Human papillomavirus infection, herpes simplex virus type 2 seropositivity, and cervical cytological abnormalities among asymptomatic women in Ghana: a cross-sectional study

¹Department of Chemical Pathology, School of Medical Sciences, College of Allied Health Sciences, University of Cape Coast, Cape Coast, Ghana, ²Pharmacogenomics and Genomic Medicine Group, School of Medical Sciences, College of Health and Allied Sciences, University of Cape Coast, Cape Coast, Ghana, ³Department of Pathology, Faculty of Health Sciences, University of Cape Town, Anzio Road, Observatory, Cape Town, South Africa, ⁴Department of Medical Biochemistry, School of Medical Sciences, College of Health and Allied Sciences, University of Cape Coast, Cape Coast, Ghana, ⁵National Public Health and Reference Laboratory, Ghana Health Service, Accra, Ghana, ⁶University of Energy and Natural Resources, Sunyani, Ghana

^&Corresponding author
Oksana Ryabinina, Department of Chemical Pathology, School of Medical Sciences, College of Allied Health Sciences, University of Cape Coast, Cape Coast, Ghana

Introduction    

Cervical cancer remains a major public health concern globally and is a leading cause of cancer-related morbidity and mortality among women, particularly in low- and middle- income countries [1,2]. In Ghana, it ranks among the most common cancers affecting women, with an estimated 2,797 new cases and 1,699 deaths reported annually [3]. Despite ongoing prevention efforts, the burden of disease remains high, reflecting gaps in screening, early detection, and understanding of disease progression [1].

Persistent infection with Human papillomavirus (HPV) is well established as the main etiological factor for invasive and pre-invasive cervical cancer [4,5]. However, HPV infection alone may not be sufficient to cause malignant transformation, as many infected women do not develop cervical abnormalities or cancer [4,5]. This suggests that additional cofactors contribute to HPV persistence and progression to cervical neoplasia. Identified HPV co-factors include smoking, alcohol abuse, multiparity, long duration of oral contraceptive use, and other sexually transmitted pathogens [6]. Among these, herpes simplex virus type 2 (HSV-2), the principal cause of genital herpes, has been proposed as a potential cofactor in cervical carcinogenesis [7,8]. Biological mechanisms supporting this hypothesis include epithelial disruption caused by herpetic lesions, which may facilitate HPV entry into basal cells, as well as HSV-2-induced inflammatory responses that may impair immune-mediated clearance of HPV [9-11]. In addition, HSV-2 infection has been suggested to enhance HPV replication and promote integration of viral DNA into host cells, thereby increasing oncogenic potential [7,12,13]. Despite these plausible mechanisms, epidemiological evidence linking HSV-2 to cervical cytological abnormalities remains inconsistent [8,14].

Herpes simplex virus type 2 infection is highly prevalent worldwide, affecting an estimated 13% of individuals aged 15-49 years, with disproportionately higher rates observed among women and in sub-Saharan Africa [15,16]. Notably, most HSV-2 infections are asymptomatic, contributing to ongoing transmission and making detection challenging without serological testing [15,17]. In Ghana, while HPV prevalence has been reported in some populations, data on HSV-2 infection remain limited and are often derived from small or high-risk cohorts, restricting generalizability [18-20].

Although several studies have explored the relationship between human papillomavirus (HPV) and herpes simplex virus type 2 (HSV-2), most have focused on viral acquisition, transmission dynamics, or molecular interactions rather than clinically relevant outcomes such as cervical cytological abnormalities [5,7,9,10,21]. In addition, existing evidence has largely been derived from selected or high-risk populations, with limited data from asymptomatic women in general screening settings. The potential role of HSV-2 seropositivity as a biomarker for identifying women at increased risk of HPV persistence and cervical disease progression therefore remains insufficiently understood, particularly in low-resource settings where integrated screening strategies are needed. This study assessed the association between HPV infection, HSV-2 seropositivity, and cervical cytological abnormalities among asymptomatic women in Ghana, with the aim of determining whether HSV-2 provides additional value beyond HPV infection alone in identifying women at increased risk of cervical disease.

Methods     

We designed, conducted, and reported this cross-sectional study in accordance with the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines.

Study design and study sites: the study was a hospital based cross-sectional descriptive study, covering the period of 6 months (October 2014 - March 2015). In all, two hundred and forty (240) women attending routine Cervicare Clinics at the Kumasi South Regional Hospital, Kumasi, Ashanti Region, were enrolled in the study.

Study population: the participants were women who had come to Cervicare Clinics for visual inspection with acetic acid and/or to perform the pap smear test. There were no women with symptoms of cervical ulcer at the time of gynecological examination on recruitment into the study.

Inclusion criteria: participants were women aged 18 years and above, non-pregnant, who provided written informed consent and completed a pre-consent interview.

Exclusion criteria: women younger than 18 years, those who declined to provide written informed consent, or those unable to undergo the pre-consent interview were excluded. Pregnant women were excluded due to pregnancy-related physiological and immunological changes that may influence cervical cytology interpretation and HPV/HSV detection, potentially introducing bias in the assessment of associations. Women with a history of HPV vaccination were excluded to avoid potential confounding effects on HPV prevalence and genotype distribution. In addition, women with prior cervical surgical procedures were excluded because such interventions may alter cervical anatomy and affect cytological interpretation. Women without a cervix were also excluded, as cervical cytology assessment was not feasible.

Sample size: the sample size for this study was determined using Cochran's formula for estimating a single population proportion. Where n is the minimum sample size, Z is the standard normal deviate at 95% confidence level (1.96), p is the estimated prevalence of HPV-HSV coinfection, and d is the margin of error set at 5% (0.05). Because there is limited published data specifically reporting HPV-HSV coinfection in cervical cancer patients in Ghana, an estimated prevalence of 15% (p = 0.15) was adopted. This estimate is informed by evidence showing high HPV prevalence in cervical cancer cases in sub-Saharan Africa (approximately 85%-95%) and reported HSV-2 seroprevalence among women in the region ranging between 20% and 40%, suggesting a lower but meaningful overlap in coinfection patterns in cervical disease populations [19,22-25]. Substituting these values into the formula and accounting for potential non-response, missing laboratory results, or incomplete data, a 10% adjustment was applied. The minimum required sample size for this study was 218 participants.

Variables: the primary outcome variable in this study was cervical cytological status, classified based on pap smear results into normal cytology, cervicitis/inflammatory changes, and abnormal cytology (including low-grade and high-grade squamous intraepithelial lesions, etc.). For regression analysis, cytological abnormalities were dichotomized as normal/cervicitis versus abnormal cytology. The main exposure variables were HPV infection and HSV-2 seropositivity. HPV infection was defined as the detection of HPV DNA from cervical specimens and was further characterized by genotypes where applicable. HSV-2 seropositivity was determined by the presence of HSV-2-specific IgG antibodies detected, indicating prior or existing infection.

A key composite exposure variable, infection phenotype, was created to classify participants into mutually exclusive categories based on infection status (HPV only, HSV-2 only, co-infection, or no infection). Potential covariates considered in the analysis included age, sexual history variables (such as number of sexual partners), reproductive history, and other socio-demographic characteristics, which were assessed as potential confounders of the relationship between viral infections and cervical cytological abnormalities. All variables were defined a priori based on biological plausibility and existing literature on viral co-infections and cervical carcinogenesis.

Sampling and data collection: the study employed consecutive sampling techniques. At recruitment, all volunteers gave informed consent. A questionnaire (Annex 1) was designed purposely for the study and was pre-tested. Trained research assistants administered questionnaires through one-on-one interviews for data collection on sociodemographic and gynecological characteristics, sexual exposure, and medical history.

Sample collection: venous blood (5 ml) was drawn from all subjects for the analyses of HSV-2 IgG. The samples were allowed to clot before centrifugation. Serum obtained by centrifugation was aliquoted into Eppendorf tubes for storage at -70° C till analyzed.

Cytological specimens were obtained from all subjects. Samples of exfoliated cells from the ectocervix and endocervix were collected using the Ayre spatula and swab stick by trained nurses, using the Pap Pak cytology kit. One of the cervical specimens was suspended in DNAgard (Biomatrica Co., San Diego, USA) for DNA preservation at room temperature (for up to 3-6 months) until DNA extraction. Another cervical specimen was used to prepare the Papanicolau (Pap) smear. Cervical smears were prepared and examined independently by two experienced cytologists. Ten percent (10%) of randomly selected slides with normal cytology were reviewed by an experienced pathologist. All slides with epithelial cell abnormalities were referred to a qualified pathologist for final interpretation and report. Results from cervical cytology specimens were reported according to the 2001 Bethesda System Classification for reporting cervical cytology.

Visual Inspection with Acetic Acid (VIA) was performed as part of routine cervical cancer screening in a low-resource setting where cytology-based screening was also available. VIA was performed as a cervical cancer screening method. A trained healthcare provider applied 3-5% acetic acid (vinegar solution) to the cervix and observed the transformation zone after approximately one minute under adequate light, in accordance with WHO-recommended guidelines [26]. The test was considered positive when well-defined, dense acetowhite lesions were observed in the transformation zone or on the cervix, suggestive of possible precancerous changes. The test was considered negative when no acetowhite changes were seen. Only acetic acid was used. Lugol's iodine was not applied in this study. All findings were recorded immediately after examination.

Deoxyribonucleic acid (DNA) extraction: genomic DNA from cervical swabs was extracted using commercial spin-column based QIAamp Mini kit (QIAGEN, Hilden, Germany). The DNA was aliquot in duplicate (25 μl into each separate 2 ml tubes) and stored at -70°C until further analysis. The concentration of extracted DNA was determined by spectrophotometry at 260 nm. All samples were pre-screened with the human β-globin to assess sample integrity. Strict contamination control measures were observed throughout DNA extraction and polymerase chain reaction (PCR) procedures, and negative controls were included in each amplification run.

Herpes simplex virus type 2 IgG determination: the serum HSV-2 Ig G were determined by ELISA method using commercial test kits from Calbiotech Inc., CA, USA. The manufacturer's instructions were followed for analysis. Briefly, serum samples, calibrators, and positive and negative controls were tested in duplicate. After incubation and washing steps, enzyme conjugate and substrate were added sequentially, and optical density (OD) was measured at 450 nm using a reference wavelength of 600-650 nm within 15 minutes of stopping the reaction. Antibody index values were calculated based on the ratio of sample OD to the assay cut-off value provided by the manufacturer. Samples with an index >1.1 were considered positive, 0.9-1.1 equivocal, and <0.9 negative. Equivocal samples were retested using the same kit lot to ensure result consistency.

Deoxyribonucleic acid (DNA) genotyping: human papillomavirus-DNA detection and identification of the 18 genotypes was carried out by nested multiplex polymerase chain reaction (NMPCR) as previously described by Sotlar et al. [27], with minor laboratory adaptations. A consensus primer system targeting the E6/E7 region of HPV was used for general HPV DNA amplification, followed by type-specific multiplex PCR for genotyping. The second-round PCR enabled identification of high-risk HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59), probable high-risk types (66 and 68), and low-risk types (6/11, 42, 43, and 44). The amplified products were detected by 2% agarose gel electrophoresis for genotype determination.

Quality assurance: all laboratory procedures were conducted by trained personnel using standardized and validated protocols. HPV DNA extraction and genotyping were performed in a molecular laboratory using nested multiplex polymerase chain reaction (PCR) techniques with appropriate quality controls. HSV-2 serology was assessed using a validated enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions. To minimize measurement bias, laboratory personnel conducting HPV and HSV-2 analyses were blinded to participants' cytological results, and cytological evaluation was performed independently without knowledge of HPV or HSV-2 infection status. Standardized reporting criteria based on the Bethesda system were used for cytological interpretation. Internal quality control measures were applied throughout laboratory procedures to ensure consistency and reliability of results.

Bias and confounding: several potential sources of bias were considered in this study. Selection bias may have occurred, as participants were recruited from women attending cervical screening services and may not be fully representative of the general population. To minimize this, all eligible women presenting during the study period were consecutively enrolled. Information bias was reduced using standardized data collection procedures and validated laboratory assays for HPV and HSV-2 detection. Misclassification bias in cytological assessment was minimized by applying established diagnostic criteria based on the Bethesda system. However, some degree of misclassification cannot be excluded, particularly in cases where the transformation zone was not adequately sampled.

Due to the small number of abnormal cytology cases, multivariable analysis was limited to avoid model overfitting. As a result, residual confounding from unmeasured or incompletely measured factors may still be present. Additionally, the cross-sectional design limits the ability to establish temporal relationships between HPV infection, HSV-2 seropositivity, and cervical cytological abnormalities.

Ethical approval: the study was approved by the Committee on Human Research Publication and Ethics (CHRPE), Kwame Nkrumah University of Science and Technology, School of Medical Sciences (KNUST-SMS) and Komfo Anokye Teaching Hospital (KATH) (CHRPE/AP/115/14), Kumasi, Ghana and Ghana Health Service Ethical Review Committee, Research and Development Division (GHS-ERC:07/03/14) in accordance with the revised Helsinki Declaration of 1964 (revised 2013) on human experiments with the ethical clearance. Voluntary written informed consent was sought from the participants.

Statistical analysis: data were analysed and managed using Stata version 17 (Stata Corp, College Station, TX, USA). Categorical variables were summarized using frequencies and percentages, while continuous variables were described using means and standard deviations. Seroprevalence of HSV-2 was calculated as the proportion of positive cases, and participants were grouped according to HSV-2 infection status. Associations between HPV and HSV-2 infections (individually and jointly) and cervical cytological outcomes were assessed using Chi-square or Fisher's exact test, as appropriate. Multivariable analysis was performed using Firth penalized logistic regression to estimate adjusted odds ratios (aORs) and 95% confidence intervals (CIs), accounting for small-sample bias and mutual adjustment between HPV and HSV-2 infections. Sensitivity analyses were conducted using exact logistic regression and an interaction model to assess the robustness of findings and potential synergistic effects between HPV and HSV-2 infections. A p-value < 0.05 was considered statistically significant.

Results     

Sociodemographic, obstetric and behavioral characteristics of study participants: participants had a mean age of 41.1 years (SD ± 11.1; range 22-76) (Table 1). Most participants were married or cohabiting (63.3%), and a high proportion were literate (87.5%). The majority were economically active (84.6%), mainly engaged in trading (n = 116) and self-employment (n = 35). Within the participants 41.7% had gravidae 5 and more. The large proportion of women had their first pregnancy before 25 years (n = 131, 75.4%). The mean number of pregnancies was 4.3 (SD ± 2.8), while the mean number of children per woman was 2.7 (SD ± 1.9). Menopause was reported in 17.9% (43/240), with a mean age at menopause of 48.1 years (SD ± 9.1).

Most of participants had their first time of sexual intercourse before the age of 20 years (66.7%) with the mean age of 19.1 years (SD ± 3.7). Multiple lifetime sexual partners (>3) were reported by 44.2% of participants. Tobacco use was rare (n = 4), while 35.8% reported alcohol consumption. Contraceptive use was reported by 50.8%, with condom use in 30.4% and oral contraceptive use in 26.7%.

Prevalence of cervical cytology abnormalities: out of the 240 women who underwent cervical cancer screening using visual inspection with acetic acid (VIA) 92.1% (221/240; 95% CI: 88.0-95.0) tested negative and 7.9% (19/240; 95% CI: 5.0-12.0) tested positive (Table 2). All cervical cytology samples were adequate for interpretation. Most participants had normal cytology (NILM) (91.3%, 219/240; 95% CI: 86.9-94.5), while cervicitis/inflammatory changes were identified in 5.4% (13/240; 95% CI: 2.9-9.1) and abnormal cytology in 3.3% (8/240; 95% CI: 1.4-6.5).

Abnormal cytological findings were classified according to the Bethesda system [28]. Among abnormal cases, 3 (1.3%) were low-grade squamous intraepithelial lesions (LSIL), 1 (0.4%) was high-grade squamous intraepithelial lesion (HSIL), 2 (0.8%) were squamous cell carcinoma (SCC), and 2 (0.8%) were atypical squamous cells of undetermined significance (ASCUS). Bacterial vaginosis alone was observed in 2.5% (6/240) of participants, candidiasis alone in 3.3% (8/240), and co-infection with both conditions in 1.3% (3/240). The mean age of women with normal cytology was 41.0 years, compared to 43.3 years among those with abnormal cytology.

Agreement between VIA and cytology showed 92.1% overall agreement with a Cohen's kappa of 0.26 (SE = 0.06; p < 0.001), indicating fair agreement. Among VIA-positive women, 21.1% (4/19) had abnormal cytology, while 78.9% were cytologically normal. Additionally, four abnormal cytology cases were VIA-negative.

Prevalence and distribution of HPV DNA and HSV-2 IgG in cervical disease: human papillomavirus DNA was detected in 32.1% (77/240; 95% CI: 26.3-38.5) of participants. HPV prevalence varied by cytological category, being highest among women with abnormal cytology (50.0%), followed by normal cytology (32.0%) and cervicitis (23.1%). There was no statistically significant association between HPV infection and cytological outcome (χ² = 1.664; df = 2; p = 0.435) (Table 3).

High-risk HPV genotypes were predominantly observed among women with normal cytology, particularly HPV-52, HPV-56, HPV-58, and HPV-35. Although most high-risk HPV infections were detected in women with normal cytology, abnormal cytology was more frequently associated with specific oncogenic genotypes, including HPV-16 and HPV-33 (each 100% of detected cases), HPV-45 (50.0%), HPV-35 (18.2%), HPV-39 (14.3%), HPV-56 (12.5%), HPV-58 (10.0%), and HPV-52 (9.5%). Low-risk and probably high-risk HPV genotypes were largely confined to normal cytology and cervicitis, with no abnormal cytology cases observed in these categories. A detailed distribution of HPV genotypes by cytological outcome is provided in Annex 1. Overall, HSV-2 seropositivity was 84.5%. Seropositivity was 76.9% (10/13) among women with cervicitis/inflammatory changes and 87.5% (7/8) among those with abnormal cytology, compared with 85.4% (185/219) among women with normal cytology (NILM). However, this difference was not statistically significant (χ² = 0.594, df = 2, p = 0.743).

Infection phenotype distribution: participants were classified into four infection phenotypes: no infection, HPV-only infection, HSV-2-only infection, and HPV-HSV-2 co-infection. Overall, 55.8% (134/240) had HSV-2-only infection, 28.3% (68/240) had HPV-HSV-2 co-infection, 12.1% (29/240) had no infection, and 3.8% (9/240) had HPV-only infection (Table 3). Among abnormal cytology cases, distribution was 50.0% had HPV-HSV-2 coinfection, 37.5% had HSV-2-only infection, 12.5% had no infection and none had HPV-only infection. There was no statistically significant association between infection phenotype and cytological outcome (p = 0.557).

Association between infections and abnormal cervical cytology: the association between HPV and HSV-2 infections and abnormal cervical cytology was assessed using Firth penalized logistic regression (Table 4). HPV infection showed a positive but non-significant association with abnormal cervical cytology compared to HPV-negative women (aOR = 2.14, 95% CI: 0.56-8.14; p = 0.27). HSV-2 seropositivity was not independently associated with abnormal cytology (aOR = 0.89, 95% CI: 0.15-5.34; p = 0.896). The wide confidence intervals observed reflect the limited number of abnormal cases (n = 8) and corresponding statistical uncertainty.

Sensitivity analysis using exact logistic regression produced consistent findings. HPV infection was not significantly associated with abnormal cytology (OR = 2.17, 95% CI: 0.39-11.99; p = 0.442), and HSV-2 seropositivity also showed no significant association (OR = 1.33, 95% CI: 0.16-61.46; p = 1.000).

No statistically significant multiplicative interaction between HPV and HSV-2 infections was observed in Firth penalized logistic regression analysis (interaction OR = 2.62, 95% CI: 0.07-94.12; p = 0.598). Sensitivity analysis using exact logistic regression for co-infection similarly showed no significant association with abnormal cytology (OR = 2.61, 95% CI: 0.47-14.47; p = 0.228) (Annex 1).

Discussion     

This study found a high prevalence of HPV infection alongside a low prevalence of cervical cytological abnormalities among asymptomatic women in Ghana, with no significant association between HPV, HSV-2 seropositivity, and cytological outcomes. Within the framework of the World Health Organization cervical cancer elimination strategy, these findings underscore the challenges of effective risk stratification in resource-limited settings where screening coverage remains suboptimal [29,30].

In this study, the prevalence of cervical cytological abnormalities was low, consistent with other studies in Ghana [31,32]. The predominance of normal cytology reflects the asymptomatic nature of the study population. However, the presence of cervicitis and inflammatory changes suggests ongoing reproductive tract infection that may proceed or coexist with cervical epithelial abnormalities. The relatively high prevalence of HPV infection among women with normal cytology further reinforces the well-established observation that HPV infection may exist in the absence of cytological abnormalities.

The VIA results showed a higher proportion of positive findings compared to cytology-based abnormalities. This discrepancy likely reflects the higher sensitivity but lower specificity of VIA in detecting cervical epithelial changes, particularly inflammatory lesions that are not necessarily neoplastic [33,34]. In resource-limited settings, VIA remains a valuable screening tool; however, its limitations highlight the importance of confirmatory cytological or molecular testing to improve diagnostic accuracy [35,36].

The absence of transformation zone components in some Pap smear samples may have reduced diagnostic sensitivity, as inadequate sampling of this region is known to contribute to false-negative results [28,37]. This limitation is particularly relevant across age groups where anatomical variation may affect adequate sampling [38]. These findings support the use of combined screening approaches to improve detection performance in asymptomatic populations.

Human papillomavirus infection was common in this study population, and high-risk genotypes were frequently detected even among women with normal cytology. This pattern is consistent with the natural history of HPV infection, where viral presence often precedes the development of cytological abnormalities [39,40]. The predominance of oncogenic genotypes such as HPV-16 and HPV-18 among abnormal cases aligns with their established role in cervical carcinogenesis [22,41,42]. The presence of multiple HPV infections in some participants with abnormal cytology is consistent with evidence suggesting that multiple infections may contribute to viral persistence, although this relationship remains incompletely understood [22,41,42].

Herpes simplex virus type 2 seroprevalence was high in this study, consistent with regional epidemiological patterns in sub-Saharan Africa [15,16,43]. Although HSV-2 has been biologically proposed as a cofactor in cervical carcinogenesis through mechanisms such as epithelial disruption, chronic inflammation, and immune modulation [8,21,44,45], this study did not demonstrate a statistically significant association between HSV-2 seropositivity and cervical cytological abnormalities. This finding aligns with several previous studies that have not identified HSV-2 as an independent risk factor for cervical cancer development [14]. The lack of association may be explained by the fact that HSV-2 IgG seropositivity reflects past exposure rather than active infection [17,46], which may be more relevant in influencing HPV persistence and disease progression. Additionally, the low prevalence of abnormal cytology may have limited statistical power to detect modest associations.

Although HPV and HSV-2 co-infection was observed in a considerable proportion of participants, no significant association with abnormal cytology was found. This finding is consistent with studies suggesting that HSV-2 may facilitate HPV acquisition through mucosal disruption but does not independently drive cervical cytological changes [14,21,44]. However, the low frequency of co-infection in this study limits firm conclusions regarding combined effects. Regression analysis using Firth penalized logistic regression indicated increased odds of abnormal cervical cytology among women with HPV infection, although this was not statistically significant. HSV-2 seropositivity was not independently associated with cervical cytological outcomes. The wide confidence intervals observed reflect the small number of abnormal cases and the resulting statistical uncertainty. These findings are consistent with the established role of HPV as the primary etiological agent in cervical carcinogenesis [47], and highlight the limitations of cross-sectional data in assessing risk relationships. Sensitivity analysis using exact logistic regression produced results consistent with those obtained from the Firth logistic regression model, further supporting the robustness of the findings despite sparse data. The agreement between both approaches suggests that the observed null associations are unlikely to be driven by small-sample bias.

No statistically significant multiplicative interaction between HPV and HSV-2 infections was observed, suggesting no evidence of effect modification by HSV-2 serostatus within this study population. Similar findings have been reported in other population-based studies where co-infection did not significantly modify cervical disease risk [14,48,49]. These findings support the interpretation that HPV and HSV-2 infections exert independent effects on cervical cytological outcomes within this study population.

The findings suggest that HSV-2 serostatus does not add predictive value for identifying women at risk of cervical cytological abnormalities beyond HPV infection. This has important public health implications, as it supports prioritizing HPV-based screening and prevention strategies in cervical cancer control programs in Ghana and similar settings.

This study has several limitations. First, the cross-sectional design limits the ability to establish temporal relationships between HPV infection, HSV-2 seropositivity, and cervical cytological abnormalities. Second, the relatively small number of abnormal cytology cases reduced statistical power and limited more detailed multivariable modelling. Third, recruitment from a hospital-based screening population may introduce selection bias, limiting generalizability to the wider population. In addition, HSV-2 seropositivity was assessed using IgG antibodies, which reflect prior exposure rather than active infection and may not accurately capture the temporal dynamics relevant to HPV persistence and disease progression. Although standardized laboratory procedures were used, some degree of cytological misclassification cannot be excluded, particularly in cases with inadequate transformation zone sampling.

Despite these limitations, this study has notable strengths. It provides data from asymptomatic women, a population that is often underrepresented in studies of HPV and HSV-2 co-infection. The simultaneous assessment of HPV DNA, HSV-2 serostatus, and cytological outcomes provides a comprehensive evaluation of infection patterns. The use of Firth penalized logistic regression strengthens the robustness of estimates in the context of rare outcomes. Additionally, the classification of infection phenotypes and inclusion of HPV genotyping enhance the epidemiological value of the findings.

Conclusion     

Human papillomavirus infection remains positively associated with cervical cytological abnormalities, although not statistically significant in this study. HSV-2 seropositivity was not independently associated with cytological abnormalities, either alone or in combination with HPV infection. These findings suggest that HSV-2 serostatus does not contribute additional predictive value for cervical disease detection in this population. The findings highlight the established role of HPV in cervical carcinogenesis and support continued emphasis on HPV-based screening and prevention strategies. Further longitudinal studies are needed to clarify whether HSV-2 plays any role in HPV persistence or cervical disease progression under specific conditions.

Competing interests     

The authors declare no competing interests.

Authors' contributions     

Oksana Ryabinina contributed to the conception and design of the study, data acquisition, analysis, interpretation of data, and drafting of the manuscript; Gifty Boateng and Emmanuel Timmy Donkoh contributed to data acquisition and analysis; Nicholas Ekow Thomford and Samuel Badu Nyarko also contributed to interpretation of data; Nicholas Ekow Thomford, Samuel Badu Nyarko, Gifty Boateng, and Emmanuel Timmy Donkoh contributed to critical revision of the manuscript for important intellectual content. All the authors contributed to the final review of the manuscript, read and approved the final version of this manuscript to be published, and agree to be accountable for all aspects of the work, ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Acknowledgments     

We wish to acknowledge all the study participants.

Tables     

Table 1: demographic, obstetric and behavioral characteristics of study participants, recruited from the Cervicare Clinics at the Kumasi South Regional Hospital, Kumasi (Ghana), from October 2014 - March 2015 (N=240)

Table 2: results of visual inspection with acetic acid (VIA) and pap smear screening among women recruited from Cervicare Clinics at Kumasi South Regional Hospital, Kumasi, Ghana, from October 2014 to March 2015 (N = 240)

Table 3: distribution of human papillomavirus (HPV) and herpes simplex virus type 2 (HSV-2) infection patterns according to cervical cytology outcomes among women recruited from Cervicare Clinics at Kumasi South Regional Hospital, Kumasi, Ghana, from October 2014 to March 2015 (N = 240)

Table 4: association between human papillomavirus (HPV) and herpes simplex virus type 2 (HSV-2) infections and abnormal cervical cytology among women recruited from Cervicare Clinics at Kumasi South Regional Hospital, Kumasi, Ghana, from October 2014 to March 2015 (N = 240)

Annex     

Annex 1: supplementary materials (PDF 305KB)

References