Introduction

Sexually transmitted diseases (STDs) are a global public health concern, with a significant impact on individuals, communities, and healthcare systems. STDs encompass a wide range of infections transmitted primarily through sexual contact, including bacterial, viral, and parasitic etiologies [1]. These infections pose substantial challenges due to their prevalence, potential for long-term complications, and their role in facilitating the transmission of other infections, such as HIV [2]. Effective prevention, diagnosis, and treatment strategies are crucial to mitigate the burden of STDs and protect the overall health and well-being of individuals and populations [3]. In 2023, the efforts have mainly been concentrated on overcoming the catastrophic burden of the COVID-19 pandemic, but sexually transmitted infections still remain one of the most prevalent health issues worldwide [4].

Syphilis, a sexually transmitted infection caused by Treponema pallidum have been reported for centuries affecting millions of people worldwide [5]. The transmission of syphilis primarily occurs through various forms of sexual contact, encompassing vaginal, anal, and oral routes. Vertical transmission from an infected mother to her fetus during pregnancy or during the birthing process is another significant mode of transmission [6]. Syphilis infection manifests in a progressive manner, characterized by distinct clinical stages: primary, secondary, latent, and tertiary syphilis. Each stage presents with characteristic clinical features, ranging from localized genital ulcers and skin rashes to multiple organ system involvement [5]. Congenital syphilis, resulting from the vertical transmission of Treponema pallidum, poses significant risk to the fetus during pregnancy [7]. In the absence of appropriate treatment, a high proportion of infants born to mothers with untreated early syphilis, ranging from 70% to 100%, may acquire the infection. Tragically, up to one-third of these infected infants may experience stillbirths, signifying the need for effective screening and proper management of syphilis in pregnant women [8].

The prevalence of syphilis among pregnant women in India was documented as 0.38% [9], while the combined prevalence of syphilis among pregnant women in sub-Saharan Africa was found to be 2.9% [10]. The World Health Organization (WHO) has developed guidelines emphasizing the importance of routine testing and treatment for both infections as part of comprehensive ante-natal care (ANC) services (WHO, 2016). By integrating syphilis screening into routine ANC check-ups, healthcare systems can make significant strides towards achieving the goal of elimination [11].

The identification of syphilis depends on assessment of symptoms, clinical examination, and serological analysis. One of the widely utilized serological tests is the Venereal Disease Research Laboratory (VDRL) test, which is employed for both screening and monitoring syphilis infection [12]. Although the VDRL test is highly sensitive in detecting early-stage syphilis, there are instances when it may produce false positive results, posing a significant diagnostic challenge that necessitates additional investigation [13]. In recent years, the Treponema pallidum hemagglutination assay (TPHA) has emerged as an alternative serological test with improved specificity and sensitivity in syphilis diagnosis [14].

The biological false positive (BFP) test for syphilis is characterized by a reactive nontreponemal test result but a non-reactive treponemal test result. In other words, it exhibits positive results in the initial nontreponemal tests, such as the VDRL test or Rapid Plasma Reagin (RPR) test, but shows negative results in the subsequent treponemal-specific tests, such as TPHA [15]. This phenomenon arises from the cross-reactivity of phospholipid antibodies, which are targeted by nontreponemal tests, with diverse non-syphilis-related conditions like systemic lupus erythematosus, rheumatoid arthritis, malaria, tuberculosis, viral hepatitis etc. Apart from these conditions, technical anomalies during testing procedures and inherent biological variations also contribute to BFPs [16]. Detecting BFP in pregnancy is of utmost importance as BFP can lead to unnecessary treatment, increased psychological stress and potential harm to both the pregnant woman and the fetus [17].

Understanding the occurrence and factors contributing to BFP results in pregnant women can aid in developing strategies to improve the specificity of syphilis testing, ensuring appropriate treatment decisions, and optimizing the health outcomes for both the mother and the unborn child. The aim of this study was to determine the frequency of biological false-positive among VDRL reactive pregnant females.

Materials and Methods

This was a retrospective study conducted in the serology laboratory under the Department of Microbiology. In this study, we analyzed the results of parallel VDRL and TPHA testing of a total of 11, 198 blood samples from female patients between January 2021 and December 2022. Patients' sera with incomplete data on stage and sex and duplicate sera were excluded from this study. The sera obtained from female patients were further grouped into antenatal cases (n = 3402) and non-ANC which included outpatient department (OPD) along with STIs clinic OPD (n = 7040) and different indoor patient ward (n = 756). Blood samples were collected under aseptic precaution in plain vials without any anticoagulant. The clotted blood samples were subjected to centrifugation. The sera of blood samples were separated and VDRL was performed as per manufacturer’s instructions (Trepolipin- Modified VDRL, Tulip diagnostics Pvt Ltd, India). Specimens which were reactive by the VDRL screening test were further subjected to quantitative VDRL test with successive 2-fold dilutions of the serum in 0.9% saline. TPHA test was performed on all the sera reactive in qualitative VDRL test irrespective of their VDRL titer for confirmation of presence of anti-treponemal antibody by using TPHA Test Kit (Plasmatec Laboratory Products Ltd., Dorset). All the results of VDRL test were grouped in two categories, i.e., VDRL titer ≥1:8 and <1:8 for evaluation of TPHA results and for assessment of biological false positive results.

The retrospective nature of this study utilized existing data collected as part of routine investigation protocol. All data analyzed in this study were obtained from pre-existing datasets or anonymized records, and the study did not involve any direct interaction with human subjects.

Data Management and Statistical Analysis

Descriptive statistics were performed to summarize the VDRL reactivity status among the pregnant and non-pregnant females. The prevalence of VDRL reactivity was determined by calculating the percentage of positive results within each group. Statistical analysis, including chi-square tests or Fisher's exact tests, was performed to assess the association between variables, such as VDRL titer and TPHA reactivity. The P-values were calculated to determine the statistical significance (P-value < 0.05) of the observed associations.

Results

A total of 11,198 females were tested for the presence of syphilis by VDRL test over a period of two years. Among the total samples analyzed, a total of 3,402 samples were obtained from the pregnant females, while 7,796 samples were from the other non-pregnant females. Table 1 depicts the VDRL reactivity status among the pregnant and the non-pregnant females.

Among the pregnant females, 41 (1.21%) tested positive for VDRL. In contrast, VDRL reactivity was observed in 70 (0.9%) of the non-pregnant females. The comparison of TPHA reactivity in VDRL-reactive pregnant females across different age groups has been shown in Table 2.

Notably, the highest prevalence of VDRL reactivity was observed in the 20-29 age group, with 11 individuals (1.21%) testing reactive. Table 3 depicts the comparison of TPHA reactivity in VDRL-reactive non- pregnant females across different age groups (n=70).

Table 4 shows the relationship between the VDRL titer and TPHA reactivity in pregnant and non- pregnant females. Among the pregnant females, we observed that 7.2% tested positive for TPHA with a VDRL titer <1:8, while 19.8% tested negative for TPHA indicating BFP with the same VDRL titer. Similarly, among the pregnant females with a VDRL titer ≥1:8, 5.4% were positive for TPHA, and 4.5% were negative for TPHA indicating BFP.

Discussion

Syphilis continues to pose a substantial public health challenge, especially in the context of pregnancy, due to its potential to result in detrimental consequences for both the maternal and foetal health if not adequately addressed. Our findings revealed important insights into the burden of syphilis infection and the biological false positive reactivity in VDRL test among pregnant females. Our study included a large sample size of 11,198 females tested for syphilis over a two- year period. Among these samples, 3,402 were obtained from pregnant females, while the remaining 7,796 were from non-pregnant females. The results demonstrated a higher proportion of pregnant females (1.21%) testing reactive for VDRL test compared to non-pregnant females (0.9%). This indicates a slightly higher burden of syphilis among pregnant individuals in our population that could be attributed to several factors. Physiological changes during pregnancy, such as alterations in the immune system, hormonal fluctuations, and changes in sexual behaviour may influence the susceptibility to syphilis infection and subsequent VDRL reactivity [18]. To gain a comprehensive understanding of the underlying mechanisms and implications of the higher prevalence of VDRL reactivity among pregnant females, additional research is warranted.

It is important to note that the VDRL test, while widely used for syphilis screening, has limitations in terms of specificity and potential false-positive results. Therefore, a reactive VDRL test alone is not sufficient for a definitive diagnosis of syphilis [19]. Confirmatory tests, such as TPHA are typically performed to confirm syphilis infection. The analysis of VDRL reactivity in our study among the pregnant females in different age groups revealed varying prevalence rates. The higher prevalence of VDRL reactivity in the 20-29 age group in our study is consistent with previous studies that have reported increased rates of syphilis among young adults [20]. A study conducted in a similar population in North-West Ethiopia reported a syphilis prevalence rate of 1.5% among pregnant females in the 20-29 age group, which is in line with our findings [21]. Several factors could contribute to the higher prevalence of VDRL reactivity in the young age- group. Firstly, this age group often engages in increased sexual activity and may have a higher number of sexual partners, which increases the likelihood of exposure to syphilis [22]. Additionally, inconsistent condom usage and limited knowledge about sexually transmitted infections may further contribute to the higher risk of syphilis in this population [23]. The variations in VDRL reactivity rates across different age groups highlight the importance of age- specific screening and intervention strategies for syphilis during pregnancy.

The results presented in Table 4 provide valuable insights into the relationship between VDRL titer and TPHA positivity and their significance in the detection of syphilis infection. However, statistical analysis using the chi-square test revealed a P-value of 0.262, indicating no significant association between VDRL titer and TPHA reactivity in both pregnant and non-pregnant females. Among the pregnant females, we observed that 7.2% tested positive for TPHA with a VDRL titer <1:8. This indicates the presence of true positive results for syphilis infection in this subgroup. However, it is important to note that a substantial proportion (19.8%) of pregnant females with VDRL titer <1:8 tested negative for TPHA, suggesting the possibility of BFP.

Similarly, among the pregnant females with a VDRL titer ≥1:8, 5.4% were positive for TPHA, indicating true positive results for syphilis. However, it is noteworthy that 4.5% of pregnant females with a VDRL titer ≥1:8 tested negative for TPHA, indicating the presence of BFP. The findings from this study indicate a significant difference in the percentage of BFP reactivity between VDRL titers. Samples with VDRL titers less than 1:8 showed a higher rate of BFP reactivity (19.8%), suggesting a potential limitation of the VDRL test at lower titers. On the other hand, samples with titers ≥1:8 exhibited a lower BFP reactivity rate (4.5%). A false positive VDRL result in these cases can occur due to various factors, such as cross-reactivity with other conditions or non-specific immune responses [16]. In contrast, comparing the results with the study by Bala et al, it becomes evident that the VDRL test's specificity varies with different titers. In their study, the overall reactivity rate was 1.4%, with 0.2% of samples having a titer ≥1:8 and 1.2% of samples with a titer <1:8. Interestingly, all 12 strongly reactive samples (titer ≥1:8) in their study were confirmed as true syphilis cases using TPHA. However, they found two cases with very high VDRL titers (1:64 and 1:256) among the strongly reactive samples, indicating advanced syphilis infection [20]. Variability in the results could be attributed to the difference in the population being studied along with the socio-demographic as well as geographical differences.

The occurrence of BFP in syphilis testing is a known challenge in the diagnosis and management of the infection, particularly in pregnant females. The observed difference in BFP reactivity rates emphasizes the need for caution in diagnosing syphilis solely based on the VDRL test, particularly at lower titers. The discrepancies between VDRL and TPHA results emphasize the need for comprehensive serological testing algorithms to accurately diagnose and differentiate true positive syphilis cases from BFP cases [20]. It is crucial to consider both the VDRL titer and TPHA reactivity to make informed clinical decisions regarding the diagnosis and treatment of syphilis in pregnant females. Further research is warranted to explore the underlying factors contributing to BFP reactivity and to optimize syphilis screening algorithms for better patient care.

Conclusion

In conclusion, our study highlights the association between VDRL titer and TPHA reactivity in pregnant females. The results indicate the presence of BFP in a significant proportion of pregnant females with both lower and higher VDRL titers. These findings emphasize the importance of implementing comprehensive testing strategies that incorporate both non-treponemal and treponemal tests to improve the accuracy of syphilis diagnosis, particularly in pregnant populations.

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