Background: Cervical cancer screening strategies using visual inspection or cytology may have suboptimal diagnostic accuracy for detection of precancer in women living with HIV (WLHIV). The optimal screen and screen–triage strategy, age to initiate, and frequency of screening for WLHIV remain unclear. This study evaluated the sensitivity, specificity, and positive predictive value of different cervical cancer strategies in WLHIV in Africa. Methods and findings: WLHIV aged 25–50 years attending HIV treatment centres in Burkina Faso (BF) and South Africa (SA) from 5 December 2011 to 30 October 2012 were enrolled in a prospective evaluation study of visual inspection using acetic acid (VIA) or visual inspection using Lugol’s iodine (VILI), high-risk human papillomavirus DNA test (Hybrid Capture 2 [HC2] or careHPV), and cytology for histology-verified high-grade cervical intraepithelial neoplasia (CIN2+/CIN3+) at baseline and endline, a median 16 months later. Among 1,238 women (BF: 615; SA: 623), median age was 36 and 34 years (p < 0.001), 28.6% and 49.6% ever had prior cervical cancer screening (p < 0.001), and 69.9% and 64.2% were taking ART at enrolment (p = 0.045) in BF and SA, respectively. CIN2+ prevalence was 5.8% and 22.4% in BF and SA (p < 0.001), respectively. VIA had low sensitivity for CIN2+ (44.7%, 95% confidence interval [CI] 36.9%–52.7%) and CIN3+ (56.1%, 95% CI 43.3%–68.3%) in both countries, with specificity for ≤CIN1 of 78.7% (95% CI 76.0%–81.3%). HC2 had sensitivity of 88.8% (95% CI 82.9%–93.2%) for CIN2+ and 86.4% (95% CI 75.7%–93.6%) for CIN3+. Specificity for ≤CIN1 was 55.4% (95% CI 52.2%–58.6%), and screen positivity was 51.3%. Specificity was higher with a restricted genotype (HPV16/18/31/33/35/45/52/58) approach (73.5%, 95% CI 70.6%–76.2%), with lower screen positivity (33.7%), although there was lower sensitivity for CIN3+ (77.3%, 95% CI 65.3%–86.7%). In BF, HC2 was more sensitive for CIN2+/CIN3+ compared to VIA/VILI (relative sensitivity for CIN2+ = 1.72, 95% CI 1.28–2.32; CIN3+: 1.18, 95% CI 0.94–1.49). Triage of HC2-positive women with VIA/VILI reduced the number of colposcopy referrals, but with loss in sensitivity for CIN2+ (58.1%) but not for CIN3+ (84.6%). In SA, cytology high-grade squamous intraepithelial lesion or greater (HSIL+) had best combination of sensitivity (CIN2+: 70.1%, 95% CI 61.3%–77.9%; CIN3+: 80.8%, 95% CI 67.5%–90.4%) and specificity (81.6%, 95% CI 77.6%–85.1%). HC2 had similar sensitivity for CIN3+ (83.0%, 95% CI 70.2%–91.9%) but lower specificity compared to HSIL+ (42.7%, 95% CI 38.4%–47.1%; relative specificity = 0.57, 95% CI 0.52–0.63), resulting in almost twice as many referrals. Compared to HC2, triage of HC2-positive women with HSIL+ resulted in a 40% reduction in colposcopy referrals but was associated with some loss in sensitivity. CIN2+ incidence over a median 16 months was highest among VIA baseline screen-negative women (2.2%, 95% CI 1.3%–3.7%) and women who were baseline double-negative with HC2 and VIA (2.1%, 95% CI 1.3%–3.5%) and lowest among HC2 baseline screen-negative women (0.5%, 95% CI 0.1%–1.8%). Limitations of our study are that WLHIV included in the study may not reflect a contemporary cohort of WLHIV initiating ART in the universal ART era and that we did not evaluate HPV tests available in study settings today. Conclusions: In this cohort study among WLHIV in Africa, a human papillomavirus (HPV) test targeting 14 high-risk (HR) types had higher sensitivity to detect CIN2+ compared to visual inspection but had low specificity, although a restricted genotype approach targeting 8 HR types decreased the number of unnecessary colposcopy referrals. Cytology HSIL+ had optimal performance for CIN2+/CIN3+ detection in SA. Triage of HPV-positive women with HSIL+ maintained high specificity but with some loss in sensitivity compared to HC2 alone.

Squamous cervical cancers generally arise as a result of persistent infection with high-risk human papillomaviruses (hrHPVs) and occur near the squamocolumnar junction (SCJ) and within the transformation zone (TZ). The susceptibility of the TZ to HPV-related carcinogenesis appears linked to epithelial cell plasticity, with squamous metaplasia originating from a specialized stem cell population at this site. Two alternative cell populations have been implicated: keratin (K)7+ve cuboidal cells located at the SCJ vs a more broadly distributed K17+ve cervical reserve cell population. To distinguish between the hypotheses, we utilized multiplex immunofluorescence and large-scale digital imaging to map cell populations at the TZ of 165 women with and without hrHPV infections. Our results did not reveal a distinct population of K7+ cuboidal cells at the SCJ but found instead that the cuboidal and columnar cells of the TZ express K7 and K8 throughout and lack the p63 transcription factor required for epithelial stratification. Squamous metaplasia and reserve cells, which are defined by their subcolumnar location and pattern of biomarker expression (K5/K17/P63), were conspicuous at cervical crypt entrances within the TZ extending proximally toward the endocervix. In HPV-infected tissue, crypt-entrance regions with thin high-grade squamous intraepithelial lesion pathology showed prominent expression of hrHPV E6/E7 mRNA, as detected by fluorescence in situ hybridization, and p16/MCM expression, with infection also apparent in neighboring reserve cells. In some instances, normal/uninfected reserve cells (E6/E7 mRNA-ve) and squamous metaplasia were not only seen close to these regions of hrHPV infection but also extended well beyond the infected area both laterally and by depth. Our results confirm that the reserve cells underneath the columnar epithelia at TZ have the potential to undergo malignant squamous transformation via reserve cell proliferation, in agreement with previous histopathological studies. These translational findings highlight the importance of understanding the molecular biology of the epithelial sites where HPV cancers develop and suggest that in high-risk individuals, treatment strategies should target a wider area than previously thought.

PURPOSE: Diagnosis of AIDS lymphoma in low-resource settings, like South Africa, is often delayed, leaving patients with limited treatment options. In tuberculosis (TB) endemic regions, overlapping signs and symptoms often lead to diagnostic delays. Assessment of plasma cell-free DNA (cfDNA) by next-generation sequencing (NGS) may expedite the diagnosis of lymphoma but requires high-quality cfDNA.METHODS: People living with HIV with newly diagnosed aggressive B-cell lymphoma and those with newly diagnosed TB seeking care at Chris Hani Baragwanath Academic Hospital and its surrounding clinics, in Soweto, South Africa, were enrolled in this study. Each participant provided a whole blood specimen collected in cell-stabilizing tubes. Quantity and quality of plasma cfDNA were assessed. NGS of the immunoglobulin heavy chain was performed.RESULTS: Nine HIV+ patients with untreated lymphoma and eight HIV+ patients with TB, but without lymphoma, were enrolled. All cfDNA quantity and quality metrics were similar between the two groups, except that cfDNA accounted for a larger fraction of recovered plasma DNA in patients with lymphoma. The concentration of cfDNA in plasma also trended higher in patients with lymphoma. NGS of immunoglobulin heavy chain showed robust amplification of DNA, with large amplicons (> 250 bp) being more readily detected in patients with lymphoma. Clonal sequences were detected in five of nine patients with lymphoma, and none of the patients with TB.CONCLUSION: This proof-of-principle study demonstrates that whole blood collected for cfDNA in a low-resource setting is suitable for sophisticated sequencing analyses, including clonal immunoglobulin NGS. The detection of clonal sequences in more than half of patients with lymphoma shows promise as a diagnostic marker that may be explored in future studies.