Cytological and molecular screening of Chlamydia trachomatis in infertile women attending a maternity teaching hospital in

CT) is a sexually transmitted Background: Chlamydia trachomatis pathogen that threatens reproductive health worldwide. This study aims to screen CT urogenital infection using cytology and molecular methods in women suffering infertility. In total, 415 women suffering infertility, attending Wad Madani Methods: Maternity Hospital were included in this study and then classified into two groups: primary infertile women and secondary infertile women. Both urine (n= 415) and vaginal swab samples (n= 130) were collected and tested using Giemsa stain and Polymerase Chain Reaction (PCR) for detection of CT. CT was detected in 33.7% (140/415) of urine samples and 73.1% Results: (95/130) of vaginal swab samples using Giemsa stain, compared with 44.6% (185/415) and 84.6% (110/130) using PCR, respectively. In the primary infertile group (n= 265), chlamydia was detected in 35.8% (95/265) of urine and 75% (60/80) of swab samples by Giemsa stain compared with 50.9% (135/265) and 75% (60/80) of the samples by PCR. In the secondary infertile group (n= 150), chlamydia was detected in 30% (45/150) of urine and 70% (35/50) of swab samples by Giemsa stain compared with 33.3% (50/150) and 100% (50/50) of the samples by PCR. The associated risk factors were age, lower abdominal pain, and urethritis (p< 0.05). The sensitivity, specificity, positive predictive value, and negative predictive value of Giemsa stain in detecting chlamydia compared to PCR were 86.4%, 100%, 100%, and 83.6%, respectively. Giemsa stain can be used as a screening test for detection Conclusions: 1 2 3


Introduction
Chlamydia is the most common bacterial sexually transmitted infection (STIs), with more than 100 million new cases per year, and is caused by an intracellular Gram-negative bacterium named Chlamydia trachomatis (CT) [1][2][3] . CT infections of the lower female genital tract are frequently asymptomatic. However, if these infections do not resolve or persist untreated, the organisms can ascend to the upper genital tract, potentially causing salpingitis and functional damage to the fallopian tubes 4 . The World Health Organization (WHO) estimated that in 2012 there were 357.4 million new global cases of STIs, including CT (130.9 million cases) and Neisseria gonorrhoeae (78.3 million cases) 5 . Improvements in screening tests hold promises for increasing screening rates and preventing consequences of silent untreated infections. Until recently, asymptomatic infection and the lack of a simple and sensitive screening test has been a barrier to the accurate detection of CT infection. The prevailing concept was that chlamydia tests were conducted using cervical swabs for women and urethral swabs for men, but, due to the greater sensitivity and specificity of molecular tests, urine for both sexes can be used as a less invasive sampling technique 3 . Due to the asymptomatic nature of many chlamydia infections, screening is recognized as the most effective approach for reducing the sequela of this disease. Thus, the development of alternative, low-cost, easy-to-use, point-of-care methods for the detection and simultaneous screening of CT infections in clinical settings in a useful time frame remains a critical strategy for improving reproductive genital tract health worldwide 1 .
Sudan is one of the largest countries in Africa and most of its population live in rural areas. This country faces manifold problems including; low socioeconomic status, poor transportation and education, and health problems. One of these health problems is infertility. In Gezira State, Sudan, CT infections are neglected infections and there is no local preventive screening program or exhaustive information about the prevalence of these infections. In addition, to the best of our knowledge, no previous studies have been carried out to determine the size of the problem. This prompted the following study to be performed, which focusses on urogenital CT infections among women suffering infertility, which may be indicator for higher risk for more serious sequela of sexually transmitted diseases.

Study design and setting
A cross-sectional hospital-based study was conducted from May 2017 to May 2018, to screen CT in urine and high vaginal swab samples from infertile women attending Wad Medani Maternity Hospital, Medani, Sudan.
Two permissions were granted to carry out this study; firstly from the Faculty of Medical Laboratory Sciences, University of Gezira, and secondly from the Ministry of Health, Gezira State. Written informed consent was obtained from all study subjects after they had been informed about the study objectives (for participants under the age of 18 years, consent was obtained from the husbands as per Sudanese policy).

Participants and selection criteria
In total 415 Sudanese, married women, suffering infertility were enrolled during the routine clinic of Wad Madani Maternity Hospital. The women were classified into two groups as follow: primary infertile women (n= 265), those failed to become pregnant for at least one year after marriage; and secondary infertile women (n= 150), those failed to become pregnant after a history of abortion and/or successful pregnancy/ies before. Women suffering vaginal bleeding were excluded from this study.
The study sample size (415) was calculated according to the following equation: (n = (z) 2 p (1 -p ) / d 2 ), where n stands for number, z equal 1.96 at 95% level of confidence, p for estimated proportion of the population, and d for margin of error 0.05.

Data collection
A questionnaire (Extended data 6 ) was designed and filled out before clinical sample collection in order to obtain participant's demographic characteristics and relevant clinical symptoms.
Socioeconomic status was classified according to the participant's income, or husband's monthly income if the participant was not in employment as follows: high status, >30000 SDG per month; moderate, 30000-8000 SDG; low, <8000 SDG.
Education level was classed according to the last degree achieved by the participant as follows: high for undergraduate and post graduate degrees certificate; medium for secondary school degree certificate; and low for primary and pre-school level.

Sampling
Participants were instructed on the proper self-collection of the first-void urine (i.e., the initial 10-30 mL of voided urine) using a sterile plastic wide neck leak-proof cup and transported to the laboratory within two hours. Also, high vaginal swabs were taken using a Dacron swab (COPAN DIAGNOSTICS INC.) by a trained medical officer.
Cytological technique. The urine sample was centrifuged to obtain a pellet to make a smear slide. Another smear was performed from the swab sample by rolling the swab gently on a glass slide. All smears were fixed using alcohol and then stained using weak solution of Giemsa stain for a longer staining time. The presence of intracellular inclusions of chlamydia was tested microscopically.

Molecular technique.
The DNA was extracted from urine and high vaginal swab samples using the manual chloroform phenol method, as described by Mohammed 7 . In brief, 500 µl of the sample was added to 500 µl of guanidine chloride, 5 µl proteinase K, and 150 µl ammonium acetate. The mixture was incubated overnight at 37°C, then boiled and cooled at room temperature. One milliliter (1 mL) of pre-chilled chloroform was added, vortexed, and centrifuged for 5 minutes at 3000 rpm. The upper layer was transferred to another tube, and 3 ml of cold absolute ethanol was added and kept at -20°C for 2 hrs. Then the tube was centrifuged at 3000 rpm for 15 minutes. The supernatant was discarded, the tube dried, the pellet was re-suspended with  Table 5).

Discussion
Chlamydia is the most prevalent sexually transmitted bacterial infection worldwide 2,7-9 . Screening of the causative pathogen is important, not only to identify symptomatic individuals for the management of the infection and preventing the more serious   sequela caused by the organism, but also to identify asymptomatic individuals who serve as reservoirs for the disease.
Age is shown to be a risk factor for chlamydia infection among the sexually active population. In total, 29 of 34 studies in women have shown a significant relationship between age and chlamydia infection 10 . This is supported by results reported by Kucinskiene et al. 11 21 . In addition, in our study, all women with secondary infertility were found to be suffering from chlamydia infection by PCR, which is also higher than that reported by Malik et al. (30.6%) 22 . This might suggest the role of chlamydia in the infertility cases seen in our study.
Traditionally, detection and diagnosis of CT depends mainly on culturing cervical and urethral swab samples taken from women and men, respectively 23 . The culture technique is time consuming and requires well trained personnel and access to specialized facilities, which is not easy to be offered as a routine screening technique in Gezira State 24 . On the other hand, the sampling of both cervical and urethral swab have not been accepted by most patients due to the patients reported pain caused by the invasive nature of the spatula and the swab. In addition, there is resistance due to traditional beliefs (especially in communities that advocate circumcision in women). In order to overcome these drawbacks, we used urine samples in this study as a non-invasive sample to detect CT, which had been reported, evaluated and validated for use in most new techniques directed towards the detection of chlamydia trachomatis [25][26][27][28][29][30] .
Cytology was used as one of the non-cultural methods to detect intracellular inclusion bodies of CT 31-34 . This technique is especially useful and easy to perform and is accessible. Ultimately, this technique has established itself as a primary methods 35 . Giemsa staining is used and accepted by many authors to detect intracellular inclusion bodies of CT in cell lines used for culturing techniques 30,35,36 . In the current study, the Giemsa staining cytology reported high specificity and sensitivity when compared to PCR, which may suggest this technique as a reliable screening test for use in Gezira State.

Conclusions
From the results of this study, chlamydia may be one of the causes of infertility inf women at Gezira State. The sensitivity and specificity of a cytology technique using Giemsa stain from a urine or vaginal sample can be used to screen urogenital chlamydia infection where PCR may be difficult to perform.
CT screening, especially for Sudanese women, is of the utmost importance and should be performed through local or national screening programs. Screening programs have been shown to significantly decrease CT prevalence in some regions of the United States and Sweden 37,38 , and subsequently the severe sequela of the infection, including genital damage and infertility.