Analytical performance of a low-cost multiplex polymerase chain reaction human papillomavirus genotyping assay for use in Sub-Saharan Africa.

We have tested a multiplex polymerase chain reaction (PCR) human papillomavirus (HPV) genotyping assay to fill the need for rapid and low-cost HPV detection in Sub-Saharan Africa. This method allows high throughput genotyping and simultaneous detection of 14 high-risk and two low-risk HPV types, by PCR amplification of HPV DNAs in a single reaction tube. In this study, we describe stepwise experiments to validate the multiplex HPV PCR assay for determination of HPV genotypes from 104 cervical brush samples from Tanzanian women. Assay performance was evaluated by determination of intra-laboratory reproducibility, sensitivity, and specificity. Further performance was assessed by comparison with the widely accepted and validated HPV My09/My11 amplification and hybridization assay. Statistics; the Cohen kappa (κ) and McNemar P values were used to analyze interobserver and intermethod agreement. Overall concordance between the multiplex and line blot hybridization assays was 99% (per sample) with a κ value equal to 0.95; and 96.49% (per detection event) with a κ value of 0.92. Interobserver reproducibility of the assay per sample was 95.76% with κ of 0.91. These results demonstrate that the multiplex HPV PCR assay has high analytical sensitivity and specificity in detecting as many as 16 different HPV genotypes and that its simplicity and low cost makes it well suited for sub-Saharan Africa.

Clinicopathological Characteristics and Outcomes of Anal Squamous Cell Carcinoma Patients With and Without HIV Infection in Sub-Saharan Africa.

PURPOSE: In the past 20 years, the burden of anal cancer (AC) increased by 60% in the United States and over three-fold in Africa. Rates of AC have increased by 20× in people living with HIV and the highest (50×) in men with HIV who have sex with men. However, in sub-Saharan Africa (SSA) where HIV is endemic, data on clinicopathological characteristics and outcomes of patients with AC are lacking. To address this, we have investigated AC disease presentation, treatment outcomes, and its predictors in a cohort of patients who were either HIV-infected or HIV-uninfected in SSA. METHODS: We conducted a retrospective cohort study of patients with anal squamous cell carcinoma (SCC) treated at Ocean Road Cancer Institute in Dar es Salaam, Tanzania from January 2014 to December 2019. Associations between the study outcomes and their predictors were analyzed using univariate and multivariate analysis models. RESULTS: A total of 59 patients with anal SCC were retrieved and had at least 2-year follow-up. The mean age was 53.9 (standard deviation ±10.5) years. While none of the patients presented with stage I disease, 64.4% had locally advanced disease. HIV infection was the major comorbidity (64.4%). The rate of complete remission at the end of treatment was at 49% while the 2-year overall survival (OS) and local recurrence-free survival were 86.4% and 91.3%, respectively. Despite high HIV coinfection in the cohort, AC treatment outcomes were not significantly associated with HIV status. Disease stage (P = .012) and grade (P = .030) were significantly associated with 2-year OS. CONCLUSION: Patients with anal SCC in Tanzania present mainly with locally advanced disease associated with high HIV prevalence. In this cohort, the SCC grade was independently associated with treatment outcomes unlike other factors such as HIV coinfection.

Epstein-Barr Virus, But Not Human Papillomavirus, Is Associated With Preinvasive and Invasive Ocular Surface Squamous Neoplasias in Zambian Patients.

Background: The etiopathogenesis of ocular surface squamous neoplasia (OSSN) is not fully understood. We assessed the frequency of oncogenic viruses in OSSN by immunohistochemistry (IHC) and polymerase chain reaction (PCR) for human papillomavirus (HPV), Epstein-Barr virus (EBV), Merkel cell polyomavirus (MCPyV), Kaposi sarcoma virus, and adenovirus. Cases from Zambia were prospectively enrolled using a cross-sectional study design between November 2017 and March 2020. Methods: Demographic and clinical data [age, sex, HIV status, antiretroviral therapy (ART) history, CD4 count, plasma viral load] and tumor biopsies were collected from 243 consenting patients. Tumor samples were bisected, and half was used for DNA isolation, while the other half was formalin fixed and paraffin embedded (FFPE) for histopathology analysis. The expressions of latent EBV nuclear antigen 1 (EBNA1), CDKN2A/p16INK4A (p16), and MCPyV large T-antigen (LT) were tested by IHC. Multiplex PCR was used to detect 16 HPV genotypes and four other DNA tumor viruses [Kaposi's sarcoma-associated herpesvirus (KSHV), EBV, MCPyV, and adenovirus]. Relationships between HIV status, viral DNA and protein expression, and tumor grades were determined by statistical analysis. Results: OSSN tumors from patients were 29.6% preinvasive and 70.4% invasive. Patients presented with unilateral tumors that were 70.4% late stage (T3/T4). OSSN patients were HIV positive (72.8%). IHC on 243 FFPE biopsies resulted in the detection of EBNA1 (EBV), p16 high-risk HPV (HR-HPV), and MCPyV LT expression in 89.0%, 4.9%, and 0.0%, respectively. EBNA1 was expressed in all grades of preinvasive [cornea-conjunctiva intraepithelial neoplasia (CIN)1, 100%; CIN2, 85.7%; CIN3, 95.8%; and carcinoma in situ (CIS), 83.8%] and in invasive (89.2%) OSSN. PCR on 178 samples detected EBV, HR-HPV, and MCPyV in 80.3%, 9.0%, and 13.5% of tumors, respectively. EBV was detected in all grades of preinvasive and invasive OSSN. EBV detection was associated with high HIV viral loads (p = 0.022). HR-HPV was detected in 0.0% CIN1, 0.0% CIN2, 5.6% CIN3, 13.0% CIS, and 7.0% invasive OSSN. Conclusions: Our findings of EBV DNA and EBNA1 protein in all the grades of preinvasive and especially invasive OSSN are consistent with a potential causal role for EBV in OSSN. A role of HPV in OSSN was not clearly established in this study.

Mycoplasma Co-Infection Is Associated with Cervical Cancer Risk.

Tanzania faces one of the highest cervical cancer burdens in the world. Recent work has suggested that the bacterial family Mycoplasmataceae is associated with higher levels of human papillomavirus (HPV), human immunodeficiency virus (HIV), and pre-cancerous cervical lesions. Mycoplasmataceae infection in Tanzania is not well understood, especially when considering the differences between sexually transmitted species of Mycoplasmataceae. To establish the prevalence of common Mycoplasmataceae cervical infections and evaluate their relationship with risk factors for cervical cancer, 1160 Tanzanian women responded to an epidemiological questionnaire and were tested for HIV, HPV, cervical lesions, Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma spp., and Lactobacillus iners. A subset of 134 women were used for 16s metagenomic sequencing of cervical DNA to establish the relative abundance of Mycoplasmataceae and Lactobacillus present. PCR detection of bacteria at the cervix found Ureaplasma spp. in 51.4% of women, M. hominis in 34%, M. genitalium in 2.3%, and L. iners in 75.6%. M. hominis and M. genitalium infection were significantly more prevalent among women with HPV and HIV. M. hominis prevalence was similar despite severity of cervical lesions; however, abundance of M. hominis increased significantly in women with cervical lesions. These results emphasize the importance of understanding the relationship between M. hominis and HPV-related cervical pathogenesis.

Relationship between the Cervical Microbiome, HIV Status, and Precancerous Lesions.

Nearly all cervical cancers are causally associated with human papillomavirus (HPV). The burden of HPV-associated dysplasias in sub-Saharan Africa is influenced by HIV. To investigate the role of the bacterial microbiome in cervical dysplasia, cytobrush samples were collected directly from cervical lesions of 144 Tanzanian women. The V4 hypervariable region of the 16S rRNA gene was amplified and deep sequenced. Alpha diversity metrics (Chao1, PD whole tree, and operational taxonomic unit [OTU] estimates) displayed significantly higher bacterial richness in HIV-positive patients (P = 0.01) than in HIV-negative patients. In HIV-positive patients, there was higher bacterial richness in patients with high-grade squamous intraepithelial lesions (HSIL) (P = 0.13) than those without lesions. The most abundant OTUs associated with high-grade squamous intraepithelial lesions were Mycoplasmatales, Pseudomonadales, and Staphylococcus We suggest that a chronic mycoplasma infection of the cervix may contribute to HPV-dependent dysplasia by sustained inflammatory signals.IMPORTANCE HPV is known to be the causal agent in the majority of cervical cancers. However, the role of the cervical bacterial microbiome in cervical cancer is not clear. To investigate that possibility, we collected cervical cytobrush samples from 144 Tanzanian women and performed deep sequencing of bacterial 16S rRNA genes. We found that HIV-positive patients had greater bacterial richness (P = 0.01) than HIV-negative patients. We also observed that women with high-grade squamous intraepithelial lesions (HSIL) had greater cervical bacterial diversity than women with cytologically normal cervices. Data from our precise sampling of cervical lesions leads us to propose that Mycoplasma contributes to a cervical microbiome status that promotes HPV-related cervical lesions. These results suggest a greater influence of the bacterial microbiota on the outcome of HPV infection than previously thought.