High-risk human papillomavirus and HBV, HCV, and T. pallidum association among women living with HIV in selected health facilities of Mekelle, Tigray, Northern Ethiopia.

The study included 390 women living with HIV giving a response rate of 96.5% (390/404). Majority of the participants were urban dwellers 88.5% (345/390), had a normal body mass index 59.2% (231/390), and 35.1% of them were married (137/390). As to their level of education is concerned, 34.9% (136/390) of the respondents had completed elementary education, and 43.1% (168/390) identified themselves as housewives. The median age of the participants was 42 years, with an interquartile range of 14 (Table  1 ). Out of the 390 women living with HIV and tested HR-HPV, HBV, HCV and T. pallidum , 39.7% (155/390) of the study population were positive for HR-HPVs. Additionally, 18.97% (74/390) of the study population were positive for the other STI pathogens (HBV, HCV and T. pallidum ), collectively. Each prevalence rates were found to be 10.3% (40/390, 95% CI: 7.4–13.1) for T. pallidum , 7.2% (28/390, 95% CI: 4.6–9.7) for HBV and 1.5% (6/390, 95% CI: 0.5–2.8) for HCV. All women (100%, 6/6) with serological evidence of anti-HCV antibodies have had HCV RNA levels of < 12 IU/mL by RT-PCR, indicating an active HCV infection with a low viral load for quantification. Additionally, 100% agreement between non-terponemal and treponemal antibody tests for syphilis was observed, indicating active/recent T. pallidum infections. Figure  2 illustrates the frequency of each pathogen in the total population (Fig.  2 ). Fig. 2 The frequency of HCV, HBV, T. pallidum and HR-HPV among women living with HIV in Mekelle, Tigray, Northern Ethiopia, 2025 ( n  = 390). The frequency of HCV, HBV, T. pallidum and HR-HPV among women living with HIV in Mekelle, Tigray, Northern Ethiopia, 2025 ( n  = 390). Regarding the prevalence of STI pathogens in relation to sociodemographic characteristics, 10.7% (37/345) of urban residents were seropositive for T. pallidum , while 7.2% (25/345) of them were seropositive for HBV. Notably, HBV positivity was higher among certain demographics, including women who were not married (16.7%, 8/48), those who can read and write (12.2%, 6/49), self-employed women (16.7%, 1/6), the overweight (11.3%, 8/71), and women aged 35 to 49 years (8.7%, 18/207). Additionally, 11.3% (8/71) of women with obesity tested positive for HCV. T. pallidum positivity was elevated among respondents with no formal education (13.0%, 12/92), farmers (15%, 3/20), those with obesity (27.3%, 3/11), and women aged 50 to 66 years (12.1%, 14/116) (Table  1 ). Table 1 Sociodemographic characteristics of the study participants and distribution of STI pathogens among women living with HIV in Mekelle, Tigray, Northern Ethiopia, 2025 ( n  = 390). Characteristics Number (%) HBV HCV T. pallidum P (%) N (%) T P (%) N (%) T P (%) N (%) T Residence Urban 345 (88.5) 25 (7.2) 320 (92.8) 345 (100) 5 (1.4) 340 (98.6) 345 (100) 37 (10.7) 308 (89.3) 345 (100) Rural 45 (11.5) 3 (6.7) 42 (93.3) 45 (100) 1 (2.2) 44 (97.8) 45 (100) 3 (6.7) 42 (93.3) 45 (100) Marital status Married 137 (35.1) 9 (6.6) 128 (93.4) 137 (100) 2 (1.5) 135 (98.5) 137 (100) 15 (10.9) 122 (89.1) 137 (100) Not married 48 (12.3) 8 (16.7) 40 (83.3) 48 (100) 1 (2.1) 47 (97.9) 48 (100) 5 (10.4) 43 (89.6) 48 (100) Divorced 119 (30.5) 9 (7.6) 110 (92.4) 119 (100) 3 (2.5) 116 (97.5) 119 (100) 11 (9.2) 108 (90.8) 119 (100) Widowed 86 (22.1) 2 (2.3) 84 (97.7) 86 (100) 0 86 (100) 86 (100) 9 (10.5) 77 (89.5) 86 (100) Educational level No formal education 92 (23.6) 7 (7.6) 85 (92.4) 92 (100) 0 92 (100) 92 (100) 12 (13) 80 (87) 92 (100) Read and write 49 (12.6) 6 (12.2) 43 (87.8) 49 (100) 1(2) 48 (98) 49 (100) 5 (10.2) 44 (89.8) 49 (100) Elementary 136 (34.9) 11 (8.1) 125 (91.9) 136 (100) 2 (1.5) 134 (98.5) 136 (100) 15 (11) 121 (89) 136 (100) Secondary school 61 (15.6) 3 (4.9) 58 (95.1) 61 (100) 1 (1.6) 60 (98.4) 61 (100) 5 (8.2) 56 (91.8) 61 (100) College and above 52 (13.3) 1 (1.9) 51 (98.1) 52 (100) 2 (3.8) 50 (96.2) 52 (100) 3 (5.8) 49 (94.2) 52 (100) Employment status House wife 168 (43.1) 12 (7.1) 156 (92.9) 168 (100) 1 (0.6) 167 (99.4) 168 (100) 20 (11.9) 148 (88.1) 168 (100) Farmer 20 (5.1) 0 20 (100) 20 (100) 1 (5) 19 (95) 20 (100) 3 (15) 17 (85) 20 (100) Merchant 92 (23.6) 8 (8.7) 84 (91.3) 92 (100) 1 (1.1) 91 (98.9) 92 (100) 8 (8.7) 84 (91.3) 92 (100) Governmental employee 42 (10.8) 2 (4.8) 40 (95.2) 42 (100) 3 (7.1) 39 (92.9) 42 (100) 3 (7.1) 39 (92.9) 42 (100) Non-governmental employee 16 (4.1) 0 16 (100) 16 (100) 0 16 (100) 16 (100) 2 (12.5) 14 (87.5) 16 (100) Daily laborer 46 (11.8) 5 (10.9) 41 (89.1) 46 (100) 0 46 (100) 46 (100) 4(8.7) 42 (91.3) 46 (100) Self-employed 6 (1.5) 1 (16.7) 5 (83.3) 6 (100) 0 6 (100) 6 (100) 0 6 (100) 6 (100) Body mass index (BMI) Underweight 77 (19.7) 6 (7.8) 71 (92.2) 77 (100) 6 (7.8) 71 (92.2) 77 (100) 6 (7.8) 71 (92.2) 77 (100) Normal weight 231(59.2) 14 (6.1) 217 (93.9) 231 (100) 14 (6.1) 217 (93.9) 231 (100) 22 (9.5) 209 (90.5) 231 (100) Overweight 71 (18.2) 8 (11.3) 63 (88.7) 71 (100) 8 (11.3) 63 (88.7) 71 (100) 9 (12.7) 62 (87.3) 71 (100) Obesity 11 (2.8) 0 11 (100) 11 (100) 0 11 (100) 11 (100) 3 (27.3) 8 (72.7) 11 (100) Age in years 18–34 67 (17.2) 3 (4.5) 64 (95.5) 67 (100) 1 (1.5) 66 (98.5) 67 (100) 7 (10.4) 60 (89.6) 67 (100) 35–49 207 (53.1) 18 (8.7) 189 (91.3) 207 (100) 3 (1.7) 204 (98.3) 207 (100) 19 (9.2) 188 (90.8) 207 (100) 50–66 116 (29.7) 7 (6) 109 (94) 116 (100) 2 (1.7) 114 (98.3) 116 (100) 14 (12.1) 102 (87.9) 116 (100) N: Negative, P: Positive, T: Total. Sociodemographic characteristics of the study participants and distribution of STI pathogens among women living with HIV in Mekelle, Tigray, Northern Ethiopia, 2025 ( n  = 390). N: Negative, P: Positive, T: Total. Among the 155 h-HPV-positive women, the combined prevalence of HBV, HCV, or T. pallidum positivity was 34.19% (53/155). Specifically, 2.6% (4/155), 14.2% (22/155), and 17.4% (27/155) of the HR-HPV positive respondents were seropositive for HCV, HBV, and T. pallidum , respectively (Table  2 ). However, of the total population (390), the prevalence of HBV/HR-HPV, HCV/HR-HPV, and T. pallidum /HR-HPV co-infections among the study population was 5.6% (22/390), 1.02% (4/390), and 6.9% (27/390), respectively (Table  2 ). Furthermore, while no triple infections and no HCV/ T. pallidum con-infections observed, there were HCV/HBV (0.25% (1/390)) and HBV/ T. pallidum (0.76% (3/390)) co-infections. Table 2 Prevalence of HR-HPV/HBV/HCV/ T. pallidum co-infections among women living with HIV in Mekelle, Tigray, Northern Ethiopia, 2025 ( n  = 390). Other STIs status HR-HPV status, n (%) Positive Negative Total HBV, n (%) Positive 22 (14.2) 6 (2.6) 28 (7.2) Negative 133 (85.8) 229 (97.4) 362 (92.8) Total 155 (100) 235 (100) 390 (100) HCV, n (%) Positive 4 (2.6) 2 (0.9) 6 (1.5) Negative 151 (97.4) 233 (99.1) 384 (98.5) Total 155 (100) 235 (100) 390 (100) T. pallidum , n (%) Positive 27 (17.4) 13 (5.5) 40 (10.3) Negative 128 (82.6) 222 (94.5) 350 (89.7) Total 155 (100) 235 (100) 390 (100) Prevalence of HR-HPV/HBV/HCV/ T. pallidum co-infections among women living with HIV in Mekelle, Tigray, Northern Ethiopia, 2025 ( n  = 390). Of the total study participants (390), the he highest co-infection rates were observed between T. pallidum and other (non-18/16) HR-HPV genotypes at 4.8% (19/390), followed by HBV with other HR-HPV genotypes at 3.8% (15/390). T. pallidum and HPV-16 co-infection reached 3.1% (12/390), while HBV- HPV-16 co-infection were noted at 2.5% (10/390). The lowest coinfection rate was identified between HCV-HPV-18 at 0.25% (1/390) and HBV-HPV-18 at 0.25% (1/390). Among the HR-HPV positive group, the highest coinfection percentages were recorded for HPV-16 with T. pallidum at 28.5% (12/42), and HPV-16 with HBV at 23.8% (10/42) (Fig.  3 ). Fig. 3 The percentages of HCV, HBV, and T. pallidum coinfections with HR-HPV types among women living with HIV in Mekelle, Tigray, Northern Ethiopia, 2025( n  = 390). The percentages of HCV, HBV, and T. pallidum coinfections with HR-HPV types among women living with HIV in Mekelle, Tigray, Northern Ethiopia, 2025( n  = 390). The chi-square test showed a statistically significant association between HBV-HR-HPV ( p  < 0.001) and T. pallidum -HR-HPV combinations ( p  < 0.001). However, in Fisher’s exact test, there was no statistically significant association between HCV and HR-HPV ( p  = 0.221). Binary logistic regression analysis revealed a strong association between HR-HPV positivity and both HBV and T. pallidum . Those women living with HIV and reactive for HBsAg have had 6.52 times higher odds of HR-HPV DNA positivity (AOR = 6.52, 95% CI: 2.55–16.67, p  < 0.001). Additionally, the odds of HR-HPV positivity were 3.84 times (AOR = 3.84, 95% CI: 1.89–7.79, p  < 0.001) higher among T. pallidum seropositive women living with HIV compared to those women seronegative for T. pallidum (Table  3 ). Table 3 Bivariate and multivariate analysis of the association between HBV, HCV, T. pallidum and HR-HPV positivity among women living with HIV in Mekelle, Northern Ethiopia, 2025 ( n  = 390). Other STIs HR-HPV status, n (%) Bivariate analysis Multivariate analysis Positive Negative Total COR[95%CI] P AOR[95%CI], P HBV status, n (%) Positive 22 (14.2) 6 (2.6) 28 (7.2) 6.31 [2.49–15.96] < 0.001 6.52 [2.55–16.67] < 0.001* Negative 133 (85.8) 229 (97.4) 362 (92.8) 1 Total 155 (100) 235 (100) 390 (100) HCV status, n (%) Positive 4 (2.6) 2 (0.9) 6 (1.5) Not included Not included Negative 151 (97.4) 233 (99.1) 384 (98.5) Total 155 (100) 235 (100) 390 (100) T. pallidum , n (%) Positive 27 (17.4) 13 (5.5) 40 (10.3) 3.60 [1.79–7.95] < 0.001 3.84 [1.89–7.79] < 0.001* Negative 128 (82.6) 222 (94.5) 350 (89.7) 1 Total 155 (100) 235 (100) 390 (100) CI: Confidence interval, COR: Crude Odds Ratio, AOR: Adjusted Odds Ratio, *indicated statistical significance at p  < 0.05; 1 indicates the reference category. Bivariate and multivariate analysis of the association between HBV, HCV, T. pallidum and HR-HPV positivity among women living with HIV in Mekelle, Northern Ethiopia, 2025 ( n  = 390). CI: Confidence interval, COR: Crude Odds Ratio, AOR: Adjusted Odds Ratio, *indicated statistical significance at p  < 0.05; 1 indicates the reference category. In this section, the statistical analysis is intended to explore the presence of statistically significant associations between HBV/HCV/ T. pallidum and the specific HR-HPV types/groups designated as HPV-16, HPV-18 and other HR-HPVs (pool of non-16/18 HR-HPVs). In the Chi-square/Fisher’s exact test analysis, significant associations at p  < 0.05 were observed in HBV-HPV-16 ( p  < 0.001), HBV-other HR-HPVs ( p  = 0.020), HCV-HPV-16 (Fisher’s exact test, p  = 0.019), T. pallidum -HPV-16 ( p  < 0.001), and T. pallidum -other HR-HPVs ( p  = 0.04) combinations (Table  4 ). Table 4 Association of HBV, HCV, and T. pallidum with specific HR-HPV types using Chi-square/Fisher’s Exact Test among women living with HIV in Mekelle, Tigray, Northern Ethiopia, 2025 ( n  = 390). HPV-16 HPV-18 Other HR-HPVs P (%) Neg. (%) T (%) P-value P (%) N (%) T (%) P-value P (%) N (%) T (%) P-value HBV Positive 10 (35.7) 18 (64.3) 28 (100) < 0.001* 1(3.6) 27 (96.4) 28 (100) 1.000 15 (53.6) 13 (46.4) 28 (100) 0.020* Negative 32 (8.0) 330 (91.2) 362 (100) 1 13(3.6) 349 (96.4) 362 (100) 1 112 (30.9) 250 (69.1) 362 (100) 1 HCV Positive 3 (50) 3 (50) 6 (100) 0.019* 1(16.7) 5 (83.3) 6 (100) 0.198 2 (33.3) 4 (66.7) 6 (100) 1.000 Negative 39 (10.2) 345 (89.8) 384 (100) 1 13(3.4) 371 (96.6) 384 (100) 1 125 (32.6) 259 (67.4) 384 (100) 1 T. pallidum Positive 12 (30.0) 28 (70.0) 40 (100) < 0.001* 3(7.5) 37 (92.5) 40 (100) 0.162 19 (47.5) 21 (52.5) 40 (100) 0.049* Negative 30 (8.6) 320 (91.4) 350 (100) 1 11(3.1) 339 (96.9) 350 (100) 1 108 (30.9) 242 (69.1) 350 (100) 1 P values: generated from Chi-square or Fisher’s exact test; * p-value < 0.05 indicates statistical significance; N=Negative; P=Positive; T=Total; “ 1” is the reference group. Association of HBV, HCV, and T. pallidum with specific HR-HPV types using Chi-square/Fisher’s Exact Test among women living with HIV in Mekelle, Tigray, Northern Ethiopia, 2025 ( n  = 390). P values: generated from Chi-square or Fisher’s exact test; * p-value < 0.05 indicates statistical significance; N=Negative; P=Positive; T=Total; “ 1” is the reference group. In the bivariate logistic regression analyses, both HBV and T. pallidum were found to be significantly associated ( p  < 0.05 ) with HPV-16 and other HR-HPVs (Table  5 ). Although a significant association between HCV and HPV-16 was noted via Fisher’s exact test, it did not remain significant in bivariate logistic regression after bootstrap analysis, leading to its exclusion from the multivariate analysis. In the multivariate analysis, HBV and T. pallidum were both significantly associated with HBV-16 and other HR-HPV genotypes. HBV positive women living with HIV are significantly more likely to test positive for HPV-16 and other high-risk HPVs compared to HBV negative individuals, with an odds ratio of 6.33 and 2.59, respectively. Furthermore, those who were seropositive for T. pallidum also show increased odds of HPV-16 and other high-risk HPVs positivity at 5.01 times and 2.04 times compared to seronegative respondents (Tables  5 and 6 ). Table 5 Bivariate and multivariate analysis of the association between HBV/ T. pallidum and HPV-16 among women living with HIV in Mekelle, Tigray, Northern Ethiopia, 2025 ( n  = 390). HPV-16 Bivariate analysis Multivariate analysis Positive (%) Negative (%) Total (%) COR[95%CI] P value AOR[95%CI] P value HBV Positive 10 (35.7) 18 (64.3) 28 (100) 5.72 (2.43–13.45) < 0.001 6.33 (2.60-15.41) 0.001* Negative 32 (39.0) 330 (91.2) 362 (100) 1 1 T. pallidum Positive 12 (30.0) 28 (70.0 40 (100) 4.57 (2.11–9.90) < 0.001 5.01 (2.24–11.20) < 0.001* Negative 30 (8.6) 320 (91.4) 350 (100) 1 1 AOR: Adjusted odds ratio, COR: Crude odds ratio; * P < 0.05 indicates statistical significance; “ 1” is the reference group. Bivariate and multivariate analysis of the association between HBV/ T. pallidum and HPV-16 among women living with HIV in Mekelle, Tigray, Northern Ethiopia, 2025 ( n  = 390). AOR: Adjusted odds ratio, COR: Crude odds ratio; * P < 0.05 indicates statistical significance; “ 1” is the reference group. Table 6 Bivariate and multivariate analysis of the association between HBV/ T. pallidum and other HR-HPV types among women living with HIV in Tigray, Northern Ethiopia, 2025 ( n  = 390). Other HR-HPVs Bivariate analysis Multivariate analysis Positive (%) Negative (%) Total (%) COR[95%CI] P value AOR[95%CI] P value HBV Positive 15(53.6) 13(46.4) 28(100) 2.57(1.18–5.59) 0.017 2.59(1.19–5.66) 0.017* Negative 112(30.9) 250(69.1) 362(100) 1 1 T. pallidum Positive 19(47.5) 21(52.5) 40(100) 2.02(1.04–3.92) 0.036 2.04(1.05–3.97) 0.036* N egative 108(30.9) 242(69.1) 350(100) 1 1 AOR: Adjusted odds ratio, COR: Crude odds ratio; Other HR-HPVs = non HPV-16 or HBV-18 pool of high-risk HPV types; * P < 0.05 indicates statistical significance; “ 1” is the reference group. Bivariate and multivariate analysis of the association between HBV/ T. pallidum and other HR-HPV types among women living with HIV in Tigray, Northern Ethiopia, 2025 ( n  = 390). AOR: Adjusted odds ratio, COR: Crude odds ratio; Other HR-HPVs = non HPV-16 or HBV-18 pool of high-risk HPV types; * P < 0.05 indicates statistical significance; “ 1” is the reference group.

A facility-based cross-sectional study was conducted from December 10, 2024, to July 10, 2025, in various governmental healthcare facilities in Mekelle City, Northern Ethiopia (administratively a Special Zone). Mekelle, the capital city of the Tigray Region, is located roughly 783.18 km north of Addis Ababa. The City comprises 15 governmental health facilities, including a comprehensive specialized teaching hospital, two general hospitals, two primary hospitals, and ten health centers. According to the Tigray Health Bureau Report, there are approximately34, 511 individuals living with HIV who are receiving treatment through the highly active antiretroviral therapy (HAART) program in Tigray, with 15,638 HAART clients being utilizing the governmental health facilities in City/Special Zone. The current study considered women living with HIV in Mekelle Special Zone as a source population, while the study population were women living with HIV who attended their HAART follow-up during the study period and tested for HPV in the selected governmental health centers and hospitals of Mekelle Special Zone. All women aged 18 years and older living with HIV, receiving HAART follow-up, non-pregnant, and qualified for HPV screening were part of the study. The study excluded those with no prior sexual experience, individuals who had undergone ablative or surgical procedures for precancerous or cancerous cervical lesions, those who had received a complete hysterectomy, participants vaccinated for HBV within the last 15 days, and anyone unable to provide cervical or blood samples for valid medical reasons. All women living with HIV who underwent cervical swab collection for HR-HPV screening ( n  = 390) were part of the study. To achieve the largest possible sample size, we considered the estimated prevalence of each pathogen within the study population, due to the absence of published data on the prevalence of coinfections with HR-HPV. In comparison to the prevalence of HBV/HCV/ T. pallidum among women living with HIV, the estimated prevalence of HR-HPV-HIV coinfection (35.2%) in Southern Ethiopia 40 allowed for a larger sample size for this investigation. Therefore, the sample size was determined using a single population proportion formula (at a 95% confidence interval and precision (d) of 0.05; Z at \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\raisebox{1ex}{$\alpha$}\!\left/\!\raisebox{-1ex}{$2$}\right.$$\end{document} =1.96): \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\boldsymbol{n}=\frac{{\boldsymbol{Z}}_{\raisebox{1ex}{$\boldsymbol{\alpha}$}\!\left/\!\raisebox{-1ex}{$2$}\right.}^{2}\boldsymbol{P}(1-\boldsymbol{P})}{{\boldsymbol{d}}^{2}}=350$$\end{document} . After incorporating a 15% contingency, the final sample size reached 404. Proportional allocations were designated to each of the five conveniently chosen health centers and hospitals based on the nearest three-month average number of HAART visits of the eligible HIV-positive women ( N  = 4838), which was taken from an official unpublished report of Tigray Regional Health Bureau (TRHB). Ultimately, consecutive sampling was employed to collect data and samples from 390 women who attended the HAART centers (Fig.  1 ). Fig. 1 Sampling frame and proportional allocation to sample size. Sampling frame and proportional allocation to sample size. The primary outcome variable was high-risk HPV (HR-HPV) infection status, classified as “positive” or “negative” based on real-time PCR assay results. Independent variables included coinfections of HBV, HCV, or T. pallidum , recorded as “seropositive” or “seronegative”. Sociodemographic factors were assessed as continuous (age and body mass index (BMI)), and categorical variables, including residence (rural or urban), marital status (married, not married, divorced, or widowed), educational level (no formal education, read and write, primary, secondary, or college and above), and current occupational status (e.g., housewife, farmer, merchant, driver, governmental/non-governmental employee, daily laborers and self-employed). Trained midwife nurses used a pretested semi-structured questionnaire (adapted from previous studies 40 – 42 to assess sociodemographic variables through direct interviews of the eligible and consented respondents. After the questionnaire-based assessments, each respondent was directed to provide cervical and venous blood samples. With proper orientation and following standard operating procedures of the kit, a trained midwife nurse for HR-HPV testing collected cervical material using the Abbot Collection and transportation kit. Additionally, Medical Laboratory Technologists collected 10 ml venous blood into one EDTA anti-coagulated and second anticoagulant-free test tube for detecting HBV, HCV, and syphilis. All specimens got labels of the client’s unique ART codes and medical record numbers. The test tubes containing blood were left upright for 10–15 min in a tube rack on a flat surface to settle without disturbance; then, high-speed centrifugation for up to 5000 revolutions per minute for 10 min was carried out to separate the serum. The serum and plasma were transferred into a new 2 ml capacity Nunk tube, clearly labelled and stored at negative 20 degrees Celsius until transportation. On the same day of collection, all samples were transported immediately to the Tigray Health Research Institute (THRI) for analysis in a cold chain and with a paper-based referral linkage form. Parallel to this, cervical material in transport kits was transported to THRI in a triple package cold chain. All specimens were stored at -80 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$℃$$\end{document} until analysis. The principal investigator, and according to the manufacturer’s directions for each rapid test kit, performed the serological testing towards detecting HBV, HCV and T. pallidum (syphilis) infection. Hepatitis B surface antigen (HBsAg) was detected in a 100uL of serum using a highly sensitive (97.1%) and specific (99.4%) rapid immunochromatographic assay (One-step HBsAg whole blood/serum/plasma test, Guangzhou Wondfo Biotech Co., Ltd). The serological investigation of syphilis was performed using a combination of non-treponemal (Rapid Plasm Reagin (RPR)) and treponemal tests in parallel. After bringing all reagents and the patient’s serum to room temperature, a non-treponemal antibody test was performed using RPR in 50 µL of serum. Additionally, T. pallidum specific antibodies were detected in a 10 µL of serum using the syphilis anti- T. pallidum Test kit (Bioline™ Syphilis 3.0, Abbott Diagnostics Korea Inc.). The test uses recombinant T. pallidum antigens, making it highly accurate (99.0%) in detecting anti- T. pallidum antibodies of IgG, IgA, and IgM nature. The test has a sensitivity and specificity of 99.3% and 99.5%, respectively. Final confirmation of the T. pallidum-specific antibodies was done in Mekelle Blood Bank Center using a Wantai anti- T. pallidum Enzyme-Linked Immunosorbent Assay (ELISA) in repeated measurement and as per the standard protocols of the kit (Beijing Wantai Biological Pharmacy Enterprise Co., Ltd.). HCV-specific antibody detection was done from a 10-µL serum using the Bioline™ HCV test kit, Abbott Diagnostics Korea Inc. This highly sensitive (100%) and specific (99.4%) immunochromatographic test kit uses pre-coated recombinant HCV capture antigens of core and non-structural proteins (NS4 and NS55). All samples with serological evidence of HCV were further confirmed by nucleic acid test (HCV RNA) in plasma using the Real-time HCV assay (Abbot Molecular Inc., Des Plaines, IL 60018, USA), and as per the kit protocols. Additionally, the researcher, under the verification of the senior technical team at THRI, used the Abbot Real-time high-risk HPV assay in the m2000sp automated nucleic acid extractor and m2000rt RT-PCR (Abbot Park, Illinois, USA) to detect HR-HPV. This assay showed reproducible and consistent analytical performance 43 , and the extraction and amplification were done according to standard molecular protocols, as described previously 44 – 47 . The data were coded and entered into Epi Info™ version 7.2.7 and then exported to SPSS version 27 for analysis. Descriptive statistics computed frequencies and percentages of HBV, HCV, T. pallidum , and HR-HPV positivity, and participants’ sociodemographic characteristics. Coinfection rates were also assessed using the cross-tabulations of HBV-HR-HPV, HCV-HR-HPV, and T. pallidum -HR-HPV, with a further cross-tabulation of the HR-HPV types. To analyze the statistical correlation of each non-HPV STI pathogen with HR-HPV, the chi-square test was used to identify independent associations between each categorical variable (combinations) 18 . When the number of cells with event counts of less than five surpassed 20%, Fisher’s exact test was applied instead of the chi-square test 48 . Therefore, the chi-square/Fisher’s exact test was primarily required to screen for the presence of possible associations between two categorical variables and to handle cells with a small number of events in the two-by-two table, which were not suitable for the logistic regression model (for instance, HCV and HPV-18). Significantly associated ( p  < 0.05) variables in the chi-square/Fisher’s exact test were reanalyzed using a bivariate and multivariate logistic regression model to generate adjusted odds ratios (AOR) and assess the strength of association between each pathogen and HR-HPV. Model fitness was verified via the Hosmer-Lemeshow model fitness test ( p  > 0.05). Multi-collinearity between independent variables (HBV/HCV/ T. pallidum ) was assessed using the chi-square (Phi/Cramer’s V values) or Fisher’s exact test (no significant correlation affecting the regression model was observed). Hence, fulfilling these assumptions, the logistic regression model generated unbiased odds ratios and confidence intervals to declare the strength and direction of the association. Variables demonstrating significance at p  < 0.05, with 95% confidence intervals not crossing “1,” progressed forward to multivariate analysis. Statistically significant associations were established at p  < 0.05, along with their respective 95% confidence interval. Ensuring data quality began with piloting the questionnaire (5%), training data collectors and supervisors, specimen quality, proper cold chain shipping, and internal quality control of testing kits using known positive and negative sera from both THRI and Mekelle Blood Bank center. In case of any abnormal lines (ghost, partial or invisible) during serologic testing, the test was repeated with careful pipetting and timing. The adequacy and quality of each sample, code correspondence, and questionnaire legibility were all validated on the spot by senior supervisors in the field. To prevent centrifugation, pipetting, testing, and interpretation errors, specific standard operating procedures in the field and the manufacturer’s directions for each kit were permanently displayed at the testing sites. Finally, an expert from the THRI team verified all laboratory results.

The study highlights significant associations between HR-HPV, HBV and T. pallidum among women living with HIV. However, the association between HCV and HR-HPV may require further investigation using large sample size. High co-infection rates of HBV and T. pallidum with HR-HPVs indicate a significant health challenge in the HIV positive women population, which may be complicating disease management and accelerating disease progression. These findings emphasize the necessity of integrated and regular screening for these sexually transmitted infections, as well as prompt treatment, counselling on safe sexual practices and partner tracing. Further prospective research should concentrate on the effects of concurrent infections.

The present study examined and provided evidence on the prevalence of HCV, HBV, and T. pallidum co-infections alongside high-risk HPVs in women living with HIV, while evaluating statistically significant relationships between these types of STI pathogens and HR-HPV infections. The study revealed that the highest prevalence of co-infection was observed between T. pallidum and HR-HPV (6.9%), followed by HBV and HR-HPV (5.6%). In contrast, the lowest prevalence of co-infection was found with HCV and HR-HPV at 1.02%. The study reveals that 17.4% of HR-HPV-positive women are seropositive for T. pallidum , slightly exceeding the rates observed in Mozambique (13.7%) 18 , but comparable with the findings in Brazil, 15-17.2% 49 , 50 . A Chinese study among HIV-positive women reported a higher co-infection rate of HR-HPV and Syphilis (39.5%) 51 . Regarding the risk association, the odds of HR-HPV positivity was 3.84 times (AOR = 3.84) higher among T. pallidum seropositive women living with HIV compared to those seronegative for T. pallidum . However, the evidence regarding the association between syphilis and HR-HPV positivity is heterogeneous in the existing literature. Previous studies in Ethiopia 52 , Rwanda 53 and Brazil 50 found no significant associations between T. pallidum and HR-HPV positivity, whereas a study in Mozambique 18 demonstrated statistically significant associations. Contrastingly, a study in South Africa reported no T. pallidum co-infection despite the HR-HPV and HIV associations, likely due to the detection method (PCR-based detection of T. pallidum ) used and duration with HIV 19 . Variations in population and sample size may account for differing results, as some studies (Rwanda) focused solely on HIV negative women 53 , or included enrolled women attending gynecologic clinics (Ethiopia) regardless of their HIV status 52 . Other recent findings in Ethiopia’s Shashemene (AOR = 3.12) and Bahir Dar (AOR = 17.06) suggested that the odds of HR-HPV positivity was significantly higher among women with a history of STI compared to those without a history of STI women with of positivity, though the specific STI etiologies, including T. pallidum not detailed 40 , 54 . In light of our findings, there is supporting evidence in the published literature; T. pallidum infection can significantly influence the risk of HPV, both directly and indirectly. Individuals with syphilis face double risk of acquiring HIV and other STIs 55 . Co-infection with Syphilis complicates immune recovery compared to HIV alone and accelerates disease progression 8 , 56 . Furthermore, both HIV and Syphilis notably increase the likelihood of HR-HPV infections, with HIV raising the risk by 11 times and Syphilis by 5.5 times. This synergistic interaction can lead to the development of genital lesions, with the co-presence of T. pallidum , HR-HPV and HIV raising the risk of high-grade cervical intraepithelial neoplasms by 18 times 57 . Additionally, weakened immune responses in women with HIV can lead to persistent HR-HPV infections, increasing the susceptibility to T. pallidum infection 28 . These findings imply the importance of focused prevention and management strategies in regions with a high burden of T. pallidum and, particularly, sub-Saharan Africa, which could simultaneously benefit efforts in controlling both infections 58 – 60 . A high percentage of HBV infection (14.2%) was observed compared to HCV (2.6%) among the HR-HPV-positive women living with HIV. The study revealed a higher co-infection rate of HBV with HR-HPV (5.6%) in contrast to HCV with HR-HPV (1.02%). Supporting these findings, 18.5% of HBV-positive women living with HIV developed HR-HPV infection in China 61 , while 11% of women living with HIV were co-infected with HBV and HPV in Cameroon; these women show increased liver cancer markers 62 . Furthermore, a study in Burkina Faso supports our finding in that 18% of individuals treated for chronic HBV/HCV infection were found to be positive for HR-HPV strains, with HIV serving as a significant risk factor 48 . Recent studies from South Africa and Gabon reported rates of HBV (3.9% in South Africa) and HR-HPV (51.2% in South Africa; 72% in Gabon) independently among HIV-positive women, though the co-occurrence was not distinctly addressed; The context of the respondents was also different (HIV \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm$$\end{document} pregnancy) 63 , 64 . Additionally, clustered occurrence of HIV-HBV-HCV-HPV (5.8%) was identified in Brazil, though the co-infection rates were not aggregated by gender 65 . Overall, the evidence indicated a substantial burden of these infections (HBV and HR-HPV) among women with HIV in Africa, highlighting the necessity for integrated screening and management strategies for these triple STIs 66 , 67 . The low rate of HCV and HPV observed in the current study may be attributed to the lower prevalence of HCV compared to HBV in the region. However, it may have high clinical significance. Notably, HR-HPV-positive women had a 24 times (AOR = 24.00) significantly higher odds of liver disease attributable to HCV, especially in the context of liver transplantation candidates in the USA 26 . HIV infection may facilitate persistent HPV infection due to immunosuppression and superinfection potential 28 , 68 , while chronic hepatitis infections may similarly mediate immune response weakening through the regulatory T-cell-mediated downregulation of cellular immunity. This can lead to concomitant HPV infections 26 . Hence, in the study area, the synergistic effect of HIV and HBV in the context of conflict-associated poor adherence to HAART 69 could have contributed to the high rate of HBV-HR-HPV infection. The current study indicated that HBV-infected women living with HIV have had a 6.31 times (AOR = 6.31) higher odds of HR-HPV positivity, consistent with the findings in China linking HBV co-infection with elevated HR-HPV risk (AOR = 3.48) 61 . The association between HR-HPV and other STIs varied by HR-HPV types. The odds of HPV-16 positivity was 4.57 times (AOR = 4.75) significantly higher among women co-infected with HBV compared to HBV negative women. Similarly, T. pallidum seropositive women have had 4.57 times (AOR = 4.57) significantly higher odds of HPV-16 positivity compared to seronegative women. This aligns with studies highlighting a significantly (19 times) higher risk of high-grade cervical lesions in HIV-HPV-16-affected women in China 70 . A similar mechanism could apply to HPV-18, but lower co-infection rates may have affected the statistical significance of HPV-18. The current study also revealed that odds of positivity for non-16/18 HR-HPV types was significantly higher among women with HBV (AOR = 5.01) and T. pallidum (AOR = 2.04), compared to women with negative test results for HBV and T. pallidum , respectively. This finding emphasizes the need for targeted genotyping and risk assessments for various HPV strains. For instance, the association of T. pallidum with HPV-31, HPV-33, HPV-35, HPV-51, HPV-52, HPV-58, HPV-66, HPV-68, and HPV-73 were reported by others 18 . In the context of HIV, the correlation of T. pallidum , HCV, and HBV can potentially increase the risk of acquiring other STIs, as well as HPV-related cervical aberrations and malignancies due to compromised immunity. For instance, a case-control study in China revealed that compared to women with benign tumors, the odds of cervical cancer were 58.5 times (AOR = 58.5) higher for those with HPV alone, and 67.1 times (AOR = 67.1) higher for those co-infected with HBV and HPV 71 . This indicates heightened vulnerability to cervical cancer in case of HBV-HPV coinfections 23 , 71 . Furthermore, associations have been identified between HCV and non-Hodgkin’s lymphoma, as well as the impact of HBV infections on the survival rates of Hodgkin’s lymphoma in HIV infected individuals 72 . A study conducted in Taiwan highlighted that among individuals with HIV, the presence of HCV (50%), HBV (44.2%) and syphilis (35.9%) was associated with increased odds of testing positive for other bacterial STIs 73 . The study also highlighted that the odds of the additional STIs were higher among individuals with HBV (AOR = 2.76) and HCV infections (AOR = 5.62) 73 . Such associations can potentially cause abrasions of the genitalia and may facilitate HPV acquisition. Furthermore, a study in Gondar, Ethiopia, revealed that 16.6% of cervical cancer suspects tested positive for HIV, HBV, HCV and T. pallidum infection, suggesting shared risk factors and a possible link to HPV 35 . Women living with HIV and with a history of STI exhibited higher odds of HR-HPV across studies in Ethiopia 40 , 44 , 45 . These findings emphasize the urgent need for further research and policy initiatives addressing the interplay of these viral STIs (HCV, HBV, and HPV) and T. pallidum , among women living with HIV, particularly given the connections between increased HPV prevalence and various bacterial infections 74 – 76 . The study demonstrates strengths, including the provision of evidence regarding co-infections supported by statistical associations, which holds great public health and clinical relevance, especially in evidence-scarce or developing settings. However, the study also presents limitations inherent to cross-sectional designs, notably the difficulty of establishing a temporal relationship between variables, such as co-infection types and HR-HPV positivity. Additionally, the research was restricted to conveniently selected health facilities and enrolled the respondents consecutively.

Sexually transmitted infections (STIs) pose a major worldwide health concern, carrying serious social, economic and health consequences 1 . STIs directly affect sexual and reproductive health by contributing to discrimination, malignancies, obstetric complications and death 2 . Globally, an estimated 289 million people contracted STIs in 2021, with increasing trends of infection in females 3 . T. pallidum , Hepatitis B virus, Hepatitis C virus, and Human Papillomavirus (HPV) are among the repertoire of STI etiologies, especially among people engaged in risky sexual behaviors, including unsafe sexual intercourse, and circumstances posing trauma or lesions in the genitalia 1 . According to the World Health Organization (WHO), in 2022, T. pallidum accounted for around eight million adult infections, HPV was attributed to around 300 million infections and 311,000 cervical cancer-related mortalities among women, and HBV were responsible for 254 million chronic hepatitis cases and over a million deaths 2 . Similarly, the WHO estimated that around 50 million people experienced chronic HCV infection in 2022, with approximately one million new infections occurring each year, and 242,000 mortalities 4 . Globally, there were increasing incidences (1990 through 2019) of acute (46% increase) and chronic HCV infections (72% increase) among women of reproductive age group, sub-Saharan Africa taking the highest shares 5 . These alarming figures contrast with the WHO’s ambition to combat and eliminate HBV and HCV by 2030 6 . HIV is among the top viral STIs; HIV positive individuals could have a different picture of other STIs due to weak immunity and risky engagements. Untreated STIs can speed up the progression of HIV disease, whereas HIV infection can make the diagnosis and treatment of STIs more complex 7 . In sub-Saharan Africa, people living with HIV experience a high burden of STIs, including syphilis 8 . Global figures indicated that the highest prevalence of T. pallidum -HIV coinfection (10.5%) was documented in the East Africa region, and compared to HIV negative women, HIV positive women had a 4.7 times higher risk of T. pallidum infection 9 . Furthermore, in Ethiopia, around 6.6% of people living with HIV were affected by HCV 10 , which reflects a demand for an integrated response towards STIs in key populations 10 , 11 . Evidence regarding the relationship between HBV, HCV, T. pallidum , and high-risk HPV (HR-HPV) infection among women living with HIV is limited, despite the established presence of co-infections such as HIV-HBV, HIV-HCV, and HIV- T. pallidum , HIV-HPV, and their associated adverse disease features 12 – 15 . STI co-infections with HR-HPV in females can potentially amplify the risk and progression of cervical abnormalities through various mechanisms, including abrasions of the mucosal epithelium, sustained HPV infections as a result of weak immunity, and delayed immune clearance 16 , 17 . According to a study in Africa, the association of HPV with other STI etiologies, including HIV and T. pallidum , was 50% among women with gynecologic complaints with statistically significant correlations 18 . Furthermore, HIV positivity, a history of frequent sexual intercourse, and an STI syndrome (vaginal discharge) were shown to increase the odds of HR-HPV, which could indicate the co-occurrence of other STI etiologies and massive cervical inflammation 19 . Because HPV is linked with increased pelvic inflammatory disease, endometriosis, tubal infertility 20 , and multiple cancers of the genitalia and extra genital sites, simultaneous occurrence of other oncogenic virus infections can aggravate disease complications and the risk of HPV associated malignancies 21 , 22 . For instance, a significant association between the two oncogenic viruses HBV and HPV infections was reported, and a synergistic effect on the odds of all types of cervical dysplasia; compared to HPV mono-infections, co-infected women exhibited 11.49 times higher odds of cancerous cervical lesions 23 . HCV was also significantly correlated with HPV-associated head and neck cancers 24 , and it has been proposed that HCV’s nonstructural protein (NS 5 B) could be involved in the damage of the tumor suppressor protein RB (retinoblastoma protein) through recruiting HPV oncoprotein E6 25 . On the other hand, HR-HPV can significantly increase the odds of HCV-attributable liver disease, in which HCV infection could have dampened cellular immunity, favoring persistent HR-HPV infections 26 . Hence, this can have severe outcomes in the context of women living with HIV. Overall, about 40% of HIV associated cancers are attributable to oncogenic viruses, including HPV, HBV, and HCV 27 . Consequently, HIV-positive women have a three to six times increased risk of contracting HPV and developing severe cervical lesions 28 . On top of this, the presence of other cancer-causing viruses as co-infections may further enhance the likelihood of various cancers. This heightened risk can be linked to low CD 4 + counts, persistent inflammatory responses, exposure to viral proteins, weakened epithelial structures, and the cumulative effects of mixed infections 28 , 29 . This implicates the necessity of investigating STI co-infections for optimal HIV care and reducing the probabilities of HPV-linked cervical cancer in the HIV positive women population. In Ethiopia, HIV-HCV, HIV-HBV, and HIV- T. pallidum co-infections were documented in various regions of the country with a relatively higher rate of HBV-HIV and HIV- T. pallidum coinfections compared to HCV-HIV co-infection 30 – 34 . However, there is a paucity of data on the HBV/HCV/ T. pallidum coinfections with HR-HPV. A study conducted in north west Ethiopia indicated that around 16.6% of women with cervical cancer suspects were infected by STIs, collectively HIV, HBV and HCV 35 . Furthermore, on average, 15% of HIV positive women in Ethiopia experienced precancerous cervical lesions 36 , and around 72% of those with signs of cervical dysplasia had abnormal pathologic features 37 , which might have also been affected by HPV. Another nationwide study implicated that the rate of HBV-HIV co-infection among females was 4.8%, and the triple rate of infection (HBV- T. pallidum and HIV) was at 1.08%. Most importantly, these co-infections were significantly associated with inflammation, immunosuppression and unsuppressed viral load 34 , which might be fertile grounds for persistent HPV infections. Generally, there is low knowledge and practice of cervical cancer screening among HIV positive women in Ethiopia 38 . Hence, HR-HPV and other STI co-infections might have remained underreported. In Tigray, the prevalence of HBV-HIV and HCV-HIV infections stands at 10% and 3.6%, respectively, with HIV viral load suppression and a history of STIs identified as significant predictors for these infections 39 . Despite the importance of understanding co-infections, no studies have yet investigated the association of HBV, HCV, and T. pallidum with HR-HPV among women living with HIV. This knowledge is crucial for reducing malignancies in the HIV-positive female population, especially in regions affected by conflict. Therefore, the current study seeks to explore this association in selected governmental health facilities in Mekelle, Tigray, Northern Ethiopia.