More deaths occur in African women from invasive cervical cancer (ICC) than from any other malignancy. ICC is caused by infection with oncogenic types of human papillomavirus (HPV). Co-infection with the human immunodeficiency virus (HIV) accelerates the natural history of ICC, and may influence the HPV type distribution. Because HPV vaccines are available, this malignancy is theoretically preventable, but the vaccines are largely type-specific in protection against infection. Data on specific HPV types causing ICC in African women is limited, and many studies utilized swab samples rather than actual cancer tissue. A previous study using archived, ICC tissue from women in Botswana identified an unusual HPV type distribution. A similar study was therefore performed in a second sub-Saharan country to provide additional information on the HPV type distribution in ICC.Archived, formalin-fixed, paraffin-embedded ICCs were acquired from women in the United States, Kenya, or Botswana. DNA was extracted and HPV genotyping performed by Roche Linear Array. HIV sequences were identified in ICCs by PCR.p < 0.0001). Non-HPV 16/18 types were detected in 10.9 % of HPV-positive cancers from the U.S., 17.2 % from Kenya, and 47.8 % from Botswana (p < 0.0001). HIV was detected in 2.2, 31.5, and 32.4 % from ICCs from the U.S., Kenya, or Botswana, respectively (p = 0.0002). The distribution of HPV types was not significantly different between HIVinfected or HIV-uninfected women. The percentages of ICCs theoretically covered by the bivalent/quadrivalent HPV vaccines were 93.5, 93.9, and 61.8 % from the U.S., Kenya and Botswana, respectively, and increased to 100, 98, and 77.8 % for the nanovalent vaccine.HPV types 16 or 18 (HPV 16/18) were identified in 93.5 % of HPV-positive ICCs from the U.S., 93.8 % from Kenya, and 61.8 % from Botswana  contains supplementary material, which is available to authorized users. An estimated 528,000 cases of invasive cervical cancer (ICC) occurred worldwide in 2012 [Many obstacles exist for sub-Saharan countries in the fight against cervical cancer, where this malignancy is diagnosed most often at ages 35 to 45 years, claiming the lives of women when they may be raising children and contributing to the economy of their communities . All butInfection with oncogenic human papillomaviruses (HPV) is the cause of ICC . CervicaAs sub-Saharan African countries consider implementing HPV vaccination, it is important to understand the range of HPV types responsible for ICC cases in a country. Surveillance and better understanding of local epidemiology must be encouraged before selecting the HPV vaccine of choice for a specific country. The object of the study was therefore to compare HPV typing data from a previously published study of ICC cases in the United States and Botswana  to additApproval for the study was granted by the Institutional Review Board at Indiana University School of Medicine, University of Pennsylvania, the Botswana Ministry of Health, Gaborone, Botswana, and by the Institutional Research and Ethics Committee at Moi University Teaching and Referral Hospital. All specimens were de-identified prior to transport to the HPV Laboratory.Acquisition of archived, paraffin-embedded blocks of formalin-fixed cervical cancer specimens from women residing in Indianapolis, Indiana (U.S.) and from Botswana was previously described . Case seAdditional ICC specimens, obtained from 2010 to 2013 were identified at the Moi University Teaching and Referral Hospital in Eldoret, Kenya. Section (5 μm) from paraffin-embedded specimens were cut using a new blade for each block. The first and last sections were stained with hematoxylin and eosin for verification of ICC. Specimens without invasive cancer tissue were eliminated from the study.The remainder of each block was transported to Indiana University School of Medicine (Indianapolis), where tissues were re-embedded due to apparent dehydration of paraffin. Additional sections were prepared, and the first and last sections were stained with hematoxylin and eosin to verify the presence of ICC, Those without invasive cancer tissue were eliminated from the study. For specimens containing cancer tissue representing less than an estimated 5 % of the total specimen, macro-dissection was performed by identifying cancerous tissue using a light microscope, marking the areas cancer tissue on the undersurface of the slide, and removing this tissue with a sterile scalpel.Thin-section polymerase chain reaction (PCR) of specimens from Kenyan women was performed exactly as described in a prior report of ICC cases from women living in Botswana and the U.S. . BrieflyHPV detection and genotyping was performed using the Linear Array HPV Genotyping Test  as described . The GH2Sera were not available from women, as this study used archived ICC specimens. In addition, medical records were not available for most subjects. Therefore, HIV was detected by thin-section PCR using DNA extracted from paraffin-embedded cervical cancer specimens as previously described . BrieflyHIV PCR products were analyzed on 1.8 % agarose gels. To confirm the amplification of desired products, two reactions from cancers with an amplicon of expected size were extracted from gels using the QIAquick Gel Extraction Kit (Qiagen) and sequenced bi-directionally. Homology of the sequenced samples with known HIV gag gene sequences was verified using the Basic Local Sequencing Alignment Search Tool (BLAST) search (NCBI).This was a comparative study, not a clinic trial, as specimens were archived. Adequacy of extracted DNA was based on β-globin-positivity among ICC specimens. ICC specimens positive for the β-globin human control gene in the Roche Linear Array Assay were included in the analysis of HPV types. HPV type-specific prevalence  and non-oncogenic, or “low-risk (LR) types) was compared among β-globin-positive ICCs from the three countries, and between specimens that were from HIV-infected or HIV-uninfected women from Kenya or Botswana. In addition, HPV types were divided by their respective classification into Alpha 9 (A9 or HPV 16-related) and Alpha 7 (A7 or HPV 18-related). The A9 types included HPV 16, 31, 33, 35, 52, and 58, and A7 types included HPV 18, 39, 45, 59, and 68. Prevalence of A9 and A7 types between countries were compared using Fisher’s exact tests or Chi-square tests. Analyses were performed using SAS .p = 0.01).Paraffin-embedded tissue blocks were previously obtained from 51 women from the U.S.  (Table 1p = 0.49) (Table p = 0.128)  adequate specimens from the U.S., 171 of 182 (94.0 %) from Botswana, and 178 of 187 (95.2 %) from Kenya (p = 0.085) (Table p = 0.007) (Table HR-HPV types were detected in 46 of 46 (100 %) HPV-positive specimens from the U.S., 130 of 136 (95.6 %) from Botswana, and 145 of 146 (99.3 %) from Kenya (5) Table . LR-HPV 7) Table .Table 2HP < 0.001) (Table p = 0.089) (Table p < 0.0001) (not shown). Other HPV types detected in ICC specimens are shown in Table HPV 16 was the most frequently detected type in ICC specimens from all three countries, detected in 40 of 46 (87.0 %) HPV-positive specimens from the U.S., 58 of 136 42.7 %) from Botswana, and 118 of 146 (80.8 %) from Kenya (.7 % fromp < 0.001) (Table p < 0.001) (Table Types other than HPV 16 and 18 (non-HPV 16/18 types) were detected in 5 of 46 (10.9 %) HPV-positive specimens from the U.S., 65 of 136 (47.8 %) from Botswana, and 25 of 146 (17.1 %) from Kenya (p < 0.001) (Table p = 0.002) (Table A9 HPV types were detected in 41 of 46 (89.1 %) HPV-positive specimens from the U.S., 79 of 136 (58.1 %) from Botswana, and 120 of 146 (82.2 %) from Kenya (p = 0.022) (Table p = 0.259) (Table A7 HPV types were detected in 7 of 46 (15.2 %) HPV-positive specimens from the U.S., 49 of 136 (36.0 %) from Botswana, and 40 of 146 (27.4 %) from Kenya (p = 0.0003). Looking at only those specimens in which an HPV type was detected, HIV sequences were detected by thin-section PCR in 1 of 46 (2.2 %) specimens from the U.S., 44 of 136 (32.4 %) from Botswana, and 46 of 146 (31.5 %) from Kenya (p = 0.0002). As we had previously done for ICCs from women in Botswana, an analysis was performed on ICCs from Kenyan women in which an HPV type was detected to determine if there were differences between the HPV types in cases from HIV-infected women and HIV-uninfected women. No differences in oncogenic HPV types or groups of oncogenic HPV types were found between ICC cases from HIV-infected or HIV-uninfected Kenyan women  β-globin-positive specimens from the U.S., 54 of 171 (31.6 %) from Botswana, and 55 of 178 (30.9 %) from Kenya  ICC specimens that contained any HPV type. There was no significant difference in the percentage of ICC specimens containing more than a single HPV type between the three countries (data not shown). In addition, because HIV infection has been associated with detection of multiple HPV types in clinical specimens in some studies, an analysis was performed to determine if detection of multiple HPV types was more common in cancers from HIV-infected women compared to HIV-uninfected women. In ICC specimens from the U.S., Botswana, and Kenya, or the three countries combined, there was no significant association between HIV status and the detection of multiple HPV types (data not shown).Three HPV vaccines are currently available in the United States: the bivalent vaccine , the quadrivalent vaccine , and a recently approved nine-valent vaccine . All three provide excellent protection against HPV 16 and 18. The bivalent and quadrivalent vaccines may each provide a degree of “cross protection” against non-vaccine HR-HPV types, but this is likely to be weak and short-lived compared to efficacy against the oncogenic types actually represented in the vaccines . In contTheoretical vaccine coverage by the currently available HPV vaccines were calculated based on HPV types identified in the ICCs from the three countries Table . The bivIt is important to understand differences in the HPV types causing ICC in different regions of the world, and differences in the HPV types causing ICC between women who are infected or not infected with HIV. This information is paramount in the fight against cervical cancer, especially in the era of safe and effective vaccines against HPV. It is entirely possible that HPV type distributions could differ in ICC cases between different countries in a large continent such as Africa. In addition, because there are several available methods for both collection of cancer cells/tissue and for HPV analysis, comparing studies can be difficult.More country-specific data is needed before conclusions can be drawn. Two analyses of studies performed in African women suggested that HPV 16 was present in approximately half of ICCs, the lowest prevalence among the major regions of the world . Our ownWe wanted to expand the analysis, using identical methods of tissue collection and HPV typing to a second sub-Saharan country, Kenya. The current study was therefore designed to compare and contrast HPV types causing ICC in the U.S., Botswana, and Kenya. Using a direct approach known as thin-section PCR, we found that the distribution of HPV types in ICC specimens from western Kenyan women was similar to that in cancers from the U.S. No statistically significant differences were found for individual HPV types or for groups of types. HPV 16 was the most frequently detected type in ICCs from women living in the U.S. (87.0 %) and from women living in Kenya (82.0 %). Together, HPV types 16 or 18 were detected in approximately 94 % of ICC specimens from both the U.S. and Kenya.In contrast, the differences between HPV types in ICCs from women living in Kenya and Botswana are striking and likely not exclusively related to HIV infection prevalence. Fewer ICCs from women living in Botswana contained HPV 16. More non-HPV 16/18 oncogenic types were identified in ICCs from women living in Botswana compared to those from the U.S. or Kenya.Other groups have also examined ICC cases from sub-Saharan African women, but in each case, different methods were utilized in both sampling and in HPV detection, compared to our study and to each other. Several other studies have included data on HIV status as well, but utilized serological HIV testing, as such information was available. For example, De Vuyst et al., conducted a study of Kenyan women with ICC . Fifty-oP < 0.001) and CIN2/3 among women not receiving anti-retroviral treatment (P = 0.013). The authors concluded that the burden of HR-HPV and CIN2/3 was high in HIV-infected Kenyan women and was related to immunosuppression level. A third study from that group examined frozen tissue biopsies from women with ICC in Kenya and South Africa [Another study from this group was performed to determine associations of HPV infection and cervical intraepithelial neoplasia (CIN) among 498 HIV-infected Kenyan women who underwent HPV PCR-based testing, cytology, and cervical biopsy . Overallh Africa . One hunA recent study by Maranga et al., examined HPV types in cervical smears and carcinomas from HIV-infected and HIV-uninfected Kenyan women . Smear sHIV co-infection accelerates the natural history of cervical cancer. For Kenya and Botswana, we did not know the HIV status for the women. Using a thin-section PCR devised by our group, HIV was identified in ~32 to 34 % of ICC specimens from women living in Botswana or Kenyan, compared to ~4 % of ICCs from women living in Indiana. These numbers do not reflect the HIV prevalence of all women in these countries but illustrate the higher incidence of ICC in HIV-infected women. More studies of the thin-section PCR for HIV are needed to validate this potentially useful assay. Based on studies by other groups, we expected that ICCs from women who were HIV-infected may have a different HPV type distribution that from ICCs from HIV-uninfected women in these countries . HoweverThe current study has implications for health policies related to prevention of cervical cancer. This study indicates that HPV types 16 and 18 are the primary types causing ICC in Indiana, U.S., and in western Kenya. However, our recent study in Botswana (and shown again in this manuscript) showed a different HPV type distribution. As indicated above, three HPV vaccines are currently available: the bivalent vaccine, the quadrivalent vaccine, and the nine-valent vaccine. All are expensive, costing approximately $360 in the U.S. for the series of three required doses. All three provide excellent protection against HPV 16 and 18. The nine-valent HPV vaccine includes VLPs present in the quadrivalent vaccine  plus VLPs of five additional HR-HPV types  [When global data is examined, the five additional HR-HPV types in the nine-valent HPV vaccine account for approximately 12 % of all cervical cancers . CombineHow important would the nine-valent HPV vaccine be in Botswana? Due to the large percentage of non-HPV 16/18 types identified in the ICC cases in Botswana, the bivalent and quadrivalent vaccines would theoretically provide less protection against ICC than it would for women in Kenya or the U.S. The nine-valent vaccine would theoretically provide a higher degree of protection against ICC among women living in Botswana.There are several limitations to this study. First, only ICC cases from western Kenya were included and results cannot be generalized to the entire country of Kenya. Second, this study used paraffin-embedded cervical cancers, and as a result, the sensitivity for HPV detection could be less than if fresh cancer tissues had been available. Because the current study used archived specimens, fixation conditions could not be controlled and may have contributed to differences between countries in the frequencies of HPV detection . Third, Continued efforts must be made to improve utilization of the HPV vaccines, and with the help of the World Health Organization and other agencies, and hopefully a reduced price of the nine-valent HPV vaccine can be negotiated for countries in which non-HPV 16/18 types are frequently identified in careful studies of ICC. For now, every effort should be made to vaccinate young girls and adolescent women with any of the currently available HPV vaccines. This will help assure a cervical cancer-free future for Kenya, Botswana, and other sub-Saharan countries where cervical cancer screening is not widely available.


L1 protein comprising HPV vaccine formulations elicits high-titre neutralizing antibodies and confers protection against specific HPV subtypes. HPV L2 protein is an attractive candidate for cross-protective vaccines. HPV-33 and HPV-58 are very prevalent among Chinese women.Cervical cancer is associated with infection by certain subtypes of human papillomavirus (HPV). The L1/L2 in Sichuan China, HPV-33 and HPV-58 L1 and L2 genes were sequenced and compared with other genes submitted to GenBank. Phylogenetic trees were constructed by maximum-likelihood and the Kimura 2-parameters methods (MEGA 6). The secondary structure was analyzed by PSIPred software, and HPV-33 and HPV-58 L1 homology models were created by SWISS-MODEL software. The selection pressures acting on the L1/L2 genes were estimated by PAML 4.8.To study the gene intratypic variations and polymorphisms of HPV-33 and HPV-58 L1 sequences 20 single nucleotide mutations were observed included 8/20 non-synonymous and 12/20 synonymous mutations. The 101 HPV-33 L2 sequences included 12 single nucleotide mutations comprising 7/12 non-synonymous and 5/12 synonymous mutations. The 223 HPV-58 L1 sequences included 32 single nucleotide mutations comprising 9/32 non-synonymous and 23/32 synonymous mutations. The 201 HPV-58 L2 sequences comprised 26 single nucleotide mutations including 9/26 non-synonymous and 17/26 synonymous mutations. Selective pressure analysis showed that most of the common non-synonymous mutations showed a positive selection. HPV-33 and HPV-58 L2 were more stable than HPV-33 and HPV-58 L1.Among 124 HPV-33 L2 were better candidates as clinical diagnostic targets compared with HPV-33 and HPV-58 L1. Clinical diagnostic probes and second-generation polyvalent vaccines should be designed on the basis of the unique sequence of HPV-33 and 58 L1/L2 variations in Sichuan, to improve the accuracy of clinical detection and the protective efficiency of vaccines.HPV-33 and HPV-58 The online version of this article (doi:10.1186/s12985-016-0629-9) contains supplementary material, which is available to authorized users. Human papillomavirus (HPV) infection plays a critical role in the development of cervical cancer . The risGenital HPV subtypes are typically divided into two groups according to their presumed oncogenic potential. High-risk oncogenic HPV subtypes −16, −18, −58, −33, −52 and −45 are more common in Asia than elsewhere , 10. NevL1 and the minor capsid proteins L2 [L1 proteins form a pentamer, and 72 pentamers constitute the virus capsid. The L1 and L2 proteins self-assemble into virus-like particles (VLPs) that induce high levels of neutralizing antibodies and are highly protective [L1 only prevents infection by specific HPV subtypes because of the lack of cross-protective epitopes in different HPV subtypes. HPV L2 protein also induces neutralizing antibodies, the N-terminal of L2 protein contains cross-protective epitopes and represents the target of neutralizing antibodies [L2 may be an attractive approach for a candidate vaccine.The HPV genome is packaged within the major capsid late protein teins L2 , 13. Fivotective , 15. Thetibodies . TherefoL1 are available [L2 and other subtypes of L1 are currently under investigation [Vaccines against HPV-16, HPV-18, HPV-6 and HPV-11 vailable . The twotigation .L2 and HPV-33 L1/L2 in China are limited. The molecular variants of HPV-33 L1/L2 worldwide are not widely reported. Ethno-geographical variations are observed in distribution of HPV subtypes. Among different subtypes of HPV, there are subtypes and variants that can acquire biological advantages through fixed mutations in their genomes, and even small variations could result in small adaptive improvements that could alter the composition of an HPV-infected population [The data supporting HPV-58 pulation . Alteredpulation . IdeallyL1/L2 gene polymorphism and intratypic variations in Sichuan, China. This study can provide essential data for future research on viral prevention and therapeutics. Above all, our study provides critical data facilitating the development of diagnostic probes and design of vaccines based on HPV-33 and HPV-58 L1/L2.This study investigated the HPV-33 and HPV-58 Cervical specimens were collected from Sichuan Reproductive Health Research Center Affiliated Hospital, The Angel Women’s and Children’s Hospital, The Chengdu Western Hospital Maternity Unit, and The Peoples’ Hospital of Pengzhou, and other institutions. Between January 1, 2009, and December 31, 2014, women presenting for cervical screening underwent histology and cytology evaluations for cervical disease. Women over 14 years of age and with visible cervical lesions and/or HPV-related diseases  were eligible for inclusion. Cervical specimens were collected from participants and placed in a preservative buffer and stored at −20 °C.HPV-DNA was extracted and examined using a Human Papillomavirus Genotyping Kit  according to the manufacturer’s instructions. This kit enabled the classification of 23 HPV subtypes . In total, 478 HPV-58 and 273 HPV-33 positive samples were collected.L1/L2 genes of HPV-33 and HPV-58 were amplified using primer pairs. The primers were designed according to the GeneBank reference sequences for HPV-33 (GenBank: M12732.1) and HPV-58 (GenBank: D90400). The primer sequences were listed in Table The entire The sequences and variations were analyzed by NCBI Blast, and DNAMAN version 5.2.2. The nucleotide positions were numbered according to the GeneBank reference sequences of HPV-33 (GenBank: M12732.1) and HPV-58 (GenBank: D90400). All the data were confirmed by repeating PCR amplification and sequence analysis at least twice.L1/L2 and HPV-58 L1/L2 variation patterns were constructed with the maximum-likelihood trees using MEGA  6 software and Kimura’s two-parameter model. The tree topologies were evaluated using bootstrap resampled 1,000 times [Then, phylogenetic trees of respective HPV-33 00 times .L1/L2 gene sequences, the codeml program in the PAML  version 4.8 package was used to perform the likelihood ratio tests (LRTs) to infer non-synonymous and synonymous nucleotide divergence for coding regions by the method of Nei and Gojobor [http://bioinf.cs.ucl.ac.uk/psipred). PSIPred is a simple and accurate secondary structure prediction method, incorporating two feed-forward neural networks, which enable the analysis of output obtained from PSI-BLAST (Position Specific Iterated-BLAST). A very stringent cross validation of the method indicated that PSIPred 3.2 attained an average Q3 score of 81.6 % [To estimate the positive selections at particular sites of the HPV-33 and HPV-58  Gojobor –23. The f 81.6 % .http://swissmodel.expasy.org/) based on the crystal structure of the HPV16 L1 pentamer [HPV33 and HPV58 L1 homology models were created by SWISS-MODEL  in the 124 HPV-33 L1 sequences studied. We identified 20 single nucleotide changes among the 124 sequences studied. Specifically, 12/20 (60.00 %) were synonymous and 8/20 (40.00 %) were non-synonymous mutations. Only 1 non-synonymous mutation was observed in sequence encoding the helix. The detected mutations are summarized in Table L1 was showed in Additional file Compared with the HPV-33 reference sequence (GenBank: M12732.1), the nucleotide variation rate of HPV-33 L2 was 100.0 % (101/101) in the 101 HPV-33 L2 sequences studied. We identified 12 single nucleotide changes among the 101 L2 sequences studied. Specifically, 5/12 (41.7 %) were synonymous mutations and 7/12 (58.3 %) were non-synonymous mutations. The detected mutations are summarized in Table L2 was showed in Additional file Compared with the HPV-33 reference sequence (GenBank: M12732.1), the nucleotide variation rate of HPV-33 L1 was 96.86 % (216/223) in the 223 HPV-58 L1 sequences studied. We identified 32 single nucleotide changes among the 223 sequences studied.Compared with the HPV-58 reference sequence (GenBank: D90400), the nucleotide variation rate of HPV-58 L1 was showed in Additional file Specifically, 23/32 (71.88 %) were synonymous mutations and 9/32 (28.12 %) were non-synonymous mutations. 3 non-synonymous mutations were observed in sequences encoding the helix, 1 non-synonymous mutation was observed in sequence encoding the sheet. The detected mutations are summarized in Table L2 was 68.55 % (168/201) in the 124 HPV-58 L2 sequences studied. We identified 26 single nucleotide changes among the 201 L2 sequences studied. Specifically, 17/26 (65.38 %) were synonymous and 9/26 (34.62 %) were non-synonymous mutations. 3 non-synonymous mutations were observed in sequences encoding the sheet. The detected mutations are summarized in Table L2 was showed in Additional file Compared with the HPV-58 reference sequence (GenBank: D90400), the nucleotide variation rate of HPV-58 L1-positive selection was seen with T56N, G133S, K135R, T226K, G268E and E385D. HPV-33 L2-positive selection was seen in D77N, D350H, D360N and N372T. The HPV-58 L1-positive selection included L150F, F318Y, I325M, T375N and E377D. The HPV-58 L2-positive lineages included N231T, A416T and M446L. Results of the selective pressure analysis of HPV-58 and HPV-33 L1 and L2 genes  are summarized in Tables The variable dN/dS rate ratios were tested among the various lineages using the PAML4.8 . HPV-33 L1 were aligned , HPV58 , HPV33 , HPV52 , HPV18 , HPV56 , HPV66 , HPV31 , HPV59 , HPV68 , HPV51 , HPV35 , HPV53 , HPV45 , HPV73 , HPV39 , HPV83 , and MM4 . Low-risk genotypes included HPV6 , HPV11 , HPV43 , HPV42 , and HPV44  [In our previous study of 10682 patients, 3370 (31.5 %) were positive for HPV infection. High-risk subtypes were as follows: HPV16 . The HPV-58 was classified into four variant lineages  . In the L1 and L2 variation frequencies were higher than those of HPV-33 L1 and L2. Among these variations, C5807A, A5822G, G5984A, A6437G, T6470C, T6485C and A6695C  represented novel HPV-58 L1 mutations, which were found until now only in Sichuan, China [L2, mutations other than A4621C and A5206G, were newly reported [L1, T5960C, A5997G (K135R), T6385G, G6396A (G268E), T6463C, G6520A, T6613C, A6694G, A6951C (K453T), C7044A (P484H) and G7063A were reported for the first time, these newly reported mutations were only found in China in reports related to HPV-33 L1 [We demonstrated that HPV-58 n, China , 28–32. reported . In HPV-PV-33 L1 , 33, 34.L1 included T266K (18/124), T56N (13/124), G133S (13/124) and E385D (13/124). The most common non-synonymous mutations of HPV-33 L2 were D77N (15/101), F281Y (8/101), D350H (6/101), D360N (6/101), and N372T (6/101). The most common non-synonymous mutations of HPV-58 L1 comprised L150F (88/223), I135M (88/223), and T375N (62/223). The most common non-synonymous mutations of HPV-58 L2 were A416T (146/201), N231T (24/201), and M446L (24/201). Results of the selective pressure analysis suggest that most positive selection mutations of HPV-33 and HPV-58 L1/L2 were common non-synonymous mutations, indicating that the positive selection variations beneficial for HPV-33 and HPV-58 adapted to their environments widely. The other variations in the positive selection mutations were uncommon. However, with the inheritance of the virus, positive variations may become increasingly common and gain importance in the future. The most common synonymous mutations of HPV-33 L1 were A6664G (18/124), T6613C (14/124), T6478C (13/124) and A6673G (13/124). The most common synonymous mutations of the HPV-33 L2 included C5220T (101/101) and C4905T (9/101). The most common synonymous mutations of HPV-58 L1 were A6416G (65/223), T6434C (65/223), G6641A (65/223), and A6560G (64/223); and those of the HPV-58 L2 were T4627C (148/201), G5395C (146/201), and G5266A (86/201). Due to the high diagnostic value of L1, and its variability [L1 is often selected as a clinical diagnostic target. We considered the most sites in common mutations to design clinical diagnostic probes targeting HPV L1 and L2 genes. The intratypic variations observed in L1 and L2 enabled the analysis of known and novel HPV subtypes [L2 were less frequent than those of HPV-33 L1. The sequence and single nucleotide changes of HPV-58 L2 were less frequent than those of HPV-58 L1, while those of HPV-33 and HPV-58 L2 were more conserved than those of L1, suggesting that HPV-33 and HPV-58 L2 were better candidates as clinical diagnostic targets compared with HPV-33 and HPV-58 L1.The most common non-synonymous mutations of HPV-33 L1; N82T of HPV-58 L1) next to these sites. We believe mutations occurred on the outwardly facing surface-exposed loops deserve research attention and should be studied for vaccine design targeting HPV-33 and 58 L1.Nearly all conformational epitopes are located on one or more of the outwardly facing surface-exposed loops of BC, DE, EF, FG, and HI . Sites 5L2. G4452A (G70E) and G4470A (S76N) were identified at residues 69–81 and 108–120 of HPV-58 L2. Amino acid residues 33–52, 73–84, 89–100, and 121–140 of L2 contain non-neutralizing antibody epitopes [L2 and The G4470A (S76N) of HPV-58 L2 were discovered at residues 73–84. These mutations must be considered during vaccine design targeting HPV-33 and 58 L2.Amino acid residues 69–81 and 108–120 of L2 protein are highly conserved and contain cross-reacting epitopes that play an important role in inducing neutralizing antibodies . G4438A L1/L2 proteins of HPV-58 variants in Sichuan and that of the L1/L2 proteins of HPV-33 in China. Because of limitations related to sample size, sample copies, and sequencing technology, the present study may have had a sampling bias against integrated HPV genomes. The data presented in this study have significant implications for the understanding of intrinsic geographical and biological differences in HPV-33 and HPV-58 L1/L2, as well as contribute to the design of clinical diagnostic probes and second-generation polyvalent vaccine based on HPV-33 and HPV-58 L1/L2.This is the first study examining the role of L1 and L2 may alter the virulence of variants, and also define altered epitopes in vaccine design. The reference sequences of HPV-33 and 58 only represent minor sequence patterns of HPV-33 and 58 L1/L2. Further, the distribution of HPV-33 and 58 L1/L2 variations in Sichuan has its own peculiarities. Therefore, clinical diagnostic probes and second-generation polyvalent vaccines should be designed on the basis of the unique sequence of HPV-33 and 58 L1/L2 variations in Sichuan, whereby the accuracy of clinical detection and the protective efficiency of vaccines can be improved.Mutations in HPV


Safety and immunogenicity of the quadrivalent human papillomavirus (qHPV) vaccine were evaluated in HIV-positive Spanish MSM. The prevalence of High Squamous Intraepithelial Lesions (HSIL) and genotypes of high-risk human papillomavirus (HR-HPV) were also determined, as well as risk factors associated with the presence of HR-HPV in anal mucosa.® Pap Test), HPV PCR genotyping (Linear Array HPV Genotyping Test), and high-resolution anoscopy (Zeiss 150 fc© colposcope) were performed at V1.This is a randomised, double blind, placebo-controlled trial of the quadrivalent HPV (qHPV) vaccine. The study enrolled from May 2012 to May 2014. Vaccine and placebo were administered at 0, 2 and 6 months . Vaccine antibody titres were evaluated at 7 months. Cytology  were screened for inclusion; 14.2% had HSIL, 73.1% HR-HPV and 4.5% simultaneous infection with HPV16 and 18. Study participants (n = 129) were randomized to qHPV vaccine or placebo. The most common adverse event was injection-site pain predominating in the placebo group [the first dose ; the third dose . The vaccine did not influence either the viral load of HIV or the levels of CD4. Of those vaccinated, 76% had antibodies to HPV vs. 30.2% of those receiving placebo (p = 0.0001). In the multivariate analysis, Older age was associated with lower HR-HPV infection , and risk factor were viral load of HIV >200 copies/µL (RR 1.42 95% CI 1.17–1.73) and early commencement of sexual activity .This trial showed significantly higher anti-HR-HPV antibody titres in vaccinated individuals than in unvaccinated controls. There were no serious adverse events attributable to the vaccine. In our cohort, 1 of every 7 patients had HSIL and the prevalence of combined infection by genotypes 16 and 18 was low. This suggests that patients could benefit from receiving qHPV vaccine. Older age was the main protective factor against HR-HPV infection, and non-suppressed HIV viremia was a risk factor.http://www.isrctn.com/ISRCTN14732216).Clinical trial registration: ISRCTN14732216 ( Anal squamous cell carcinoma (ASCC) in HIV patients is, currently, one of the most-frequent non-AIDS-defining cancers . There aThe classical risk factors involved in the infection of oncogenic HPV in the anal mucosa include: young age , a largeSeveral strategies for the prevention of ASCC have been evaluated, including: screening for ASCC/HSIL with anal cytology alone , 12, anaThere have been several trials with the quadrivalent human papillomavirus (qHPV) vaccine carried-out in HIV seronegative MSM. In two of them, MSM were compared to heterosexual males (HM), and they found lower protection rates against external genital lesions (EGL) in MSM  , and a lAgencia Española del Medicamento y Productos Sanitarios; AEMPS). The recruitment period was between May 15th 2012 and May 15th 2014. Clinical trial registration: ISRCTNregistry. ISRCTN14732216. DOI 10.1186/ISRCTN14732216. Date assigned 02/08/2016. Retrospectively registered. See clinical trial protocol at http://www.isrctn.com/ISRCTN14732216. This is a randomised, double blind, placebo-controlled trial of the quadrivalent HPV (qHPV) vaccine. It was conducted according to the protocols of the Spanish Drugs and Health Products Agency  at screening for inclusion or only had condylomas and/or low squamous intraepithelial lesion (LSIL) in anal biopsy.HIV MSM patients who had simultaneous anal infection with the four genotypes addressed by the vaccine, and who at least had HPV genotypes 16 and 18.Active opportunist infection at the time of recruitment into the study.Patients who, in screening anoscopy had HSIL, or ASCC or had received treatment for these lesions (these patients were excluded because patients with HSIL have a higher probability of progression to ASCC).History of allergy to aluminium and/or yeast extract excipient.University Hospital Virgen de las Nieves”, Granada (Spain), and Ciudad de Jaén (Spain).The patients who enrolled were HIV-positive MSM that were attending the Infectious Diseases Service of the “Fundación Progreso y Salud) of the Government of Andalucia (2011 convocation). This trial was registered in Clinical trial registration: ISRCTN14732216.The purposes of the study were explained to the potential participants who then underwent screening, and enrolled if they met the inclusion criteria for the trial. They were asked to sign the fully informed consent form. The project received approval from the hospital’s Ethics Committee (Institution Review Board) “Comité de Ética de la Investigación Biomédica de la Provincia de Granada (CEI-GRANADA)” University Hospital Virgen de las Nieves. The study was conducted in compliance with ethical and moral principles stated in the Declaration of Helsinki as well as the current Spanish Laws on Biomedical Research. Data were coded to ensure anonymity. The project received funding from the Foundation for Progress and Health , the conditions and objectives of the study were explained. The details were summarised in a document, which was presented to the patient who then signed the informed consent form.Clinical-epidemiological variables At the baseline visit (V1), data collected included: age, number of different partners participating in anal intercourse in the previous 12 months, and over the whole sexual life of the participant; use of prophylactics and percentage frequency of their use; work status , education level, smoking habit (packets/year), alcohol abuse  consumed per day); intra-venous drug abuse (IVDA); HIV infection route, months since HIV diagnosis, CDC status, CD4 nadir (considered to be the lowest level of CD4 throughout evolution of HIV), months of anti-retroviral therapy (ART), current line of ART, virological failure , concomitant treatment. Other diseases included chronic viral hepatitis B infection (VHB), or hepatitis C (VHC), syphilis, other sexually transmitted diseases (STD), history of anal and/or genital condylomas and the therapy employed, current condylomas, latent active or treated tuberculosis infection.At clinical visits at 2 and 6 months (V2 and V3), the data collected (again) were number of different anal-sex partners, use of condoms, ART therapy , appearance of STD or condylomas. The adverse events assessment system employed was a questionnaire that included the most frequent local reactions such as fever, nausea, vomiting, dizziness, syncope, headache and others such as allergic reaction, pruritus, difficulty breathing and/or wheezing. Rare occurrences included lymphadenopathies, chest and lower-limb pain, confusion, chills, muscle pain. The adverse events (AE) were graded on a scale of 1–4. In case of AE grade 4, the blind of the vial administered was broken and, if the code identified the vaccine, the reaction was communicated immediately to the relevant drug-vigilance authorities.Blood analyses At visits V1, V2 and V3 full blood haemogram and blood chemistry analysis were measured, together with CD4, CD8 lymphocytes counts, and HIV viral load (VL).Antibodies against the 4 genotypes of the qHPV vaccine were determined after the 3rd dose, at 7th month. The analyses were performed in the microbiology department of the hospital by the same microbiologist of the research team (AS). The assays were performed using HPVG ELISA commercial kit , according to the manufacturer’s instructions. The ELISA kit measures antibodies against the major capsidic protein (L1) of HPV types 6, 11, 16, 18. The blood samples collected in the clinic were centrifuged in the laboratory, frozen at −20 °C and thawed for use at the time of the assay. The results were expressed qualitatively as positive or negative.High-resolution anoscopy (HRA) (at V1) All the recruited patients had an anoscopy performed and a biopsy taken following 3% acetic acid instillation and Lugol. Biopsies were taken of acetic-white change and Lugol-negative zones, and normal mucosa, with a minimum of 1 biopsy in each of the four quadrants . The colposcopy equipment employed was Zeiss 150 fc©.PCR of the HPV and anal cytology (at V1): 2 mucosa samples were taken from the anal canal with cotton swabs soaked in physiologic saline serum. The samples were stored in liquid medium (Thin Prep® Pap Test) for the detection and genotyping of the HPV using the polymerase chain reaction (PCR) qualitative technique (Linear Array HPV Genotyping Test) performed in a Gene Amp PCR System 9700 thermocycler . Cytology evaluation was with the Thin Prep® Pap Test . Both samples were sent to the anatomy–pathology laboratory where the same senior pathologist of the research team (JE) carried out the cytology evaluation, validation of PCR methodology, and histology analyses. HPV genotypes 16, 18, 26, 31, 33, 35, 39, 45, 51–53, 56, 58, 59, 66, 68, 73 and 82 were considered high-risk-HPV (HR-HPV). Genotypes 6, 11, 34, 40, 42–44, 54, 55, 57, 61, 70–72, 81, 83, 84 and 89 were considered low-risk-HPV (LR-HPV). The HPV-18 virus species was classified as genotypes 39, 45, 59, 68; and of the HPV-16 species as genotypes 31, 33, 35, 52, 58, 67 .Similarly, there were no statistically significant differences with respect to the levels of CD4  against HPV at 7 month vs. 30.2% of those receiving placebo (p = 0.0001). There was no record of the baseline (V0/screening) HPV antibody status of the patients.The following variables were observed to be protective factors against the infection of genotypes of HR-HPV in the anal mucosa: age, i.e. being older ; duration of ART ; and months since HIV diagnosis . Conversely, we found the following predictive factors: early age of sexual activity commencement ; and HIV viral load ≥200 copies/mL   and, as a risk factors the early commencement of sexual activity  and viral load >200 copies/mL   related to the vaccine administration. The commonest AE was local injection-site pain which was more frequent in the placebo group. In a contrasting result  a differWith respect to immunogenicity, in our study 76% of patients receiving the vaccine had measurable antibody levels at the 1th month following administration of the 3rd dose of the vaccine. Although this was significantly higher in the vaccine arm, the lack of baseline antibody levels precludes a definite conclusion that the vaccine is immunogenic. The prevalence of patients with detectable antibody against HPV is lower than in a previous clinical trial carried-out in HIV MSM patients in whom 98% of the patients developed antibodies against the four HPV genotypes in the quadrivalent vaccine . These dThe prevalence of HR-HPV in the anal mucosa in our group of HIV-positive MSM patients was 73.7%. Similar percentages have been communicated in previous research in seropositive MSM . HoweverWith respect to the genotypes against which the qHPV generates immunity, in our cohort we found, in the pre-vaccination analyses, that the most frequent of all was genotype 16, which was present in 1 of every 4 patients, and only 4.5% of our patients had a simultaneous infection by two oncogenic genotypes (16 and 18). On analysing the combinations of viruses pertaining to the species 16 and 18, we observed that up to 50% of the study participants were infected by the viral species HPV16 and 37% by the species HPV18. HPV16 is the genotype that has been shown most frequently, in the majority of published studies, to be associated with ano-genital pathology in both genders . HIV infWe observed that only 14.2% had HSIL lesions at V1; results similar to those communicated by other authors , 40, andIn relation to the risk-factors associated with the infection of HR-HPV in the anal mucosa, multivariate analyses identified viral load ≥200 copies/mL as risk predictor for HR-HPV infection, whilst older age was protective. A study, that analysed the incidence and clearance of anal high-risk human papillomavirus in HIV-positive MSM, found that those with low HIV-RNA-viral load had the highest clearance . ClassicThe principal limitations of our study that need to be highlighted derive from the exclusion criteria which do not permit generalisation of data; 18.7% of patients who underwent screening were excluded because of HSIL (14.2%) or because of simultaneous infection by genotypes 16 and 18 (4.5%). Baseline Ab were also not determined and this event is a potential weakness. The results of our study suggest immunogenicity, but without baseline differences to compare we can’t be certain.Consequently, a further study would be necessary to demonstrate the usefulness of the vaccine in these subsets of patients who had been excluded.This trial of qHPV vaccine conducted in Spanish HIV-positive MSM patients showed significantly higher anti-HR-HPV antibody titres in vaccinated individuals than in unvaccinated controls. There were no serious adverse events attributable to the vaccine. Although current prevalence of HPV 16/18 is low, a large proportion of men likely have had incident HPV 16/18 infections and cleared them; therefore, they may have natural immunity that protect against subsequent infection. Older age was the protective factor against HR-HPV infection and HIV no suppressed was the risk factor.


Past military service is associated with health outcomes, both positive and negative. In this study we use the 2013 National Health Interview Survey to examine the constellation of conditions referred to as musculoskeletal disorders (MSDs) for Veterans and non-veterans with health conditions that limit their daily activities. Multivariate logistic regression analysis reveal that Veterans are more likely to report MSDs like neck and back problems, fracture bone and joint problems as an activity limiting problem compared to non-veterans. The relationship between age and reports of activity limiting MSDs is moderated by Veteran status. Veterans in this sample report more activity limiting MSDs at younger ages compared to non-veterans and fewer MSDs at older ages. This research contributes to our understanding of potentially limiting health conditions at earlier ages for Veterans. There are 22 million Veterans of the United States Armed Forces , making Military service has both positive and negative health effects. Early in life military service members tend to be healthier than same-aged non-military counterparts . This isStandardized risk mortality ratios as the marker of health outcomes suggests that military service is health positive for most Veterans. If, however, health is viewed from the standpoint of morbidity, Veterans are more likely than similar aged civilians to have worse health. Studies indicate that Veterans have more cardiovascular disease , obesityMost studies of the health of U.S. service members typically include data on active-duty service members or data on Veterans enrolled in the Department of Veteran Affairs (VA) health care system , 19. TheLarge national studies comparing Veterans and non-veterans have until very recently been based on data collected before 2004 , 25. ResThe current study seeks to fill an important gap in the literature by using the National Health Interview Survey, a nationally representative data set that includes a representative sample of Veterans living in the civilian population to explore four MSDs leading to limitations of activity ; Back/NeWe used the 2013 National Health Interview Survey (NHIS) to examine the health of military Veterans in comparison to non-veterans with regard to MSDs. The NHIS is one of the nation’s largest cross-sectional household health surveys and is conducted through the Centers for Disease Control and Prevention’s National Center for Health Statistics (NCHS) . The NHIThe NHIS follows a stratified multi-stage cross sectional design that is designed to produce estimates for the nation, for each of the four census regions and within census region by metropolitan status. In the Respondents were asked whether they have ever served in the United States Armed Forces, Reserves, or National Guard. Those who identified as having ever served are called Veterans for the purposes of this study. This differs somewhat from Veteran status defined according to American legal code (38 U.S. Code § 101) as military personnel that have “served in the active military, naval, or air service” and were “discharged or released therefrom under conditions other than dishonorable”, which establishes the legal definition to determine eligibility for Veterans’ Benefits .International Classification of Disease 9thedition (ICD-9) codes used by the NHIS to create each musculoskeletal category  with their ethnicity . This recoding divides the sample into racial and ethnic categories that include White, non-Hispanic; Black/African American, non-Hispanic; and Hispanic/Latino. We chose to exclude individuals who identified themselves as Other race, American Indian, Pacific Islander, Asian American because there were so few respondents in the analytic sample who self-identified using these categories. For example, there were 16 veterans who identified as Asian American and 24 respondents who fell into all other racial/ethnic categories . Sex was coded so that 1 = males, and 0 = females. Marital status was coded so that people who were married and those living with an unmarried partner  were coded in the same category, divorced and separated respondents were coded together, and widowed and never married respondents were each in their own category. Individuals with chronic health conditions that limit their daily activities are less likely to work for pay compared to those without chronic illnesses. TherefoIn order to test the moderating effect of race/ethnicity and age on the relationship between Veteran status and MSDs, we created 2 interaction terms in the analysis. The first is product term of Veteran status and race/ethnicity  and the second is a product term for Veteran status and age (in years).All analyses were run using SPSS version 23.0 . In thisIn the adjusted models , VeteranIn the model that includes only veteran status , we see In the unadjusted models , the resIn the unadjusted models, results indicate that Veterans have a lower odds of reporting other , being 19% less likely to report other musculoskeletal conditions compared to non-veterans . In the Veteran status is a significant moderator for the relationship between age and two MSDs—back/neck problems and fracture, bone/joint injury. In order to understand the moderating effect, we have plotted the mean predicted probability of having these MSDs for each age by Veteran status, Figs Among both Veterans and non-veterans, as shown in decreasing odds of Veterans reporting a back/neck problem with advancing age compared to same-aged civilians. This suggests that Veterans have back and neck problems at younger ages, but the aging process appears to erase the higher likelihood of reporting as non-veterans eventually catch up physically to Veterans. The source of the back and neck injury is not reported, but the main difference between Veterans and non-veterans is military service. This is unsurprising given that military service-related duties consist of regular, sustained physical activity and intensive repetitive physical labor, both common sources of musculoskeletal injuries. The risks for military-related injuries begin in basic training with regular physical training that continues throughout the Veteran’s military career. State-side military post duty carries its own risks resulting from the demands of the military occupational specialty (MOS). Logistics occupations require regular lifting and movement of supplies and equipment. Communications and monitoring specialties require long periods of sitting, which may put troops at risk for musculoskeletal problems resulting from poor posture. Medical service personnel may walk long hours on hard tile floors in hospitals and clinics in addition to pushing, pulling, rolling, and transporting heavy loads of supplies and patients. Pre-deployment activities offer unique musculoskeletal risks that stem from the intensive period of readying and centralizing war equipment. During deployment neck and back injuries can result from a number of sources; long 12–16 hour workdays that translate into more lifting, sitting, or pushing, than regular stateside post duty. Protective gear like the PASGT helmet, which weighs 3 to 4 lbs depending on size, puts strain on the soft tissues of the neck and back. Life on a forward operating base sometimes means unexpectedly needing to run, jump and sprint on uneven to avoid incoming fire. Troops assigned combat patrol may regularly do this while also carrying 40 to 120 lbs. or more of supplies and equipment. Deployment also places troops at increased risk of small arms fire or blast-related musculoskeletal injuries. Evidence suggests that injuries are more likely to be related to the more mundane experiences related to MOS duties rather than from direct combat [Military service has both positive and negative health effects for Veterans. Our findings show that Veterans are almost 3 times more likely to report a back/neck problem compared to non-veterans. As indicated above, there was a t combat , 33. Onet combat .less likely to be married, divorced, or widowed in general. Younger bodies are more resistant to musculoskeletal injuries and are not as likely as older bodies to suffer injuries resulting from chronic overuse that comes with the aging process. Along these lines, other research has shown that among active duty personnel suffering from lower back pain, older service members had higher rates of back pain compared to younger service members [Our analysis also found that respondents who were married/living with a partner, widowed or divorced/separated all had a significantly higher likelihood of having back/neck problems compared to respondents who were had never been married. This likely captures an age dynamic, as younger persons are  members . We haveWe also explored the interaction between Veteran status and race/ethnicity. We found that Black Veterans specifically had lower odds of reporting back/neck problems compared to white Veterans and Hispanics. Black military personnel tend to be overrepresented in logistics support and administrative services compared to whites and Hispanics who tend to be overrepresented in combat specialties . HispaniVeterans were more than 3 times more likely to report an activity-limiting fracture, or bone/joint injury compared to non-veterans in the adjusted models. Black respondents were also less likely to report fracture, or bone/joint injury as an activity limitation compared to whites. Like the reporting of back/neck injuries, military service predisposes Veterans to injury via training, deployment, and/or combat. Damage to bones and joints is one possible result.There was no relationship in our study between Veteran status and reporting Arthritis/Rheumatism or Other Musculoskeletal Conditions as an activity limiting condition. This differs from other research that suggests Veterans report higher rates of arthritis than civilians , 38. MurOur results indicate that Veterans with back and neck injuries and fracture, bone and joint injuries that limit activity suffer activity limitations at earlier ages than non-veterans. Musculoskeletal problems represent some of the most physically disabling chronic health conditions , and disThe National Health Information Survey (NHIS) data do not allow for drilling down into the specifics of the musculoskeletal injuries. Data report the presence of a Back/Neck injury, but beyond this, we cannot determine whether the injury is to the neck or back, the upper back or lower back. We also cannot say whether musculoskeletal injuries are service-connected or occurred after military service was completed. Back/Neck and Fracture, Bone/Joint injuries are reported as activity limiting conditions by Veterans at younger ages and higher rates than non-veterans, but the specifics of where and when are inaccessible given the data.As noted above, NHIS respondents who indicated they had ever served in the United States Armed Forces were coded as “Veterans”. The NHIS does not verify Veteran status using the legal definition, which according to 38 U.S. Code § 101 of U.S. law, is someone who has “served in the active military, naval, or air service” and was “discharged or released therefrom under conditions other than dishonorable”. The only available data that does confirm Veteran status comes from the Veterans Health Administration using VHA-enrolled Veterans; to be eligible for VA benefits, Veterans must meet the legal definition for Veteran status. One problem with VHA data is that the majority of eligible Veterans are not enrolled in VA health care services . AnotherThis study established that Back/Neck and Fracture, Bone/Joint injuries are more likely to be reported as activity limiting conditions at younger ages for Veterans compared to non-veterans. Future research should explore the nature, type, and severity of reported activity limitations. For example, are activity limitations specific to activities of daily living, or ADLs—feeding, bathing, toileting, dressing, or are the reported limitations centered on instrumental activities of daily living (IADLs), such as the ability to do housework or participate in community functions? How many limitations in ADLs and IADLs are respondents experiencing? How do Veterans and non-veterans differ in their ADL and IADL limitations? Are there age-related differences in limitations of ADLs and IADLs? Such questions can more fully capture the ways that military service affects Veterans health.among Veterans by age, taking a more intersectional approach; for example sex-by-age-by-Veteran status. There is some evidence indicating that female OEF/OIF Veterans are more likely to be diagnosed with musculoskeletal problems than male OEF/OIF Veterans [We also highlighted significant differences for musculoskeletal injury-related limitations between Veterans and non-veterans by age. Future work will explore differences Veterans . ExpandiIn addition to sex-age-Veteran status interactions, looking at sex-by-race-by-Veteran status across age will provide important information on which categories of Veterans are most at risk for musculoskeletal injuries, they types of injuries they are most at-risk of suffering, and the ages at which they are most likely to report injury-related limitations.Veterans in this study are more likely at younger ages to report Back/Neck and Fracture, Bone, and Joint injuries as activity-limiting conditions compared to non-veterans. Using the National Health Information Survey provides a unique, nationally representative sample of Veterans that offers of broader picture of Veterans’ health concerns than is traditionally offered through the use of VA data alone. We offer this research as a general guide to thinking about the role that military service may play in the musculoskeletal health, and overall quality of life, of Veterans.


The cost effective maintenance of underwater pressure pipes for sewage disposal in Austria requires the detection and localization of leakages. Extrusion of wastewater in lakes can heavily influence the water and bathing quality of surrounding waters. The Distributed Temperature Sensing (DTS) technology is a widely used technique for oil and gas pipeline leakage detection. While in pipeline leakage detection, fiber optic cables are installed permanently at the outside or within the protective sheathing of the pipe; this paper aims at testing the feasibility of detecting leakages with temporary introduced fiber optic cable inside the pipe. The detection and localization were tested in a laboratory experiment. The intrusion of water from leakages into the pipe, producing a local temperature drop, served as indicator for leakages. Measurements were taken under varying measurement conditions, including the number of leakages as well as the positioning of the fiber optic cable. Experiments showed that leakages could be detected accurately with the proposed methodology, when measuring resolution, temperature gradient and measurement time were properly selected. Despite the successful application of DTS for leakage detection in this lab environment, challenges in real system applications may arise from temperature gradients within the pipe system over longer distances and the placement of the cable into the real pipe system. The organization of a comprehensive wastewater treatment network in Austria required the construction of centralized wastewater treatment plants, as well as supply pipes, transporting the sewage from the polluter to the treatment plant. Given economic constraints, the supply pipe system was planned along the shortest, technically feasible distances. Consequently, in the 1970s and 1980s, pressure pipes for sewage transport have been installed in several scenic lakes in Austria  technology provides a mean to circumvent the difficulties and limitations. DTS systems allow to detect and locate temperature changes along a fiber optic cable up to a length of 10 to 30 km in a very high spatial and temporal resolution  and inside the pipe might be generated by filling the pipe system with warmer surface water in the summer months from the warmer epilimnion. The application of negative pressure within the pipe system will cause the intrusion of cold hypolimnion water into the pipe system. The penetrating colder water will alter the local water temperature in the pipe, which will be detected and monitored by the DTS system. After finishing the tests, the cable can be removed completely.In order to test this new monitoring concept, a medium-scale laboratory experiment was designed to test the feasibility of the method. Special focus was paid on the varying cable positioning inside the pipe as well as the limits and challenges of this methodology in the practical implementation. In the following chapter, the methodology and materials used for the experiment will be explained. An overview about the DTS technology, the approach of leakage detection via DTS in nature, and the transformation of the setup to the experimental design will be given. In section TM device with a maximum spatial resolution of 0.25 m and a temporal resolution of 10 s and a Brusens® temperature FO cable  was used.The DTS technology provides temperature measurements with high temporal and spatial resolution along a FO cable  inside the pipe, simulating conditions for leakage detection in a natural environment. A schematic of the experimental design is illustrated in Fig. Under normal pumping conditions, a water extrusion into the tank water from the pipe through a leakage is to be expected; no temperature differences would be detectable inside the pipe. To generate an inversion of the flow, firstly, the flush with warm water was stopped and the valves at the beginning and the end of the pipe were closed. After a couple of minutes, an approximated uniform temperature difference of the water inside the pipe and the water in the tank was observable. An inverse flow was induced by implementing a slightly negative pressure of 0.1 bar at the end of the pipe, provoking an intrusion of colder water from the tank into the pipe. Cold water mixed with the warmer water leading to a local temperature decrease near the leakage. An overview of the workflow can be found in Fig. Temporal resolutionSpatial resolutionCable positioningNumber and size of leakagesA set of 13 measurement sequences were conducted. They differed in the following:The temporal and spatial resolution of the DTS measurements has a strong influence on the detected temperature differences as well as on the measurement uncertainties. Lower spatial and temporal resolution might lead to buffered detected temperature differences, as well as lower measurement uncertainties. Cable positioning might be affecting the measured temperature, as sections near the pipe wall might be influenced by thermal exchange between outside and inside temperature. This would result in lower temperature measurements near the wall. The influence of spatial and temporal resolution on the localization was tested by realizing different measurement runs under identical experiment conditions. To test the wall influence on the leakage detection, another measurement series was realized with a free-floating cable and other measurement series bonding the cable at the pipe internal wall. Latter have been compared with measurements, separating the cable at least 3 cm from the wall with braces made by foamed polystyrene. Finally, tests were conducted with multiple leakages of different sizes and different spatial resolutions.Tcum (°C) during the integration time (t), similar as described in Sayde et al.   as well as the temporal resolution (tr) . The spatial resolution refers to the spatial integration scales over which a single temperature value is reported, the temporal resolution refers to integration time, and the fiber temperatures are resolved  a reference temperature calculated from the measurements taken just before provoking the water intrusion into the pipe and Tkref+ the temperature measured k time steps after provoking the intrusion of water. Using the cumulative temperature Tcum reduces the probability of erroneous detected temperature changes caused by measurement uncertainties or erroneous measurements.with Th, a threshold exceedance of Tcum (|Tcum| > |Th|) at location z indicates a leakage because the probability that the temperature difference arise from uncertainties gets insignificantly small. Tz)h( is defined asWhether a cumulative temperature change arises from a leakage or from random noise of the measurements is determined by a predefined threshold Tcum the standard deviation of Tcum (°C) provoked by measurement uncertainties, ΔTbackground (°C) the background temperature shift occurring through thermal conduction (between outside and inner pipe water), and t the number of measurements for the determination of Tcum. σTcum is calculated applying a Gaussian error propagation under the assumption of normal distribution of the errors (Rice Tbackground has been determined under static flow conditions of the system between stopping the flush and setting the negative pressure (tpressure). During this period, the only temperature changes are caused by thermal conductivity. The slope of a simple linear regression of the temperature over time in this period has been used as ΔTbackground (°C/n).with σors Rice . ΔTbackTref plays a crucial role when applying the presented methodology. Tref heavily influences the outcome of Tcum as it appears in every addend of the sum. Different methods for the determination of Tref were tested. To reduce the uncertainty of Tref, a set of n measurements are used for the determination of Tref instead of one single measurement, reducing the uncertainty  and the starting point in (m), when the first value of n is taken  with varying m and n and analyzing the consequences on Th − Tcum. An ensemble of (m × n)/2 calculations of Tref has been conducted at every location z along the cable. Here,These measurements are taken just before initializing the negative pressure, identical to the determination of Δsee Fig.  is examinmax the maximum number of temperature measurements available before the negative pressure is set, mmax the latest moment for taking a reference temperature measurement before the negative pressure is set at time tpressure.with Tz)cum can be detected . Background temperature shifts are marginal as the measurement time is maximal tmax = 240 s and no relevant temperature changes through thermal convection are observable (−1.45 − 0 °C/min) in this time lag.The analysis of ted Fig. . The ampz along the cable, a set of temperature values in time was used varying m and n. In Fig. m and n variation on Tcum − Th is shown. The integration time of this measurement was 200 s with a temporal resolution of 10 s and a spatial resolution of 25 cm. Trend elimination was done by linear regression just before the start of the pressure reduction. The trend elimination diminishes the sensitivity of choosing the appropriate m and n heavily, in contrast to the calculation of the arithmetic mean of the n values (figure not shown). Latter shows higher inconsistency in leakage identification for a location z.For the reference temperature at every location Tcum as well as on the threshold Th. The former diminishes at lower resolution. This is obvious, as the intruding water at the leakages is less in relation to the measurement volume at lower spatial resolution. The latter diminishes at lower spatial resolution as well because of reduced measurement uncertainty. In Fig. Tcum and Th is shown. Regarding the detection of the leakage, both, the high as well as the low spatial resolution is capable to detect leakages. The effect of lower Tcum is compensated by the lower measurement uncertainty and consequently by the lower threshold Th. Regarding the positioning of the leakage, high spatial resolution is preferable.The effect of the spatial resolution on leakage positioning has effects on both, Tcum and Th influences on the accurate leakage detection, as well as the spatial and temporal resolution on the ability for detecting leakages. A higher number of measurements increase the sample size and reduce the effects of outliers. The spatial resolution influences on the relation Tcum to Th, especially at the beginning of the measurement when water intrusion starts and the mixing volume is still low. At lower spatial resolutions, the measurement volume of a measurement is higher than at higher resolutions. Consequently, the temperature drop from water intrusion is smaller than at higher resolution. At the beginning of the measurement, the mixing volume is still small and the temperature fluctuation is within the threshold. These effects are demonstrated in Fig. Tcum increased non-linearly with time after setting the negative pressure. The threshold Th was higher at high spatial resolutions, influencing the result especially at the beginning of the measurement. Nevertheless, the leakage was detected correctly independently of the spatial resolution, if the integration time was sufficiently long. High spatial resolutions are preferable, as the influence on the measured temperature difference is more striking than the effects of higher uncertainty on Th. The integration time should be as long as possible. In the experiments, 60 to 180 s were considered optimal. The maximum is limited by the ability to hold the negative pressure in the system and by heat conduction from the outside into the pipe system. Lower spatial resolution requires longer integration times.The number of measurements used for the calculation of Tref along the cable. Thus, small scale variations due to the unknown relative cable positioning against the pipe wall are captured and eliminated in the calculations of Tcum. However, if the cooling effect of the wall effects superimposes the effect of the temperature rise of the flushed water, the local temperature gradient between outside water temperature and the measured inside temperature might vanish or get lower than the measurement uncertainty itself. In Fig. T is bigger than the threshold Th for a single measurement, leakage detection is possible but might need an increased number of measurements for Tcum. Since this temperature difference can change at every location z along the cable, a revision of the temperature along the pipe has to be realized in advance.The influence of the cable position was tested by either fixing sections of the cable to the inside wall with duct tape or separating it by at least 3 cm with spacers made of foamed polystyrene. A reduced measured temperature, when the cable gets in contact with the wall, was not necessarily an impediment for leakage detection. A crucial step was the accurate determination of the reference temperature In a laboratory experiment, the potential of an adapted methodology of the distributed temperature sensing technology for detecting leakages in pressure pipes was tested. In comparison to traditional methods, the proposed methodology is advantageous because even small leakages can be located accurately. Furthermore, the installation of the cables in existing pipes is temporary without installation of any additional technical equipment, except the DTS itself. The temperature gradient between the pipe system and the surrounding lake water can be generated by flushing superficial lake water from the epilimnion in the summer months without any cost-intensive heating of the water.Testing several experimental designs, best results in the laboratory experiment were obtained with high spatial resolution to not overlook small leakages and short measurement intervals of 10 s. Concerning the data post-processing, the accurate determination of a reference temperature from measured DTS signals was the most crucial part. It also showed to be essential to maintain a stable temperature gradient within the pipe system. This was best achieved in the lab experiment by flushing the pipe as long as possible, before the start of the measurements. To reduce effects of uncertainty, the integration time should be chosen as long as possible in dependence of the maximum time negative pressure is uncritical for the statics of the pipe.Further challenges may arise in the practical implementation. While a suitable measurement design in terms of spatial resolution, temporal resolution and integration time of the measurement are already conditioned by the DTS technology/system itself, it has to be designed under strong consideration of the pipe length and the effective temperature gradient along the pipe system. Also, the insertion method of the DTS cable into the pipe should be tested in consideration of the emerging tensile stress through wall frictions. The effects of pressure drops along the pipe have to be evaluated. If multiple leakages lead to a significant pressure drop, an analysis and repair in sections, starting at the closest section to the pump, could be tested.While in the laboratory, a stable temperature gradient could be generated; in nature, much more heterogeneous temperature conditions in the pipe are expected. As a next step, we suggest to test the developed methodology under natural real system conditions, in order to explore the practical feasibility of our method for large-scale application. Overcoming the difficulties and exploiting the natural temperature gradient and the existing infrastructure, the methodology might provide a cost-attractive alternative to traditional methods in sewage pipe inspection.


HIV-positive patients carry an increased risk of HPV infection and associated cancers. Therefore, prevalence and patterns of HPV infection at different anatomical sites, as well as theoretical protection of nonavalent vaccine should be investigated. Aim was to describe prevalence and predictors of oral HPV detection in HIV-positive men, with attention to nonavalent vaccine-targeted HPV types. Further, co-occurrence of HPV DNA at oral cavity and at anal site was assessed.This cross-sectional, clinic-based study included 305 HIV-positive males , consecutively observed within an anal cancer screening program, after written informed consent. Indication for anal screening was given by the HIV physician during routine clinic visit. Paired oral rinse and anal samples were processed for the all HPV genotypes with QIASYMPHONY and a PCR with MY09/MY11 primers for the L1 region.p < 0.001), where HPV DNA was found in 199 cases (85.2%). Oral HPV tended to be more frequent in patients with detectable anal HPV than in those without (p = 0.08). Out of 265 HPV DNA-positive men regardless anatomic site, 59 cases (19.3%) had detectable HPV at both sites, and 51 of these showed completely different HPV types. At least one nonavalent vaccine-targeted HPV type was found in 17/64 (26.6%) of patients with oral and 199/260 (76.5%) with anal infection. At multivariable analysis, factors associated with positive oral HPV were: CD4 cells <200/μL  and >5 sexual partners in the previous 12 months .At the oral cavity, HPV DNA was detected in 64 patients (20.9%), and in 28.1% of these cases multiple HPV infections were found. Prevalence of oral HPV was significantly lower than that observed at the anal site , oral HPV DNA was detected in approximately one fifth of tested subjects, but prevalence was significantly lower than that observed at anal site. Low CD4 cell count and increasing number of recent sexual partners significantly increased the odds of positive oral HPV. The absence of co-occurrence at the two anatomical sites may suggest different routes or timing of infection. In the current era of combination antiretroviral therapy (cART), tumours have become the leading cause of mortality in HIV-positive persons , 2. AmonIn a meta-analysis of 26 publications focusing on men having sex with men (MSM), pooled prevalence of any HPV at the oral cavity was 17.1% (95% CI 7.3–26.8%) in HIV-negative and 28.9% (95% CI 19.1–38.7%) in HIV-positive MSM .Concerning multi-site HPV infections, particularly MSM were at high risk of HPV co-occurrence at multiple anatomic sites, as shown in a study that found 59.1% MSM with multi-site HPV occurrence (34.1% and 22.7% at two and three sites respectively) . Yet, deVaccines to prevent HPV infection have been developed and are approved in some countries for immunization programs in adolescent females, as well as in boys and young men , 16. CurIn Italy at present not much is known about prevalence and predictors of oral HPV DNA detection in HIV-infected patients, and literature about co-occurrence of HPV infection in oral cavity and at anal site is scanty. For this reason, only few HIV/AIDS centres investigate presence of HPV in oral rinses and offer oral cancer screening to their patients. The aim of our study was to describe prevalence and predictors of oral HPV DNA detection in HIV-positive men attending an anal cancer screening program, with attention to vaccine-targeted HPV types. Further, we investigated co-occurrence of HPV DNA at the oral cavity and at the anal site.This cross-sectional, clinic-based study was designed and conducted at the National Institute for Infectious for Disease, Rome, Italy, according to the Helsinki declaration and approved by the Institutional Review Board (n.42–18/6/2013).All HIV-positive men, aged 18 years or older, consecutively observed within an anal cancer screening program between February 2015 and June 2016 provided written informed consent. The indication for anal screening was given based on current European AIDS Clinical Society (EACS) guidelines [A questionnaire on health behaviours was created ad hoc and interviewer-administered. Questions focused on the following information: demographics, tobacco and alcohol use (more than 3 units per day), previous history of anal/genital condylomatosis, number of sexual partners in lifetime and in the previous 12 months, oral and anal sexual intercourse in lifetime and in the previous 12 months.Clinical charts were used to abstract presence and type of current cART, as well as viroimmunological parameters (CD4 cell count and HIV RNA needed to be determined within 3 months of samples collection).At the same time of anal cancer screening, participants provided a sample of oral rinse obtained by swishing and gargling in the oral cavity 10–15 ml of bottle mineral water (Fonte Tullia) for 20–30 s and spitting into a sterile specimen cup. One aliquot (5–7 ml) was sent to the Laboratory of Pathology for cytological evaluation, while the second was centrifuged for 15 min at 3000 g. Cell pellet was washed with sterile phosphate buffered saline and re-suspended in RPMI medium. Nucleic acids were extracted by automated QIASYMPHONY purification system . QuantiFast Pathogen Internal Control , was used to check presence of inhibiting factors. Furthermore, to ensure specimen adequacy, i.e. the presence of DNA, the samples were tested by amplification of a 110 bp fragment of the human β-globin gene using PCO3/PCO4 primers  followedhttps://blast.ncbi.nlm.nih.gov/Blast.cgi, nih.gov/BLAST) [For HPV DNA testing, a PCR for the L1 region was used. Products were analysed by electrophoresis in 1.7% agarose gel and stained with ethidium bromide, as previously described . HPV-posv/BLAST) . A similAnal swabs were collected in a standardized manner with a flocked swab according to the manufacturer’s instructions  . The swaSubjects with positive HPV DNA were classified in having a high-risk (HR) HPV, if at least one HR HPV-type was found, or otherwise classified having a low-risk (LR) HPV, if harbouring only low-risk HPV-types. Carcinogenic risk classification was done according to IARC monograph (2007) . DetectiAll assays were performed by trained laboratory technicians blinded to clinical information of the patient and to the assay results.Fixed cytological smears were stained according to the Papanicolau method  and adeqTwo pathologists, blinded to clinical and HPV information, independently evaluated cytological specimens. A cytological assessment of the quality of the obtained cells from each oral sample was undertaken, using standard parameters that include quality of preparation, cellularity, and types of cells present, in addition, data on micro-organisms, leucocytes/inflammatory cells and artefacts were also obtained. Cytological findings were reported using the following categories of the Bethesda 2001 guideline  modifiedp-values were calculated and a value <0.05 was considered statistically significant. A univariate logistic regression was used to analyse the strength of association between positive oral HPV detection and other variables, calculating odds ratios (OR) and their 95% confidence intervals (CI). A multivariable regression analysis, adjusting for variables associated with the outcome at univariate analysis with a p < 0.10 and forcing age, was carried out. Data management and analysis were performed using SPSS version 23 [Descriptive analysis was conducted to characterize the subjects included in the study. Median values and interquartile ranges (IQR) were used to describe numerical variables, while counts and percentages were employed for qualitative variables. The number of patients with at least one nonavalent vaccine-targeted HPV type over the total number of subjects found to be HPV DNA-positive at each anatomical site were employed to calculate the proportion of vaccine-targeted infections separately for each anatomical site. The association between variables was assessed using chi-squared test or Fisher’s exact test, as appropriate. Two-tailed rsion 23 .The study population included 305 HIV-infected males and general characteristics are shown in Table Health behaviours assessed in the interview showed that 64.9% of patients stated cigarette smoke-exposure (17.4% previously and 47.5% currently), 26.5% reported previous or current recreational drug use, and 3.6% declared current alcohol intake. Sexual history in the patient-reported questionnaire showed at that 152 men (49.8%) counted more than 100 sexual partners in lifetime. In the previous 12 months, 21 men (6.9%) declared no sexual activity, while 97 (31.8%) recalled 10 or more partners. The 284 patients who were sexually-active in the preceding 12 months, recalled the following type of sexual partners: only heterosexual in 31 (10.9%), both sexes in 44 (15.5%), and only homosexual in 209 (73.6%) cases. Median number of sexual partners in the previous 12 months were: 1 for heterosexuals (IQR: 1–2), 7 for bisexuals (IQR: 3.0–18.8), and 4 for homosexuals (IQR: 1–10). Overall, 275 (90.2%) patients reported receptive oral sex. None of the participating patients had been vaccinated for HPV.n = 2; HPV22 n = 3; HPV32 n = 8, HPV97 n = 2; HPV107 n = 1; HPV145 n = 1.HPV DNA was found in oral rinses of 64 out of 305 HIV-positive men (20.9%). HPV genotype was detected by MY09/11 primers in all but 12 cases, in which direct sequencing identified: HPV13 n = 5, HPV62 n = 4, HPV72 n = 6, HPV81 and HPV83 in 2 cases each, HPV84 n = 4), alpha-7 in 14 , and alpha-9 in 12 cases . HPV16 and HPV18 were found in three and one oral samples, respectively. Beta-2 species were detected in 5 samples . HR types were found in 50% oral rinses with detectable HPV DNA (n = 32), and were more frequently observed in presence of multiple than of single HPV types .The upper panel of Fig. n = 49 (17.6%) and H-SIL n = 1. In Table p = 0.050).Oral rinses samples were not adequate for cytological examination in 27 patients because of low cellularity. Among 278 cases, cytology was normal in 228 (82.0%) cases, while cellular atypia (ASC-US or more) was found in 50 patients: ASC-US p < 0.001). The finding of multiple HPV infection was more frequent in anal (206/260) than in oral (18/64) samples . Out of 265 HPV DNA-positive patients regardless the anatomic site, only in 59 cases (22.3%) HPV DNA was recovered in oral rinses and in anal samples at the same time . Detection of HPV at the oral cavity tended to be more frequent in men with HPV DNA detected at anal site when compared to those with negative results . Concerning specific HPV types, among the 59 patients with detectable HPV DNA at both anatomical sites, 51 (86.4%) had completely different HPV types. Out of the remaining eight, seven cases shared only one HPV type , while one patient harboured the same double infection by HR HPV39 and HPV59 types at both sites. In Fig. HPV DNA was detected at the anal site in 260 out of 305 (85.2%) male patients. Thus, the proportion of subjects with positive HPV DNA at the oral cavity was significantly lower when compared to that of persons with HPV DNA at the anal site  or more than 100 partners in lifetime . Further, a significant association between oral HPV DNA and self-reported history of anal/genital condylomatosis , use of recreational drugs , and current CD4 cell count below 200/μL when compared to higher values  was found.Table p < 0.01) and choosing to include “number of sexual partner in the previous 12 months” instead of “number of lifetime partners” to avoid collinearity, a significant independent association between detectable oral HPV DNA and the following conditions was confirmed: CD4 cell count ≤200/μL versus > 200/μL  and to report >5 sexual partners in the previous 12 months versus 0–1 partner .At multivariable analysis, after adjusting for age as well as all variables found to be associated with positive oral HPV DNA at univariate analysis  with positive oral HPV DNA, and in 199 out of 260 of patients (76.5%) with HPV positivity at the anal site. The nonavalent vaccine-targeted vaccine would have covered all identified HPV types in 11/17 (64.7%) of patients with HPV at the oral cavity and in 33/199 (16.6%) of patients with HPV at the anal site  observed within an anal cancer screening, HPV was detected in 20.9% of subjects at the oral cavity with a significantly lower prevalence than that observed at the anal site. Severe immunodepression and number of sexual partners in the previous 12 months, but not anal HPV detection, were significantly associated with a higher odd of positive oral HPV DNA. Co-occurrence of HPV DNA at the oral cavity and at the anal site was not found, and with few exceptions in patients with infection at both sites HPV types were completely different.In our study, positive HPV DNA in oral rinses was found in approximately one out of five tested HIV-infected patients, confirming that HPV detection at the oral cavity is quite common in HIV-positive men, especially if MSM. This is in accordance with previous studies  and withWith regards to factors predictive of positive oral HPV DNA, our finding of CD4 cell count below 200/μL and higher number of sexual partners in the previous 12 months, agrees with some previous studies , 12. In In our study, detection of HPV DNA at the oral cavity and at the anal site was not associated to each other. Furthermore, in patients with positive HPV DNA at both anatomical sites, HPV strains were mostly different. The absence of concordance has also been observed in another few studies conducted in HIV-positive men , 30, 31.In our study, prevalence of most frequent oral HPV types was HPV66 and HPV33 among HR, and HPV32, HPV72, HPV11 and HPV61 among LR. HPV16 was found in only 1.0% of cases. This finding differed from previous studies, that showed HPV16 as the predominant type .In our study population, if the patient had been vaccinated before exposure, the nonavalent vaccine would have targeted at least one HPV strain in only about 27% patient with detectable HPV DNA at the oral cavity and in 77% at the anal site. Notably though, nonavalent vaccine would have covered all identified HPV types in more than 60% of patients with oral HPV DNA, but only less than 20% of patients with HPV DNA at the anal site. Based on these findings, it may be reasonable to investigate the efficacy of nonavalent vaccines in preventing oral HPV infections.Our study has some limitations that should be mentioned. Since the tested men have been send at anal cancer screening program by the caring HIV physician, selection bias may have occurred by choosing patients at higher risk of sexually transmitted diseases in general and HPV specifically. Moreover, since a non-standardized interviewer-administered questionnaire was used to collect information regarding sexual history and practices, interview bias may have occurred with distortion of responses by the interviewer or elusion and lies in the answers of the patients. However, our interview was mostly build on fixed-wording questions and multiple-choice answers, reducing the risk of bias.In this study on Italian HIV-positive men (predominantly MSM), oral HPV DNA was detected in approximately one fifth of tested men, but prevalence was significantly lower than that observed at the anal site. Low CD4 cell count and increasing number of recent sexual partners significantly increased the odds of positive oral HPV detection. The absence of co-occurrence of HPV DNA at the oral cavity and at the anal site may suggest different routes or timing of infection.