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The prevalence of Chlamydia trachomtis infection among women attending health clinics in ShirazIslamic Republic of Iran Negin Hadi, MD MPH*, Fariba Barazandeh, MD**, Fariborz Azad, DVM*** *Associate professor of Community Medicine, Department of community Medicine, Shiraz Medical School, Shiraz University of Medical Sciences, Shiraz , Islamic Republic of Iran **General Physician, Shiraz Emergency Medical Services, Shiraz, Islamic Republic of Iran ***Chief of Immunology Lab, Department of Pathology, Shiraz Medical School, Shiraz, Islamic Republic of Iran Correspondence Author: Dr. Negin Hadi, Associate professor of Community Medicine, Department of community Medicine, Shiraz Medical School, Shiraz University of Medical Sciences, Shiraz , Islamic Republic of Iran, E-mail: [email protected] 1 Abstract Introduction: Chlamydia trachomitis (C.T) is one of the most prevalent sexual transmitted infections (STI) and micro-organisms detected in smears of discharge in sexually active women. This population based study was under taken for the first time in Shiraz (South of Iran) to determine the prevalence of genital C.T in women. The infection usually has no symptoms; although it may have long complications such as pelvic inflammatory disease, ectopic pregnancy, chronic pelvic pain, tubal infertility and cervical cancer. Methods : The number of 402 women, which referred to randomized selected health centers in Shiraz, for routine pap smear screening test were tested using IMAGEN Chlamydia test, a direct immunofluoresence test for detection of Chlamydia in human urogenital specimen. The results of this test were compared to socio-demographic condition of each participant as well as their STD symptoms at present and in the past. Results : The prevalence rate of Chlamydia infection was 8 %( 32/402). No correlation was found between this infection and the age, marital age, number of children, education and occupational status of the participants. On the other hand, the correlation between the presence of infection and a positive history of STD symptoms at present and in the past was significant. Conclusion : Chlamydia infection is quite frequent in our society, where usually the knowledge about STD infections is little. Considering the sequela of this infection and the fact that this is a quiet disease, it seems rational to screen sexually active women and inform them more about this issue. Keywords: sexually transmitted infection – Chlamydia trachomatis – prevalence – direct immunofluorensence – endocervical specimen – Iran 2 Introduction Different microbial agents cause considerable morbidity and mortality worldwide (1). Among these microorganisms, Chlamydia trachomatis infection (CTI), an obligate intracellular bacterium, with millions of cases reported annually throughout the world is the most common cause of bacterial sexually transmitted infections (STI) with the highest incidence in developing countries, (2-3-4-5) which could be transmitted in homosexuals even in women who have sex with women (6). During the last decade the incidence rate of CTI has increased significantly in Denmark, Norway, the United Kingdom and northern Ireland, Finland and Sweden, also in eastern Europe(7). Amount of costs related to treatment of CTI complications are estimated as the second grade after HIV (7). Demographic risk factors for Chlamydia infection are young age, unmarried status, and lower socioeconomic conditions. Other risk factors are anatomic (ectopia), behavioral (number of sexual partners), microbiologic (concurrent gonorrhea), and hormonal (use of oral contraception) (8). Up to 70-80% of women infected with Chlamydia have no symptoms (2-4-9) and up to 50% of infected men are asymptomatic (10). Some others are not easily recognized with this infection on clinical grounds alone (8). However when left untreated, this infection can lead to sequela complications such as pelvic inflammatory disease, ectopic pregnancy, chronic pelvic pain and tubal scarring and infertility (3-4-8-11). Most recent studies concluded that CTI may be a risk factor of cervical cancer (7). Due to this, effective prevention efforts must include screening programs to detect and treat asymptomatic infections (2). These screening tests are designed to detect disease before clinical symptoms develop and at the point when early intervention can improve long-term outcomes (5). According to Cook Rl and Ostergaard L, young women who are sexually active, are a perfect group for screening for genitourinary infections with C. trachomatis as this infection with its sequela is common in this group especially among females aged 15-19, early treatment will improve clinical outcomes and screening tests are available (5-9). 3 Besides suggestive signs and symptoms and the presumptive diagnosis(which is made in suspicious cases when gonococci are not found), confirmatory tests are urethral, cervical or rectal cultures, urine or cervical NAAT( nucleic acid amplification test), Polymerase chain reaction(PCR), Ligase chain reaction(LCR), enzyme-linked immunosorbent assay(ELISA) and direct immunofluoroscent antibody slide test (DFA)(12). Based on the screening method used and the study group, the prevalence rate would be different. A review of the articles between 2001 and 2008 shows the prevalence of Chlamydia infection to be between 2% to 8.5% in normal population referring for routine tests to health clinics all over the world (4-2). In Iran the Chlamydia prevalence studies are mainly focusing on specific groups such as those with PID, cervicitis, tubal infertility and pregnant women (313-14-15-16-17). In a study in 2007, Firoozjahi A., Bakhtiari A., showed a prevalence rate of 11.6% for Chlamydia infection among women attending health care centers in Babol(18). The population based study about prevalence of CTI in women in Iran are rarely performed, and it has not been studied in south of Iran. This peculiar finding promoted the authors to investigate the prevalence of CTI and its risk factors in general population women in Shiraz, which can be used for early treatment and prevention of its sequela. Methods This is a cross sectional study carried out among women in fertility age attending health care centers of Shiraz University of medical sciences. Shiraz city (located in Fars province in western south of Iran) was divided into 3 areas of north, middle and south and one health center was randomly selected from each area. 402 women referred to these centers for routine Pap smear entered this study. 4 A standardized questionnaire was made including socio-demographic variables which were age, marital age, number of children, educational and professional status and a series of questions asking about any STI symptoms at the time of referral or before, and possible previous history of sexually transmitted diseases. Midwives of these 3 centers were educated for correct sampling, carrying the samples and filling out the questionnaires. IMAGEN Chlamydia test, a direct immunofluoresence test was used in this study. It’s a qualitative test for detection of Chlamydia in human urogenital specimen using a fluorescein labeled monoclonal antibody through fluorescein isothiocyanide procedure (FITC). For cervical sampling, this area was first cleansed to remove excess mucus, blood, pus, etc. Then a cotton wool swab was entered in cervical canal and approximately 1 centimeter. The swab was rotated several times at the squamocolumnar epithelial junction and then withdrew without touching vaginal surfaces. Then the swab was rolled on the microscope IMAGEN slide on a specified area. It was left to be air dried at room temperature (15-30°c) and then fixed in fresh acetone for 10 minutes and air dried again. The slides were immediately carried in cold box to the laboratory to be froze and stored at -70°c before staining. The slides were then incubated with the FITC conjugated reagent for 15 minutes at 37°c in wet chamber. Excess reagent was removed by washing with PBS(phosphate buffer saline) for 5 minutes. The stained areas were mounted and viewed using the epifluorescein illuminative microscope. The positive and negative laboratory results were then compared to the information from the related questionnaires by means of SPSS version 16 using Chi-Square and Fisher’s exact tests. The level of significance was considered as P<0.05. 5 Results The prevalence rate of Chlamydia infection was 8%(32/402) among the participants. 89.6% of the women were housewives and 68.6% of them had at least guidance school education. Other information about their occupational and educational status is presented in table 2. 64.4% of women (260/402) had a positive history of symptoms of a sexual transmitted disease in the past. There was a positive correlation between the presence of these symptoms in the past and a positive result of IMAGEN test (Pvalue=0.001). 54.48% of the participants (219/402) were symptomatic for STD at the time of referral. There was also a positive correlation between the presence of these symptoms at present and a positive Chlamydia test result (P-value=0.001). Table 3 and 4 shows the frequency of these symptoms in the past and present time and their association with CT infection. No significant correlation was found between the participants’ age, marital age, child number, occupational and educational status and the result of Chlamydia test. The frequency of positive results of Chlamydia test based on these factors is presented in table 5. Discussion The prevalence rate of Chlamydia was 8% in this study which is comparable to the results of another study done in Babol, Iran by Bakhtiari A., Firoozjahi A. in 2005 which showed Chlamydia infection in 11.6% of women attending health centers (18). 6 Other similar studies show a prevalence rate of 2% to 8.5% (2-4-8-22). However one should consider the difference between the test method used for screening and the different groups studied based on their lifestyle in comparing the results of different studies. Although the test method of this study is highly specific, we found a less prevalence rate in comparison to the results of the study by Bakhtiari, Firoozjahi (2005). This difference is may be due to low sensitivity of direct fluorescence assay in comparison to ELISA method used in the other study. Chlamydia infection prevalence rate is mostly studied in specific groups for example in those with cervicitis showing a prevalence rate of 12.5% to 20% (15-310). The prevalence rate has been 10.2% studied in a group with PID (9). These rather higher prevalence among these groups compared with those attending health clinics for routine checkups are expected as PID and cervicitis are among the sequelae of Chlamydia infection. Other studies among pregnant women have shown the prevalence rate of Chlamydia infection to be 2 to 8% (17-21), almost the same as other studies done on women without any special health condition (2-4-8). According to the results of present study, the highest frequency of positive CTI was in 26-35 age groups of women and reduced by increasing age. Although the deference between age groups was statistically significance. Other studies also had shown a decrease in infection chance with aging (4-8-18-19-20) and it is declared clearly in Harrison’s principles of internal medicine that the peak incidence of genital C. trachomatis is the late teens and early twenties (12). This age related occurrence is possibly due to epithelial changes, partial immunity and changing sexual behavior (18).We also expected to see higher rates of Chlamydia infection in women with lower marital age. In Iran extramarital sexual relationship is rare especially among women, so the risk factor of having multiple partners, as showed to be important by other studies (19) was not included in this study. Another study had showed that the 7 mean age at first intercourse is not significantly related to the risk of infection (1). Our results also showed that the marital age does not affect the tendency to infect by Chlamydia. Child number, educational and economic status was considered as determining factors of participants’ socioeconomic condition. Other studies had shown that a poor socioeconomic condition would lead to a higher probability of Chlamydia infection (4-18).We found more positive test results of CTI among women with no child, those with high school education and self employees. It is reported no significant differences in the prevalence of CTI among different occupational groups (7). 45.5% and 35.3% of the cases were asymptomatic at present and in the past respectively. Our result was different from the results of other studies which have showed about 70% of Chlamydia infected women to be asymptomatic (2-49). However as we have not excluded those participants with other possible sexual transmitted diseases, these result is expected as the symptoms would belong to a coexist infection other than Chlamydia. If symptomatic, Chlamydia infected cases are expected to have mild symptoms of discharge, bleeding, lower abdominal pain or dysuria mostly (9). Although most cases of Chlamydia would be asymptomatic, still it is expected to see this infection more among symptomatic women (8). Vaginal discharge and lower abdominal pain have been the most prevalent accompanying symptoms in Chlamydia positive women in another study done over a group of pregnant women (21). Also Firoozjahi A., Bakhtiari A.showed in their study that post coital frequent bleeding and genital itching are the most frequent symptoms among infected women (18). However according to our study, dysuria was the most common symptom in the past and at present. The other prevalent symptoms at present time were vaginal discharge and ulcer. This difference in presenting symptoms can be due to possible coexisting STD infections. We concluded, silent Chlamydia infection in general population of women, is quite frequent in our society, where usually the knowledge about STD infections 8 is little. Considering the sequelae of this infection and the fact that this is a quiet disease, it seems rational to screen sexually active women. Other randomized trials also showed 50% reduction of incidence of pelvic inflammatory disease due to CTI by used of systemic screening for genital CTI men and women with suitable coverage as in United Kingdom this coverage is 37% (23). We recommend more studies with lager sample size to perform screening programs and promote general population knowledge about this issue . 9 References 1. Schlicht M.J., Lovrich S.D., Sartin J.S., et al. High prevalence of genital mycoplasmas among sexually active young adults with uretritis or cervicitis symptoms in La Crosse, Wisconsin. J. Clin. Microbiol. 2004; 42(10): 4636-40 2. Dicker L.W., Mosure D.J., Kay R.S., et al. An ongoing burden: chlamydial infections among young American Indian women. Matern. Child Health J. 2008;12: S25-S29 3. Hashemi F.B., Pourakbari B., Zaeimi Yazdi J. Frequency of Chlamydia trachomatis in women with cervicitis in Tehran, Iran. Infectious diseases in obstetrics and gynecology. 2007 4. Hilger T.M., Smith E.M., Ault K. Predictors of Chlamydia infection among women attending rural Midwest family planning clinics. Inf. Dis. Obst. Gynecol. 2001; 9:3-8 5. Cook R.L., Ostergaard L. Current issues in screening for Chlamydia trachomatis. Current infectious disease reports. 2003; 5:153-8 6. Bailey JV, Farquhar C, Owen C, Mangtani P. Sexually transmitted infections in women who have sex with women. Sex Transm Infect. 2004 Jun;80(3):244-6. 7. Kucinskiene V, Sutaite I, Valiukeviciene S, Milasauskiene Z, Domeika M. Prevalence and risk factors of genital Chlamydia trachomatis infection. Medicina (Kaunas). 2006;42(11):885-94. 8. Einwalter L.A., Ritchie J.M., Ault K.A., et al. Gonorrhea and Chlamydia infection among women visiting family planning clinics: racial variation in prevalence and predictors. Perspectives on sexual and reproductive health. 2005; 37(3);135-140 9. Cates W., Wasserheit J.N. Genital chlamydial infections: epidemiology and reproductive sequelae. Am. J. Obstet. Gynecol. 1991(June):1771-80 10. Weir E. Upsurge of genital Chlamydia trachomatis infection. CMAJ. 2004 Oct 12;171(8):855. 11. Malik A, Jain S, Hakim S, Shukla I, Rizvi M. Chlamydia trachomatis infection & female infertility. Indian J Med Res. 2006 Jun;123(6):770-5. 10 12.Kasper, Braunwald, Fauci, et al. Harrison’s principles of internal medicine. 16th ed., McGraw-Hill, USA, 2005 13.Tabasi Z., Badami N., Atabakhshi M., et al. Evaluation of the prevalence of Chlamydia trachomatis and its associated factors in patients with pelvic inflammatory disorders in Shabihkhani hospital in Kashan in 2000. Kashan University of medical sciences Fayz J. 1380; 5(18): 43-49 14. Eini E., Mirmiran P., Mohammadi Nasrabadi F., Velayi N. The relationship of chlamyia trachomatis with symptomatic and asymptomatic cervicitis. Iranian journal of infectous diseases and tropical medicine. 1380; 15(6):6472. 15-Amin M. Study of etiologic role of Chlamydia trachomatis in infertility, Imam Khomaini clinic of infertility, Ahvaz, 1373. Scientific journal of Kurdistan university of medical sciences. 1378; 11(3): 18-21 16- Esfandiari N., Gachkar L., Hamzehie K. Role of Chlamydia trachomatis in tubal infertility. Pejouhandeh quarterly research journal. 1380;24(6):33-46 17- Behroozi R., Badami N. The prevalence of Chlamydia infection among pregnant women refereed to perinatal clinics of Tehran university of medical sciences in the year 1373. Journal of Mazandaran university of medical sciences.1378;22-23(9):26-31 18- Firoozjahi A, Bakhtiari A. Chlamydia t rachomatis nfection in women attending health centers in Babol: prevalence and risk factors. Eastern Mediterranean Journal. 2007;13(5):1124-31 19- Levidiotou S., Vrioni G., Papadogeorgaki H., et al. Chlamydia trachomatis infections in Greece: first prevalence study using nucleic acid amplification tests. Springer-Verlag online. 2005(March) 20- Adams EJ, Charlett A, Edmunds WJ, Hughes G. Chlamydia trachomatis in the United Kingdom: a systematic review and analysis of prevalence studies. Sex Transm Infect. 2004 Oct;80(5):331-3. 21- Romoren M. Sundby J., Velauthapillai M. Chalmydia and Gonorrhoea in pregnant Batswana women: time to discard the syndromic approach? BMC Infectious Diseases. 2007; 7:27 11 22- Oliveira FA, Pfleger V, Lang K, et al. Sexually transmitted infections, bacterial vaginosis, and candidiasis in women of reproductive age in rural Northeast Brazil: a population-based study. Mem Inst Oswaldo Cruz. 2007 Sep;102(6):751-6. 23- Macleod J, Salisbury C, Low N, et al. Coverage and uptake of systematic postal screening for genital Chlamydia trachomatis and prevalence of infection in the United Kingdom general population: cross sectional study. BMJ. 2005 Apr 23;330(7497):940. Epub 2005 Apr 4. 12 Table 1 shows the participants age, marital age and child number. Table 1- Age, Marital age and child number of the participants Age Marital Age Child Number 13 Minimum Maximum Mean 17 12 0 68 39 9 29.96 19.02 1.59 Standard Deviation 7.33 4.07 1.27 Table 2- Occupational and educational status of the participants Occupation Unemployed Self employee Housewife Total Education Illitrate Primary school Guidance school High school University degree Total 14 Frequency Percentage 34 8 360 8.4 2 89.6 402 100 12 75 138 138 39 3 18.7 34.3 34.3 9.7 402 100 Table 3- The frequency of STD symptoms in the past and their association with CT infection Frequency NO. Positive for CT infection % No. % Dysuria 121 46.5 19 15.7 Itching 168 64.6 21 12.5 Discharge 189 72.7 20 10.58 Ulcer 71 7.3 7 9.86 Herpetic lesion 1 0.38 0 0 Wart 1 0.38 1 100 15 Table 4- The frequency of STD symptoms at present and their association with CT infection Frequency Positive for CT infection NO. Dysuria 53 % 24.2 No. 8 % 15.09 Itching 81 37 Discharge 159 72.6 22 13.8 Ulcer 79 36.1 11 13.9 Wart 1 0.46 1 100 16 8 9.9 Table 5 – The frequency of positive Chlamydia test based on participants demographic factors Frequency Positive for CT infection No. % Age <25 122 9 7.38 26-35 196 18 9.18 36-45 70 5 7.14 >45 11 0 0 <20 235 20 8.51 20-25 137 9 6.56 26-35 24 1 4.17 >35 2 0 0 0 16 3 18.75 1-2 270 22 3-4 55 6 10.91 >5 13 0 0 Marital age Child number 17 8.15 Occupation Unemployed 34 1 2.9 Self employee 8 1 12.5 Housewife 360 30 8.3 87 5 5.7 Guidance school 138 10 7.2 High school 138 13 9.5 University degree 39 Education Illitrate and Primary school 18 3 7.7