Human Papillomavirus Type 6 and 11 Genetic Variants Found in 71 Oral and Anogenital Epithelial Samples from Australia

<div><p>Genetic variation of 49 human papillomavirus (HPV) 6 and 22 HPV11 isolates from recurrent respiratory papillomatosis (RRP) (n = 17), genital warts (n = 43), anal cancer (n = 6) and cervical neoplasia cells (n = 5), was determined by sequencing the long control region (LCR) and the E6 and E7 genes. Comparative analysis of genetic variability was examined to determine whether different disease states resulting from HPV6 or HPV11 infection cluster into distinct variant groups. Sequence variation analysis of HPV6 revealed that isolates cluster into variants within previously described HPV6 lineages, with the majority (65%) clustering to HPV6 sublineage B1 across the three genomic regions examined. Overall 72 HPV6 and 25 HPV11 single nucleotide variations, insertions and deletions were observed within samples examined. In addition, missense alterations were observed in the E6/E7 genes for 6 HPV6 and 5 HPV11 variants. No nucleotide variations were identified in any isolates at the four E2 binding sites for HPV6 or HPV11, nor were any isolates found to be identical to the HPV6 lineage A or HPV11 sublineage A1 reference genomes. Overall, a high degree of sequence conservation was observed between isolates across each of the regions investigated for both HPV6 and HPV11. Genetic variants identified a slight association with HPV6 and anogenital lesions (p = 0.04). This study provides important information on the genetic diversity of circulating HPV 6 and HPV11 variants within the Australian population and supports the observation that the majority of HPV6 isolates cluster to the HPV6 sublineage B1 with anogenital lesions demonstrating an association with this sublineage (p = 0.02). Comparative analysis of Australian isolates for both HPV6 and HPV11 to those from other geographical regions based on the LCR revealed a high degree of sequence similarity throughout the world, confirming previous observations that there are no geographically specific variants for these HPV types.</p></div

HPV11 LCR phylogenetic tree from Australia and other geographical regions.

<p>A maximum likelihood (ML) tree was inferred from molecular phylogenetic analysis of 94 aligned LCR sequences using MEGA5 based on the Tamura-Nei model with 500 bootstrap replicates <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063892#pone.0063892-Kimura1" target="_blank">[66]</a>. These 94 sequences were derived from Australia (KC329850–KC329871), Slovenia (FN870625–FN870687), China (EU918768), Hungary (HE574701, HE574705, FR872717) and Thailand (JQ773408–JQ773412). The reference sequence (M14119) from sublineage A1 is shown in grey. The 21 variants are named by the lineage they cluster to. Sublineage A1 was comprised of one Slovenian variant (A1–1) and one geographically shared variant (A1–2). Sublineage A2 was comprised of 6 Australian (A2–3, A2–6, A2–13, A2–14, A2–16, A2–17), Slovenian(A2–1, A2–7, A2–10, A2–12, A2–15), Hungarian (A2–2), Thai(A2–9), and geographically shared (A2–4, A2–5, A2–8, A2–11, A2–18, A2–19) variants. In cases where sequences were identical only one sequence from that country was use to represent each variant. In seven instances variants from different geographical regions were also identical, again one sequence was used to represent each variant; A1–2 (China and Slovenia), A2–4 (Hungary and Thailand), A2–8 (Australia, Hungary and Slovenia), A2–5, A2–11, A2–18 (Australia and Slovenia), A2–19 (Australia, Slovenia and Thailand).</p

HPV6 LCR phylogenetic tree from Australia and other geographical regions.

<p>A maximum likelihood (ML) tree was inferred from molecular phylogenetic analysis of 105 aligned LCR sequences using MEGA5 based on the Tamura-Nei model with 500 bootstrap replicates <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063892#pone.0063892-Kimura1" target="_blank">[66]</a>. These 105 sequences were derived from Australia (KC300093–KC300140, KC333888), Slovenia (FM876166–FM876210) and South Africa (JN573163–JN573174). Reference sequences are shown in grey for lineage A (X00203), and sublineages B1 (AF092932) and B3 (L41216). The 38 variants are named by the lineage and sublineage they cluster to. Lineage A was comprised of one Australian (A-2), one Slovenian (A-3) and two geographically shared (A-1, A-4) variants. Lineage B, specifically sublineage B1 was comprised of Australian (B1–4, B1–5, B1–9, B1–10, B1–12, B1–13, B1–14, B1–15, B1–16, B1–17, B1–18, B1–20, B1–21, B1–24, B1–25, B1–26, B1–27, B1–28, B1–29), Slovenian (B1–1, B1–3, B1–6, B1–7, B1–11, B1–19, B1–22) and geographically shared (B1–2, B1–8, B1–23 ) variants, and lineage B3 was comprised of Australian (B3–4, B3–5), South African (B3–1, B3–3) and geographically shared (B3–2). In cases where sequences were identical only one sequence from each counrty was use to represent each variant. In six instances variants from different geographical regions were identical; A–1, A–4 and B1–2 (Australia and Slovenia), B3–2 (Slovenia and South Africa), B1–8 and B1–23 (Australia, Slovenia and South Africa).</p

Treatment adherence and side effects.

<p>Treatment adherence and side effects.</p

Clinical and laboratory characteristics of females.

<p>Clinical and laboratory characteristics of females.</p

Combined oral and topical antimicrobial therapy for male partners of women with bacterial vaginosis: Acceptability, tolerability and impact on the genital microbiota of couples - A pilot study

<div><p>Objectives</p><p>Recurrence following recommended treatment for bacterial vaginosis is unacceptably high. While the pathogenesis of recurrence is not well understood, recent evidence indicates re-infection from sexual partners is likely to play a role. The aim of this study was to assess the acceptability and tolerability of topical and oral antimicrobial therapy in male partners of women with bacterial vaginosis (BV), and to investigate the impact of dual-partner treatment on the vaginal and penile microbiota.</p><p>Methods</p><p>Women with symptomatic BV (Nugent Score of 4–10 and ≥3 Amsel criteria) and their regular male sexual partner were recruited from Melbourne Sexual Health Centre, Melbourne, Australia. Women received oral metronidazole 400mg twice daily (or intra-vaginal 2% clindamycin cream, if contraindicated) for 7-days. Male partners received oral metronidazole 400mg twice daily and 2% clindamycin cream topically to the penile skin twice daily for 7-days. Couples provided self-collected genital specimens and completed questionnaires at enrolment and then weekly for 4-weeks. Genital microbiota composition was determined by 16S rRNA gene sequencing. Changes in genital microbiota composition were assessed by Bray-Curtis index. Bacterial diversity was measured by the Shannon Diversity Index.</p><p>Results</p><p>Twenty-two couples were recruited. Sixteen couples (76%) completed all study procedures. Adherence was high; most participants took >90% of prescribed medication. Medication, and particularly topical clindamycin in males, was well tolerated. Dual-partner treatment had an immediate and sustained effect on the composition of vaginal microbiota (median Bray-Curtis score day 0 versus day 8 = 0.03 [IQR 0–0.15], day 0 vs day 28 = 0.03 [0.02–0.11]). We observed a reduction in bacterial diversity of the vaginal microbiota and a decrease in the prevalence and abundance of BV-associated bacteria following treatment. Treatment had an immediate effect on the composition of the cutaneous penile microbiota (median Bray-Curtis score day 0 vs day 8 = 0.09 [0.04–0.17]), however this was not as pronounced at day 28 (median Bray-Curtis score day 0 vs day 28 = 0.38 [0.11–0.59]). A decrease in the prevalence and abundance of BV-associated bacteria in the cutaneous penile microbiota was observed immediately following treatment at day 8.</p><p>Conclusion</p><p>Combined oral and topical treatment of male partners of women with BV is acceptable and well tolerated. The combined acceptability and microbiological data presented in this paper supports the need for larger studies with longer follow up to characterize the sustained effect of dual partner treatment on the genital microbiota of couples and assess the impact on BV recurrence.</p></div

Treatment adherence and side effects.

<p>Treatment adherence and side effects.</p

Prevalence and changes in prevalence of the 30 most abundant taxa in vaginal specimens over the study period.

<p>Prevalence and changes in prevalence of the 30 most abundant taxa in vaginal specimens over the study period.</p

Specimen flowchart.

<p>Specimen flowchart detailing number of vaginal and penile skin specimens available for microbiota analysis at baseline, day 8 and day 28. Seventeen women provided vaginal specimens for day 0 and 8 paired comparisons, and 16 provided vaginal specimens for day 0 and 28 paired comparisons. Sixteen males provided cutaneous penile specimens for day 0 and 8 paired comparisons and 15 males provided cutaneous penile specimens for day 0 and 28 paired comparisons. The number of couples providing specimens at each time-point is also shown. ^abaseline specimen was not available for one female; ^btwo d8 penile skin specimens failed to meet the sequence depth threshold and were substituted with day 14 specimens.</p

Clinical and laboratory characteristics of females.

<p>Clinical and laboratory characteristics of females.</p