as calculated using the Fisher formula for sample size determination.[5] Endocervical swabs were collected from the patients using sterile plastic-shaft Dacron swabs. Hand hygiene was performed and the hands gloved after which the patient requested to be in the lithotomy position with the aid of an assistant, with an appropriate-sized speculum obtained from a sterile pack, the cervical os was exposed and two cotton wool swab sticks, one at a time, was introduced 1 cm into the endocervix and rotated 360° once, ensuring not to touch the vaginal walls or the speculum.

One of the swabs was placed immediately into tubes provided, by the manufacturer of the test kit, and the assay was carried out on the same day of collection. The second specimen required for the PCR assay were pooled and stored in a laboratory freezer at −70°C. The molecular analysis of specimen was carried out at the Center for Biotechnology Research and Training, Ahmadu Bello University.

The sample was defrosted and the total DNA was extracted using a commercial DNA extraction kit (DNA Extraction Kit, Zymo Research, USA). Lysate prepared from the endocervical swabs yielded DNA, which was bound to spin columns, washed, eluted, and characterized by agarose gel electrophoresis. PCR assays, using primers obtained from Inqaba Biotechnical industries, South-Africa, were carried out on each endocervical sample.

Nested PCR was used to amplify 236-base pairs (bp) and 205-bp amplicons targeting the VD2 region of C. trachomatis outer membrane protein A (ompA) gene.

Forward outer primer CTS (5'-3'AATATTGGGATCGTTTG).

Reverse outer primer CTA (5′-3'CCCATCCCAAAGCTGC).

Forward inner primer (CTSN 5'-3' TTGATGTATTTGTACAT).

Reverse inner primer (CTAN5'-3' GCTGCCGAGCCCACCT).

The amplified products were observed following electrophoresis in 1.2% agarose gel. The Medicare GELSTAN UV illuminator was used to view DNA, and bands corresponding to the gene of interest on the DNA ladder were documented as positive, where those that do not meet this criterion were recorded as negative.

The 205-bp amplicon of the VD2 region of C trachomatis (Inqaba Biotechnical industries, South-Africa) is specific, and this was determined using the DNA ladder (Inqaba Biotechnical industries, South-Africa) which was ran alongside the PCR product and served as a basis for further identification of the ompA gene. Positive and negative controls were included in the commercial pack containing the primers and DNA ladder and processed together with the amplified products.

Glycogen assay (using the Chlamydia trachomatis test kit by Autobio® China)

The Autobio C. trachomatis test is a biochemical assay for the detection of C. trachomatis, whose invasion into epithelial cells generates a glycogen structure, which reacts with anthrone under acidic condition, and the reaction displays a color change.

Materials provided by the Company test kits include reaction tubes; test tubes containing basal medium; chromogen solution (2 vials, 5.5 ml/each); color reference card. Other materials required include bench top centrifuge and heater capable of maintaining temperature at 80°C.

Assay procedure

The specimen swab was put into the tube containing the basal medium, stirred 3–5 times, and the swab pressed against the side of the tube in order to express both cell and fluid from the swab stick as maximally as possible.

The tube with the expressed media and cells was centrifuged for 10 min at 3000 revolutions/minute. The supernatant was removed and the tube placed upside down on an absorbent paper to dry. 10 drops of chromogen solution was added to the tube and thoroughly mixed. The diluent was then incubated in a water bath at 80°C for 10 min and the color change was observed and documented.

Interpretation of results

The color of the diluent was compared with the color reference card provided. When the yellow color turned green or blue, a positive result is reported and when the yellow color did not change or turned to colors other than green or blue, a negative result is reported.

Quality control

The test kit came with positive and negative controls which were ran along with the samples and used to confirm observed results. The Statistical Package for the Social Sciences, version 20 (SPSS Inc., Chicago, IL, USA) was used for data analysis.


  Results Top


Of the women studied, 95 (26%) were positive and 270 (74%) negative for genital chlamydial infection using the PCR, while 244 (66.8%) tested positive and 121 (31.2%) tested negative using the glycogen assay [Table 1].
Table 1: Comparing results obtained, for genital chlamydial infection, by the use of polymerase chain reaction and glycogen assays

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With the results generated above and using the PCR as the standard, sensitivity for the glycogen assay was 86.3%, specificity was 40%, and the predictive value for a positive result was 33.6%, while that for a negative result was 89.3%.


  Discussion Top


This research is geared toward establishing an appropriate, effective, and sensitive method of diagnosis of genital chlamydial infections within the present set-up. This will enhance the reduction of the morbidity and subsequent complications associated with C. trachomatis infection especially in women, timely diagnosis using an available and affordable rapid diagnostic test kit and the appropriate treatment of chlamydial infections will prevent further transmission and minimize the risk of adverse consequences.

Generally speaking, the documented prevalence of genital chlamydial infections differs considerably depending on the methodologies, (isolation in cell culture, antigen detection using direct fluorescent antibody assay and enzyme immunofluorescent assay), used for the identification of C. trachomatis, leading to different prevalence rates even in similar populations. Amplification methods, as used in this study, have the advantage of a better sensitivity than culture and allows the detection of infection even in individuals with a low number of infectious units, which may be difficult using other methods.[6]

Studies done by Tukur et al., revealed a prevalence of 38.3% among patients with tubal infertility in Northern Nigeria. The diagnostic kit used was the Clinpro rapid one step C. trachomatis diagnostic kit made by Clinpro International Company, Union City, USA.(based on the use of enzyme linked immunosorbent assay (ELISA) to measure immunoglobulin G binding to C. trachomatis antigen).[7]

Coker et al. analyzed sera from patients attending a sexually transmitted diseases clinic, a family planning clinic, and an antenatal clinic in Ibadan, Nigeria, for the presence of type specific antichlamydial antibodies using a modified micro-immunofluorescence test.[8] Their study yielded a prevalence rate of 35%.

Steingrímsson et al. evaluated two rapid immunoassay methods, QuickVue-Chlamydia (Quidel Corp., San Diego California) and Kodak SureCell (Kodak Corp., Rochester, New York)for the detection of C. trachomatis antigen in endocervicalswabs from high- and low-risk females. Using the cell culture of C. trachomatis to compare, the performances of the two immunoassay methods were similar. Both immunoassay methods performed well in high- and low-risk patientgroups, both for symptomatic and for asymptomatic patients.[9]

Using the PCR as the gold standard, the sensitivity and specificity of the glycogen assay for the identification of genital C. trachomatis infection was 86.3% and 40% respectively. This is in agreement with the study done by Chun and Yin,[10] where a sensitivity of 84.8% was documented but the specificity rate of 40% differs from the 96.5% obtained in the Yang study.[10]

This may be because their study used spectrophotometry in the detection of a change in color (indicating the presence of C. trachomatis), as compared to the use of visual color change utilized by the glycogen assay kits (as the means of detecting the presence of C. trachomatis) in this study.[10]

The glycogen assay kit is locally available, not expensive (compared to the very high cost of PCR), and often used in the diagnosis of genital chlamydial infection. Given that this is a resource-limited setting, with regards to PCR assays, it took about 4 weeks to pool the first batch of endocervical samples to get results using the PCR. Compared to the glycogen assay by which results were obtained within 30 min.

The researcher intended to find out if the glycogen test kit could be a useful tool in the diagnosis of genital chlamydial infection.

This will make it easier for the clinician to treat infected individuals immediately, in an attempt to reduce the potential transmission of infection, prevent disease progression and overcome the failure to treat individuals who are tested but may become lost to follow up.

An ideal diagnostic test kit should have a high sensitivity and specificity rate comparable to the gold standard. From various studies the reported sensitivity and specificity of PCR tests for C. trachomatis (using nucleic acid amplification tests [NAATs]) ranged from 88 to 96%.[5] In this study, the Glycogen assay kit, was found to have a sensitivity of 86.3% and specificity of 40%. It may therefore not serve as a good diagnostic tool because of its tendency to give high false positive results.

With a specificity of 40% from the glycogen assay, found in this study, the test kit is far from ideal as a tool for diagnosing genital C. trachomatis infection. However, in this resource limited setting, where the health seeking attitude is poor and the return rate following hospital visits is low, the glycogen assay test kit, with a positive predictive value of 33.7% and a negative predictive value of 89.3%, may just serve as a screening tool bearing in mind that false positive results, not only burdens the diagnostic facilities but may also bring discredit to the screening program. Therefore, all positive results may need to be confirmed possibly with an ELISA based antigen test kit.


  Conclusions Top


This study found out that, using the PCR as the gold standard, the sensitivity and specificity of the glycogen assay for the identification of genital C. trachomatis infection was 86.3% and 40% respectively.

The glycogen assay test kit, having a slightly lower sensitivity than PCR (86.8%) may be a useful tool in the screening of women but it is not recommended as a diagnostic kit in the detection of genital C. trachomatis infection, in resource– limited settings where the PCR is not readily available.

Many new diagnostic tests have emerged for the diagnosis of C. trachomatis infections in recent years. None of them are perfect nor are any of them ideal for use in all situations. Of all diagnostic techniques, NAATs are the most sensitive tests. They also have a high specificity comparable to culture, and therefore represent the method of choice for C. trachomatis detection, however it is very expensive.

Recommendation

More effort is needed to discover affordable rapid diagnostic test kits. These should have high sensitivity and specificity when compared with the gold standard so that infections can be detected and treated early especially in asymptomatic patients to break the cycle of genital chlamydia transmission in the community.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
  References Top

1.
Paavonen J, Eggert-Kruse W. Chlamydia trachomatis: Impact on human reproduction. Hum Reprod Update 1999;5:433-47.  Back to cited text no. 1
    
2.
World Health Organization. Prevalence and Incidence of Selected Sexually Transmitted Diseases. World Health Organization; 2011. Available from: http://www.whqlibdoc.who.int/publications/2017/9789241502450_eng.pdf. [Last accessed 2017 Nov 30].  Back to cited text no. 2
    
3.
Land JA, Van Bergen JE, Morré SA, Postma MJ. Epidemiology of Chlamydia trachomatis infection in women and the cost-effectiveness of screening. Hum Reprod Update 2010;16:189-204.  Back to cited text no. 3
    
4.
Watson EJ, Templeton A, Russell I, Paavonen J, Mardh PA, Stary A, et al. The accuracy and efficacy of screening tests for Chlamydia trachomatis: A systematic review. J Med Microbiol 2002;51:1021-31.  Back to cited text no. 4
    
5.
Araoye MO, editor. Sample size determination. In: Research Methodology with Statistics for Health and Social Sciences. Ilorin, Nigeria: Nathadex; 2003. p. 115-21.  Back to cited text no. 5
    
6.
Black CM. Current method s of laboratory diagnosis of Chlamydia trachomatis infection. Clin Microbiol Rev 1997;10:160-84.  Back to cited text no. 6
    
7.
Tukur J, Shittu SO, Abdul AM. A case control study of active genital Chlamydia trachomatis infection among patients with tubal infertility in Northern Nigeria. Trop Doct 2006;36:14-6.  Back to cited text no. 7
    
8.
Coker G, Adelusi B, Darougar S. Chlamydial genital infection in Ibadan Nigeria. Br J Vener Dis 1982;58:366-9.  Back to cited text no. 8
    
9.
Steingrímsson O, Pawlak C, Van Der Pol B, Turner BP, Hjaltalín Olafsson J, Dolphin L, et al. Multicenter comparative evaluation of two rapid immunoassay methods for the detection of Chlamydia trachomatis antigen in endocervical specimens. Clin Microbiol Infect 1997;3:663-7.  Back to cited text no. 9
    
10.
Chun Y, Yin ZD. Glycogen assay for diagnosis of female genital Chlamydia trachomatis infection. J Clin Microbiol 1998;36:1081-2.  Back to cited text no. 10
    



 
 
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