Journal of Medical Microbiology

Correlation between cervical HPV DNA detection and HPV16 seroreactivity measured with L1-only and L1+L2 viral capsid antigens

Introduction.Persistent human papillomavirus (HPV) type 16 infection is the main causal agent of cervical cancer. Most HPV infections clear spontaneously within 1–2 years. Although not all infected women develop detectable HPV antibodies, about 60–70 % seroconvert and retain their antibodies at low levels.Aim.We investigated if cervical HPV16 DNA positivity was associated with HPV16 seroreactivity measured with two different antigen formulations. We assessed if associations were influenced by co-infection with other HPV types and HPV16 viral load.Methodology.We used baseline data for women participating in the Ludwig–McGill cohort, a longitudinal investigation of the natural history of HPV infection and cervical neoplasia. The study enrolled 2462 Brazilian women from 1993 to 1997 (pre-vaccination). ELISA assays were based on L1-only or L1+L2 virus-like particles (VLPs). Seroreactivity was expressed as normalized absorbance ratios. HPV genotyping and viral load were evaluated by PCR protocols. Pearson’srwas used to measure correlations between interval-scaled variables. Serological accuracy in HPV16 DNA detection was assessed using receiver operating characteristic (ROC) curves. We analysed the association between HPV DNA positivity and HPV16 seroreactivity by linear regression.Results.Correlations between L1+L2 and L1-only VLPs for detection of HPV16 were poor (r=0.43 and 0.44 for dilutions 1 : 10 and 1 : 50, respectively). The protocol with the best accuracy was L1+L2 VLPs at serum dilution 1 : 10 (ROC area=0.73, 95 % CI: 0.65–0.85). HPV16 DNA positivity was correlated with HPV16 seroreactivity and was not influenced by co-infection or viral load. To a lesser degree, HPV16 seroreactivity was correlated with infection by other Alpha-9 papillomavirus species.Conclusion.HPV16 DNA positivity and HPV16 seroreactivity are strongly correlated. L1+L2 VLPs perform better than L1-only VLPs for detecting IgG antibodies to HPV16 in women infected with HPV16 or other Alpha-9 HPV species. This study advances our understanding of humoral immune responses against HPV16 by providing insights about the influence of VLP antigen composition to measure humoral immune response against naturally acquired HPV infection.

Cervicovaginal loads of Gardnerella spp. are increased in immunocompetent women with persistent high-risk human papillomavirus infection

Introduction. Two high-oncogenic-risk human papilomavirus (hrHPV) genotypes – HPV16 and HPV18 – cause most of the cases of cervical cancer worldwide. Bacterial vaginosis is associated with increased hrHPV persistence, although the mechanism underlying this association remains unclear. Gardnerella spp. are detected in nearly all cases of bacterial vaginosis and are the major source of cervicovaginal sialidases. The NanH1 gene is present in virtually all Gardnerella sialidase-producing strains and has been proposed as a potential marker for persistent hrHPV infection. Hypothesis. Gardnerella spp. load and the NanH1 gene are associated with hrHPV persistence. Aim. To compare the cervicovaginal load of Gardnerella spp. and the frequency of the NanH1 gene between women with persistent HPV16 and/or HPV18 infection and those who cleared the infection after 11 months. Methodology. Among a population of 1638 HPV screened, we detected 104 with positive HPV16 and/or HPV18 results. Samples were obtained at two time points (baseline and at a median of 11 months at follow-up) and tested using the Linear Array HPV Genotyping kit (Roche Molecular Systems, Pleasanton, CA, USA). Based on their HPV16/HPV18 status at enrolment and follow-up, participants were assigned to ‘persistence’ or ‘clearance’ groups. We used cervicovaginal fluid samples obtained upon enrolment to determine the load of the 23 s rRNA gene of Gardnerella spp. and the presence of the NanH1 gene using real-time polymerase chain reaction (PCR). We compared Gardnerella spp. loads and NanH1 frequency between the groups by, respectively, Mann–Whitney and chi-squared tests, with a P-value <0.05 considered to be significant. Results. Of the 104 participants who were positive for HPV16/HPV18, 73 (70.2 %) persisted with at least 1 of the baseline genotypes at follow-up, while 31 (29.8 %) cleared the infection in this time frame. Participants in the persistence group had significantly higher loads of Gardnerella spp. [5.8E+02 (0–3.0E+05) copies µl−1] than those in the clearance group [9.9E+01 (0–7.7E+04) copies µl−1] (P=0.03). The baseline frequency of NanH1 was higher in the persistence’ (n=46, 63.0 %) than in the clearance (n=14, 45.2 %) group, although this was not statistically significant (P=0.09). Conclusion. These findings reinforce the negative effect of vaginal microbiota for the clearance of hrHPV and indicate a possible association between sialidase-producing species with hrHPV persistence.

Genetic variations in the long control region of human papillomavirus type 16 isolates from India: implications for cervical carcinogenesis

Introduction. Infection with high-risk human papillomavirus (HPV) types, specifically HPV type 16 (HPV16), is considered to be the most important risk factor in the development of cervical intraepithelial neoplasia and cancer. The long control region (LCR) is a noncoding region that comprises approximately 10 % of the HPV genome and contains regulatory elements for viral transcription and replication. Sequence variations in LCR may impact on the replication efficiency and oncogenic potential of the virus. Gap statement. Studies documenting variations in LCR of HPV16 isolates pertaining to cervical neoplastic status in India are limited. Aim. The present study was designed to characterize variations in the LCR of Indian isolates of HPV16 and study their association with cervical disease grades. Methodology. The LCR was amplified and sequenced from HPV16 positive cervical samples belonging to different cervical disease grades. Sequences were aligned to identify variations and potential transcription factor binding sites (TFbs) were predicted using the JASPAR database in addition to phylogenetic studies. Results. Among the 163 HPV16 isolates analysed, 47 different nucleotide variations were detected in the LCR, of which 25 are reported for first time in Indian isolates. Point mutations were detected in 35/54 (64.8 %) samples with normal cervical status, 44/50 (88 %) samples with low-grade cervical disease and 53/59 (89.8 %) samples with high-grade cervical disease. Variations T6586C, G6657A and T6850G were significantly associated with high-grade cervical status. Thirteen LCR variations were detected in the binding sites for CEBPB, ETS1, JUN, MYB, NFIL3, PHOX2A and SOX9 transcription factors. Conclusion. The present study helped to identify unique variations in the LCRs of HPV16 Indian isolates. The variations in the A4 sub-lineage were significantly associated with high-grade disease status. The isolates belonging to the A4 and D3 sub-lineages harboured mutations in putative TFbs, implying a potential impact on viral replication and progression to cervical cancer.

Concordance analysis between Human Papillomavirus genotyping assay and PCR-reverse spot hybridization for the detection of Human Papillomavirus infection

Introduction. Human papillomavirus (HPV), the predominant viral infection affecting the anogenital tract, is closely linked to the development of intraepithelial neoplasia and malignancies in the cervix and other anal regions. Currently, 15 high-risk HPVs (HR-HPVs) and 3 potential HR-HPV types have been recognized as contributors to cervical cancer. Consequently, it is imperative to conduct HR-HPV screening using suitable tests in order to identify precancerous lesions and prevent the development of cancer. Hypothesis. The Human papillomavirus genotyping (type 23) detection kit (PCR-reverse point hybridization method) is reliable for clinical testing. Aims. The objective of this research was to assess the concordance between the Human papillomavirus genotyping (type 23) detection kit (PCR-reverse point hybridization method) and the approved HPV test. Methodology. A sample of 781 women who received HPV genotype testing during cervical cancer screening consultations at the Department of Gynecology, Fujian Maternity and Child Health Hospital, was examined. Thirty-two cases were excluded for lacking histological results or showing signs of vulvar intraepithelial rheology, leaving 749 valid histological samples. Only 181 valid pathological specimens were available after excluding those without cervical biopsy or total hysterectomy. The consistency of the test results was assessed using the kappa (K) statistic, with CIN2+ serving as the benchmark for determining sensitivity and specificity. Statistical significance was defined as differences with P values <0.05 (two-tailed). Results. The human papillomavirus genotyping (type 23) detection kit (PCR-reverse point hybridization method) and the approved HPV test demonstrated a high level of concordance with a total kappa value of 0.969 (P<0.05). The overall concordance rate was found to be 98.720%. Using cervical intraepithelial neoplasia grade 2+ (CIN2+) as the reference standard, the human papillomavirus genotyping (type 23) detection kit (PCR-reverse point hybridization method) and the approved HPV test both showed 89.655% sensitivity (P>0.05), while the specificity values were 40.590 and 40.309%, respectively (P>0.05). Conclusion. The evaluated HPV test demonstrates comparable performance to other assays available during the same time frame and exhibits strong concordance in detecting the majority of HPV genotypes.

Exploring the diversity of vaginal microbiota between healthy women and cervical cancer patients in India

Introduction. Cervicovaginal diversity has been reported as a predictive biomarker for cervical cancer risk. We recently reported the bio-therapeutic potential of vaginal probiotics from healthy Indian women against vaginal pathogens, isolated from the invasive cervical cancer (ICC) patients. Gap Statement. The cervicovaginal microflora from cervical cancer patients has not yet been reported from Indian population. Aim. The present study aimed at comparing the cervicovaginal microbiome between healthy controls (HC) and ICC patients from the Indian population. Methodology. In total, 30 vaginal swabs (15 from HC and 15 from ICC) were subjected to 16S rRNA gene sequencing. Alpha diversity was evaluated by Shannon and Chao1 index; and beta diversity by principle coordinate analysis (PCoA) of weighted and unweighted UniFrac distances. The relative abundance of the microbial taxa was done according to linear discriminant analysis effect size (LEfSe). Results. Predominance of Staphylococcus spp. in ICC and Lactobacillus gasseri in HC groups was observed. Alpha-diversity was found to be higher in ICC as compared to HC but was statistically non-significant. LEfSe analysis revealed Bacteroides fragilis and Escherichia coli as the marker genera in ICC with a marked decrease in Lactobacillus sp. Contrarily, in HC, L. gasseri, L. iners and L. fermentum were found to be abundant. Conclusion. Differences in the vaginal microbiome between healthy and ICC women could help in the early prediction of cervical cancer risk and thus in designing prevention strategies.