Cytological and molecular screening of Chlamydia trachomatis in infertile women attending a maternity teaching hospital in Gezira State, Sudan: a cross-sectional study

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)^1–3. 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 tubes⁴. 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 technique³. 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 worldwide¹.

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.

Methods

Results

A total of 415 women were tested for CT in urine and high vaginal swab samples, their age range between 17 and 52 years. The study subjects divided into 265 (63.9%) women suffering primary infertility and 150 (36.1%) women suffering secondary infertility. Their mean age was 32.47±7.80 and 31.27±8.02 years, respectively. The majority of the women were between 31–40 years (43.4%) and 21–30 years (46.7%), and were housewives, resided in an urban city, with varying educational levels, and with moderate socioeconomic status (Table 1).

A total of 415 urine samples were collected from participants, and, due to some traditional/social beliefs and the painful nature of the swab sampling, only 130 vaginal swab samples were obtained (Table 2). Using Giemsa stain, 33.7% and 73.1% were positive for CT, compared with 44.6% and 84.6% positive using PCR (Table 3). In the primary infertile group (n=265), chlamydia was detected in 35.8% (95/265) of urine samples and 75% (60/80) of swab samples using 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% (40/150) of urine samples and 70% (35/50) of swab samples by cytology, compared with 33.3% (50/150) of urine samples and 100% of swab samples (50/50) by PCR (Table 4). The risk factors associated with genital chlamydia infection were age, lower abdominal pain (PID), and urethritis (p<0.05; Table 5).

The sensitivity, specificity, PPV, and NPV of cytology in detecting chlamydia compared to PCR were 86.4%, 100%, 100%, and 83.6%, respectively (Table 6).

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 infection¹⁰. This is supported by results reported by Kucinskiene et al.¹¹ and Navarro et al.¹⁰, and other studies performed in Sudan⁶, Brazil¹², China¹³, and Peru¹⁴. In a previous study, many women who tested positive for CT reported increased risk of PID, ectopic pregnancy, and infertility (adjusted hazard ratio was 2.36, 1.87,and 1.85 respectively)¹⁵. For the current study, 81.1% of women positive for chlamydia were suffering from PID.

This study attempted to use the Giemsa staining technique as a non-invasive sample to detect CT in order to be used as an approach for screening program in Gezira state, where PCR is difficult to be applied. In our study, participant's swab samples tested by PCR showed that 84.6% of participants were positive, which is higher than that reported by Ortashi et al. (7.3%)¹⁶, Mohamed Elawad (49%)¹⁷, and Mohammed (22.5%)^7,16.

Infertility is a medical problem that affects more than 80 million people worldwide^18,19. Although few data exist on infertility in Sudan, 68.9% of Sudanese couples suffering primary infertility, due to various causes, was reported recently²⁰. Some researchers reported the link between chlamydia and both primary and secondary infertility^4,9,21. In this study, using PCR, the percentage of positive cases among primary infertile women was 75%, which is higher than that reported by Malik et al. (27%)²² and Gorini et al. (31.8%)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, respectively²³. 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 State²⁴. 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^25–30.

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 methods³⁵. 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.

Data availability