IMMUNOLOGICAL AND MOLECULAR DETECTION OF CHLAMYDIA TRACHOMATIS AMONG WOMEN AT REPRODUCTIVE AGE ATTENDING OMDURMAN MATERNITY HOSPITAL

IMMUNOLOGICAL AND MOLECULAR DETECTION OF CHLAMYDIA TRACHOMATIS AMONG WOMEN AT REPRODUCTIVE AGE ATTENDING OMDURMAN MATERNITY HOSPITAL

Lemya A. Kaddam ^{* 1}, Mazin O. Mohager ², Adam A. Adam ³ and Mohammed A. Tahan ⁴

Department of Microbiology ¹, Soba Teaching Hospital, University of Khartoum, Khartoum, Sudan.

Department of Pathology ², College of Medicine, Al-Jouf University, Skaka, Kingdom of Saudi Arabia.

Department of Microbiology ³, Faculty of Medicine, Al Neelain University, Khartoum, Sudan.

Department of Physiology and Pathology ⁴, Faculty of Medicine, Karary University, Khartoum, Sudan.

ABSTRACT: Chlamydia trachomatis is the most common bacterial sexually transmitted disease. Over 100 million men and women worldwide are infected with Chlamydia at any point in time. The aim of this study to detect the presence of C. trachomatis antibodies and antigen among women on the reproductive age and to study the relationship between the presence of C. trachomatis and age, abortion and ectopic pregnancy. Five hundred women classify into three hundred pregnant women with a gestational age between 12 and 36 weeks and two hundred nonpregnant women. Two blood samples collected individually in two different containers, one EDTA container for PCR technique and plane container for ELISA testing during the period June 2011 to August 2013. Thirty-one samples (6.2%) were positive for C. trachomtis IgG only, while 8 samples (1.6%) were positive for both IgM and IgG. Out of 31 samples, 25 samples (5%) were positive by using the PCR technique. The results revealed statistically a significant relationship between PCR and previous abortion and ectopic pregnancy with P value, 0.001 and 0.002, respectively. The age range between (30-35) years is considered as a risk factor that exposes sexually –active women to C. trachomatis infection. Chlamydia trachomatis infection in women is a cause of abortion and ectopic pregnancy.

Chlamydia trachomatis, Abortion, PCR, ELISA

INTRODUCTION: Over 100 million men and women worldwide are infected with chlamydia at any point in time ¹.

It is the most commonly reported bacterial sexually transmitted infection (STI) in developed countries, with over 1.4 million cases reported in the United States in 2011 ².

There are at least 18 serovars of C. trachomatis; the important medical serovars associated with endemic trachoma are A, B, Ba, and C while D-K serovars are associated with sexually transmitted disease and that cause lymphogranuloma venereum are L1, L2, and L3 ³.

Left untreated, Chlamydia can ascend from the endocervix to the upper genital tract in women and cause pelvic inflammatory disease (PID) which can increase the risk of developing fallopian tube scarring, potentially leading to ectopic pregnancy, tubal infertility, and chronic pelvic pain ^{4, 5}. Also, genital infection in pregnant women increases the risk of preterm delivery, can be passed on to the baby during vaginal delivery and may result in eye and lung infections in the newborn ^{4, 6}.

MATERIAL AND METHOD:

Ethical Approval: The study was approved by the Al Neelain Medical Research Committee and Omdurman Maternity Hospital ethical committee. All participants signed informed consent forms before enrolment in the study.

Study Design: This is a descriptive cross-sectional laboratory and a hospital-based study conducted in Omdurman state, to determine the frequency of C. trachmatis among women at the reproductive age during the period June 2011 to August 2013.

Study participants were filled with a self-administered questionnaire, which included data on sociodemographic, reproductive, and medical history.

Samples Collection: The samples were collected from patients attending Omdurman maternity hospital antenatal Care unit and family planning unit.Three hundred blood samples from pregnant women with a gestational age between 12 and 36 weeks and two hundred samples from non-pregnant women. Two blood samples collected individually in two different containers one EDTA container for PCR technique and plane container for ELISA testing.

ELISA Test: The ELISA test is done by using NEUROIMMUNE kit, briefly, basis of antigen preparation was BGM- cells infected with the C. trachomatis of the serotype K. The microplate well were coated with purified MOMP antigen (major outer membrane protein), which is a transmembrane protein in the outer membrane of the elementary bodies. The test kit contains microtitre strips, each with eight break-off reagent wells coated with C. trachomatis antigens. In the first reaction step, diluted patient samples were incubated with the wells. In the case of positive samples, specific IgG IgM antibodies will bind to the antigens. To detect the bound antibodies, a second incubation is carried out using an enzyme-labeled anti-human IgGIgM (enzyme conjugate) which is capable of promoting a color reaction.

Photometric measurement of color intensity was made at a wavelength of between 620 nm and 650 nm within 30 minutes of adding the stop solution. The intensity of the formed color is proportional to the concentration of antibodies against C. trachomatis antigens.

The color intensity obtained from the automated ELISA reader was interpreted by qualitative analysis by the formula extinction value of the control extinction of calibrator 2 interpretation:

Ratio<0.8: negative

Ratio ≥0.8 to > 1.1: borderline

Ratio≥ 1.1 positive

DNA Amplification: Confirmation of Chlamydia trachomatis isolates was carried out by Amplification of the major outer membrane protein (copB) target gene using GenePack DNA PCR tests (Geneon-Germany). A master mix reagent was prepared. 10µl specific dilution buffer (Gene On), 2 µl bovine serum albumin (BSA), 7µl sterile water, and 1µl template DNA were added separately to each lyophilized DNA-PCR test tube to give a final volume of 20 µl. The amplification was conducted using TC-312 Techne thermal cycle (Germany). The PCR products were analyzed by 1.5 agarose gel electrophoresis Fig. 1.

FIG. 1: CHLAMYDIA TRACHOMTIS PCR AMPLICON ON 2% AGAROSE GEL

Lane 1= Molecular weight marker; Lane 2 and Lane 3 = Positive control; Lane 4= Negative control; Lane5 – Lane 8 =Positive bands for Chlamydia trachomtis strains (550 bp).

RESULT AND DISCUSSION: Chlamydia IgG and IgM ELISA were done for all the subjects enrolled in the study. The mean age of the women enrolled in the study was 30-35 years. Out of 500 women enrolled in the study, 31 samples (6.2%) were positive for C. trachomtis IgG only, while eight samples (1.6%) were positive for both IgM and IgG. Out of 31 samples, 25 samples (5%) were positive by using PCR technique Fig. 1.

TABLE 1: DETECTION OF CHLAMYDIA TRACHOMTIS AMONG PREGNANT AND NON-PREGNANT USING DIFFERENT TECHNIQUES

Out of the 500 women participated in current study, 339 (62.8) women complained from the symptom of infection, 21 women (6.2%) were positive for C. trachomatis, while 161 (32.2%) were said they had not complained from the symptom of the infection, (6.2%) were positive for C. trachomatis antibodies Fig.  2.

FIG. 2: THE PERCENTAGE OF WOMEN WHO HAD A HISTORY OF INFECTION COMPARING WITH WOMEN WHO HAD NOT A HISTORY OF INFECTION

TABLE 2: CORRELATION BETWEEN IGG AND NUMBER OF ABORTION

P value 0.001

TABLE 3: CORRELATION BETWEEN IGG AND NUMBER OF ECTOPIC PREGNANCY

There were several studies conducted in Sudan that reported the prevalence of C. trachomatis. The percentage of C. trachomatis reported in this study was 5% using the PCR technique and 6.2% by ELISA technique, and this not a surprising result because Sudan is a traditional Islamic society, and according to the customs and traditions of Islamic societies, free sexuality is prohibited, but this result is lower in comparison with study done by Mohamed et al., who found that percentage was 60 (47.2%) and this may be due high selection criteria for the symptomatic participate ⁷.

Another Sudanese's study found that the percentage was 14.1 ⁸, this result was relatively near to finding in IRAN and Saudi Arabia 15.8%, 10.5% respectively ^{9, 10}  and greatly less than that reported in Brazil and Quter 25.7 %, 37.5% respectively ^{11, 12}.

Our findings indicated that C. trachomatis infections common among age group (30-35 year), this result was in alignment with study done in Saudi Arabia ¹⁰ where  they reported that the common age was  above 34 years, and in contrary with Eltaybe et al., ⁸ results whose stated that C. trachomatis infections common in the age group between  24-30 years. Other reported that 25 years as suspected age group ¹³.

However, Kusano and his team mentioned that junior school graduates had the highest frequency of positive cases, followed by graduates of high schools, vocational schools junior colleges, and university graduates had the lowest frequency ¹⁴, also Elia et al., reported that the lower age range of 18-25 had the highest prevalence of C. trachomatis infection ¹⁵.

In the current  study the presence of C. trachomatis was not affected by past genital tract infection, out of 339 (62.8%) women were complained from the symptom of infection only 21(6.2%) women were positive for C. trachomatis, while out of 161 (32.2%) women were said they had no  symptom of the infection only (6.2%) were positive for C. trachomatis. This result is in line with that of Fatholahzadeh et al., who stated that there was no association between genital tract infection and presence of Chlamydial infection ¹⁶. In the present analysis, there is a correlation between detection of C. trachomatis antigen and abortion p-Value 0.001, this result similar to the finding of Wilkowska et al., who reported that the percentage of C. trachomatis infection in cervical swabs was 44.4% of women with previous miscarriages ¹⁷.

Recent Polish and Iranian studies suggested that there was a significant association between the number of abortions and abnormal vaginal discharge with C. trachomatis infection ^{18, 19}. This study detected a relationship between several ectopic pregnancy and presence of C. trachomatis antigen whereas the p-value was (0.008) this was agreed with Abdolreza et al., who observed that presence of C. trachomatis antibodies with an ectopic pregnancy patients ²⁰.

CONCLUSION: The rang age between 30-35 years is considered as a risk factor that exposes sexually-active women to Chlamydia trachomatis infection. C. trachomatis infection in women is a cause of abortion and ectopic pregnancy.

ACKNOWLEDGEMENT: Authors are grateful to the health authorities of Omdurman maternity Hosptial, for providing necessary facilities to carry out the research work. Thanks for Dr. Lamies A. Kaddam for her kind assistance and support while performing the study.

CONFLICT OF INTEREST: Nil

REFERENCES:

1. World Health Organization: Global incidence and prevalence of selected curable sexually transmitted infections - 2008. Geneva: World Health Organization 2012.
2. Centers for Disease Control and Prevention: Sexually Transmitted Disease Surveillance 2011. Atlanta: U.S: Department of Health and Human Services 2012.
3. Geo F, Karen C, Ganet S, Stephen A andTimothy A: Medical Microbiology, 25th ed, New York: McGraw-Hill 2010.
4. Schachter J and Stephens RS: Biology of Chlamydia trachomatis. In Sexually Transmitted Diseases. 4^th Edited by Holmes KK, Sparling PF, Stamm, Piot P, Wasserheit JN, Corey L, Cohen MS, Watts DH. New York: McGraw-Hill 2008: 555-74
5. Haggerty CL, Gottlieb SL, Taylor BD, Low N, Xu F and Ness RB: Risk of sequelae after chlamydia trachomatis genital infection in women. J Infect Dis 2010; 201: S134-S155.
6. Rours GIJG, Duijts L, Moll HA, Arends LR, de Groot R, Jaddoe VW, Hofman A,Steegers EAP, Mackenbach JP and Ott A: Chlamydia trachomatis infection during pregnancy associated with preterm delivery: a population-based prospective cohort study. Eur J Epidemiol 2011: 26: 493-02.
7. Mohammed, MA and Omer AA: Molecular detection of Chlamydia trachomatis among gynecological patients attending Khartoum Teaching Hospital. Journal of Bacteriology Research 2012; 4(4): 42-45.
8. Abakur EH, Abdalla MO, Huli AM and Alnour TM: Serological Detection of Chlamydia trachomatis among Pregnant Women in Khartoum State. American Journal of Medicine and Medical Sciences 2013; 3(6): 121-25.
9. Hasanabad HM, Mohammadzadeh M, Bahador A, Fazel N, Rakhshani H and Majnooni A: Prevalence of Chlamydia trachomatis and Mycoplasma genitalium in pregnant women of Sabzevar- Iran. Iran J Microbiol 2011; 3(3): 123-28.
10. Alzahrani JA, Obeid EO, Hassan IH and Almulhim AA: Screening of pregnant women attending the antenatal care clinic of a tertiary in eastern Saudi Arabia for trachomatis and N. gonorrhoea Infections, Indian J sex Transm Dis 2010; 31: 81-86.
11. Ramos BR, Polettini J, Marcolino LD, Vieira EP, Marqurs MA, Tristao AR, Nunes HR, Rudge MV and Silva MG: Prevalence and risk factors of Chlamydia trachomatis cervicitis in pregnant women at the genital tract infection in obstetrics unit care at Botucatu Medical School, São Paulo State University-UNESP. Brazil J low Genit Tract Dis 2011; 15: 20-24.
12. Thani AA, Abdul-Rahim H, Alabsi E, Bsaisu HN, Haddad P, Mumtaz GR and Abu-Raddad JL: Prevalence of Chlamydia trachomatis infection in the general population of women in Qatar. Sex Transm Infect 2012; 0: 1-4.
13. Mascellino MT, Ciardi MR, Oliva A, Cecinato F, Hassemer MP and Borgese L: Chlamydia trachomatis detection in a population of asymptomatic and symptomatic women: correlation with the presence of serological Markers for this Infection, New Micobiol 2008; 31: 249-56.
14. Kusano Y, Shibata Y Katamine S, Yamamoto T, Kurokawak K, Moriuchi R, Kubota K, Masuzaki H, Honda. S, Moji K and Takemoto T: Demographic and reproductive factors for high sero prevalence of Chlamydia trachomatis among Pregnant Women in Japan. Tohoko J Exp Med 2000; 190: 1-13.
15. Ella EE, Shenge H and Ajoge HO: Prevalence of Chlamydia trachomatis among female students attending a selected University Health Services in Zaria, Kaduna State, Nigeria. The International Journal of Engineering and Science (IJES) 2013; 11: 54-57.
16. Fatholahzadeh B, Bahador A, Haghighi MH, Bazarjani F, Haghighi F: Comparative screening of Chlamydia Trachomatis infection in women population in Tehran, Iran Red Crescent Med J 2012; 14(5): 289-93.
17. Wilkowska M, Trojniel M, Zdrodowska-Stefanow B, Ostaszewska-Puchalska I, Redźko S, Przepieść J and Zdrodowski M: The influence of Chlamydia trachomatis infection on spontaneous abortions. Advances in Medical Sciences 2009; 54: 86-90.
18. Choroszy-Król I, Teryks-Wołyniec M, Frej-Mądrzak M and Gryboś M: Chlamydia trachomatis w zakażeniachukładu moczowo-płciowego u kobiet. Proceedings of the XXV Jubileuszowy Zjazd Polskiego Towarzystwa Mikrobiologicznego. Bydgoszcz, Poland. Post Mikrobiol 2004; 43: 1-130.
19. Eslami G, Goudarzi H, Taheripanah R, Taheri S, Fallah F, Moazzami B, Taherpour A, Ohadi E, Pourkaveh B and Zahirnia Z: Chlamydia trachomatis detection by nested-PCR method on females referred to medical centers of Tehran, Iran Arch Clin Infect Dis 2012; 7(4): 124-7.
20. Jahromi AS, Farjam MR, Mogharrab F, Amiryan M, Makiani MG, Madani A, Daryanavard A, Davoudi H, Hossienpour M and Manshoori G: Chlamydia trachomatis in women with full-term deliveries and women with abortion. American Journal of Infectious Diseases 2010; 6(3): 66-69.
    

    ---

    How to cite this article:

    Kaddam LA, Mohager MO, Adam AA and Tahan MA: Immunological and molecular detection of Chlamydia trachomatis among women at reproductive age attending Omdurman Maternity Hospital. Int J Pharm Sci & Res 2014; 5(9): 3664-68. doi: 10.13040/IJPSR.0975-8232.5(9).3664-68.

    ---

    All © 2013 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.

    ---