Mycoplasmataceae is associated with higher levels of human papillomavirus (HPV), human immunodeficiency virus (HIV), and pre-cancerous cervical lesions. Mycoplasmataceae infection in Tanzania is not well understood, especially when considering the differences between sexually transmitted species of Mycoplasmataceae. To establish the prevalence of common Mycoplasmataceae cervical infections and evaluate their relationship with risk factors for cervical cancer, 1160 Tanzanian women responded to an epidemiological questionnaire and were tested for HIV, HPV, cervical lesions, Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma spp., and Lactobacillus iners. A subset of 134 women were used for 16s metagenomic sequencing of cervical DNA to establish the relative abundance of Mycoplasmataceae and Lactobacillus present. PCR detection of bacteria at the cervix found Ureaplasma spp. in 51.4% of women, M. hominis in 34%, M. genitalium in 2.3%, and L. iners in 75.6%. M. hominis and M. genitalium infection were significantly more prevalent among women with HPV and HIV. M. hominis prevalence was similar despite severity of cervical lesions; however, abundance of M. hominis increased significantly in women with cervical lesions. These results emphasize the importance of understanding the relationship between M. hominis and HPV-related cervical pathogenesis.Tanzania faces one of the highest cervical cancer burdens in the world. Recent work has suggested that the bacterial family  Mycoplasmataceae was the most significant differential cervical bacteria between women with normal cervical cytology and those with pre-cancerous lesions in Tanzania [Mycoplasmataceae are the smallest known bacteria, in both physical and genomic size. During infection of the cervicovaginal epithelium, Mycoplasmataceae establish a persistent, intracellular infection which can lead to inflammatory-cytokine-mediated tissue injury. Although it is currently unknown if there is a mechanistic relationship between HPV and Mycoplasmataceae, the nature of Mycoplasma infection allows for direct interaction with HPV during co-infection of a single cell, and indirect interaction through cytokine responses.Cervical cancer mortality is higher in Eastern Africa than in any other region of the world . In TanzTanzania . MycoplaMycoplasmataceae is comprised of the genera Mycoplasma and Ureaplasma, which include several sexually transmitted species with global prevalence. Most notably, M. hominis, M. genitalium, and U. urealyticum are relatively common sexually transmitted infections (STIs) associated with cervical inflammation [Mycoplasma, only M. genitalium is sometimes included in regular STI screening, although M. hominis is believed to have a similar pathogenesis. As a result, M. hominis has received significantly less study, and its relationship with HPV, HIV, and cervical lesions remains unclear. The prevalence of M. genitalium, M. hominis, and U. urealyticum in Tanzania has not previously been established in a large and diverse cohort, nor has it been considered alongside established risk factors for cervical dysplasia.ammation ,5,6. AmoMycoplasma and other STIs in Eastern Africa is, in part, due to the commensal cervicovaginal bacteria in the region. Specifically, L. iners is the most prevalent cervicovaginal Lactobacillus in Eastern Africa, especially in HIV+ women, but has been shown to be less protective against cervicovaginal infection than other Lactobacillus [L. iners is conducive to infection and proliferation of Mycoplasmataceae, and the relationship between the bacteria and HPV pathogenesis, remains unclear. This study aims to establish the prevalence of common Mycoplasmataceae species in Tanzania and evaluate their relationship with L. iners and risk factors for cervical cancer, including HPV, HIV, and lifestyle factors.It has been suggested that high levels of cervicovaginal dysbiosis and transmission of bacillus . WhetherDNA was successfully isolated from the cervical cytobrush samples of 1060 women. Complete data of cervical cytobrush DNA, pap-smear, VIA, HIV status, and epidemiological questionnaire response was available for 1002 women. Women with incomplete data were included in analyses where the missing data were not relevant. The cohort averaged 38.3 years of age, ranging from 18 to 73. A large majority (92.3%) of the women screened reported at least one previous pregnancy, and 84.1% were sexually active within the 3 months preceding sampling. A total of 67.4% of the cohort reported the use of at least one type of birth control, although it is unclear if they had recently used birth control at time of sampling. A total of 17.6% of the cohort had tested positive for HIV and these participants were on antiretroviral therapy at the time of sampling. Using a multiplex HPV genotyping PCR, we found that 46.1% of the cohort tested positive for at least one HPV genotype, and 38.2% of HPV positive women were coinfected with at least two genotypes.There was a significant difference between the identification of cervical lesions between VIA and pap smear. Only 17% of women with pap smears graded HSIL had lesions identified by VIA. Additionally, although 88.9% of the cohort tested negative for cervical lesions by VIA, only 20.1% of the cohort was graded NILM by pap smear. The majority of women had pap smears exhibiting low-grade cervical dysplasia: ASCUS (24.8%) and LSIL (30.8%). More severe cervical dysplasia was apparent in 24.4% of women .Mycoplasmataceae prevalence within each group. PCR detection of Mycoplasmataceae at the cervix found a high prevalence of the bacterial family among Tanzanian women—66% of whom tested positive for at least one Mycoplasmataceae. Ureaplasma spp. was the most prevalent Mycoplasmataceae, detectable in 51.4% of the cohort, followed by Mycoplasma hominis in 34%, and Mycoplasma genitalium in only 2.3% of women. Lactobacillus iners was more prevalent than Mycoplasmataceae—detectable in 75.6% of women. Detection of any Mycoplasmataceae significantly increased the likelihood of detection of other Mycoplasmataceae species in that individual  4.8 and 4.2, respectively), while Ureaplasma spp. and L. iners were only slightly more common (OR 1.6 for both). This data supports previous research suggesting the HIV+ population acts as a reservoir for M. hominis infection [Ureaplasma spp., M. hominis, and M. genitalium, respectively). Being HIV+ increased the odds of detection of all M. hominis , while M. genitalium and Ureaplasma did not have a significant increase in prevalence associated with HPV . Co-infection with two or more different HPV genotypes was associated with higher prevalence of M. hominis and M. genitalium than women infected by one HPV genotype. Multiple HPV infections were much more common amongst HIV+ women; however, this increase in Mycoplasma prevalence was also apparent in HIV− women with multiple HPV when compared to HIV−, single HPV women.Women infected with at least one HPV genotype were significantly more likely to have cervical dysplasia, especially high-grade lesions . HPV+ women were also more likely to be infected with with HPV . CommensMycoplasmataceae were not significantly more or less prevalent among women with cervical dysplasia  in L. iners prevalence, decreasing from 83.7% in women 18–29 to 59.4% in women 50+. Age did not appear to be related with a shift in Mycoplasmataceae prevalence, although women aged 50+ did have somewhat lower prevalence of M. hominis and M. genitalium, possibly related to menopause or decreased sexual activity. L. iners prevalence also decreased in women with three or more previous pregnancies; however, this may have been influenced by a higher average age among high-gravidity women. Sexual history was an important factor for detection of sampling C,E. SelfMycoplasmataceae and L. iners present. Each sample was rarefied to an even depth of 1000 reads. After rarefication, women with more than 5 reads from Ureaplasma spp., M. hominis, M. genitalium, or L. iners were considered positive for that bacteria. The prevalence of each bacteria was similar to results from PCR screening, although no M. genitalium reads were present among the subset of samples tested. By using the number of reads generated, we were able to determine the relative abundance of each bacteria in each woman’s cervical microbiome. Using this, we estimated the relative abundance of the screened bacteria within cervical cytology groups by adjusting the prevalence of a bacteria by the average relative abundance of that bacteria in positive samples of each cytology grade. When looking at the relative abundance of Mycoplasmataceae in women with cervical dysplasia, it becomes apparent that a significantly larger portion of the cervical microbiota is M. hominis  demonstrates the importance of better understanding Mycoplasma species and Lactobacillus iners. A subset of 132 women were also used for 16s metagenomic sequencing.This study reports findings derived from an ongoing cross-sectional cohort study analyzing demographics of HPV and cervical cancer in HIV-positive and -negative women from rural and urban Tanzania. Between March 2015 and February 2017, female patients undergoing cervical cancer screening were approached for enrollment in the study. Those who were pregnant, menstruating, under 18, reported being sick in the past 30 days, or had a preexisting, non-HIV, immunologic defect were excluded from the study. Disease histories and physical examinations were used to rule out any clinical symptoms or visible signs for these conditions. Samples were collected at three sites in Tanzania: Ocean Road Cancer Institute (ORCI) in Dar es Salaam and rural clinics in Chalinze and Bagamoyo. After collection of cervical samples and demographic data, samples from 1060 women were screened for This study was approved for human subjects work by the University of Nebraska-Lincoln Institutional Review Board (IRB) under protocol ID: 14709. All study participants gave informed consent and were evaluated by study clinicians. A set of pretested, standardized questionnaires was used to gather demographic data. All personal identifiers were removed from samples to ensure patient confidentiality. With the permission of the patients, medical history was retrospectively retrieved from hospital medical records. More than 30 variables were identified and assessed in the questionnaire, including time since last sexual intercourse, number of sexual partners, number of pregnancies, use and type of birth control, and self-reported history of STI infections.Blood samples were collected via venipuncture into acid-citrate-dextrose tubes  and processed using centrifugation at the on-site study laboratory within 6 h of being drawn. The separated plasma was tested at the ORCI, as part of standard of care, using Standard Diagnostics HIV-1/2 3.0 detection kit . Cervical cytobrush samples and pap smears were collected from the cervical transformation zone of all patients. Pap smears were examined by at least three trained cytologists and classified according to the pap classification protocol: negative for intraepithelial lesion or malignancy (NILM); atypical squamous cells of undetermined significance (ASC-US); low-grade squamous intraepithelial lesions (LSIL); atypical squamous cells but cannot exclude high-grade lesions (ASC-H); high-grade squamous intraepithelial lesions (HSIL). Cervical cytobrush specimens were placed in lysis buffer  and then shipped to the Nebraska Center for Virology at the University of Nebraska-Lincoln (UNL) for processing.Cervical cytobrush samples were vortexed and separated from the brush with lysis buffer. DNA was extracted from the lysis buffer using the DNeasy Tissue extraction kit  according to the manufacturer’s protocol. The DNA concentration was determined by UV spectrophotometer  at 260/280 nm.To determine HPV status, DNA samples were genotyped for HR-HPVs  and LR-HPVs (types 6 and 11) using a low-cost multiplex PCR assay .M. genitalium, M. hominis, and Ureaplasma spp. was adapted from Stellrecht et al. [L. iners established in [A multiplex PCR targeting t et al. , with thished in . PrimersThe PCRs were performed in a final volume of 25 μL. The cycling conditions were as follows: an initial denaturation of 95 °C for 15 min, followed by 35 cycles, with 1 cycle consisting of denaturation at 94 °C for 15  s, and annealing and extension at 60 °C for 1 min, then a final elongation of 72 °C for 5 min. After amplification, DNA samples were run in 0.5% agarose gels containing Ethidium Bromide at 95 volts for 1 h. Gels were then imaged using a Bio Rad ChemiDoc MP Imaging System  to visualize bands.Ureaplasma spp., M. hominis, M. genitalium, L. iners, HPV, HIV, Age, time since last sexual activity, number of sex partners, number of pregnancies, self-reporting of STI infection, use of birth control, and type of birth control used) was used to identify significant differences between women with different cervical cytology, HIV, or HPV status. The birth control types considered were pills, injections, condoms, implants, loop, and natural. Odds ratios were calculated to identify groups with significantly increased odds of HPV, HIV, or Mycoplasmataceae. A p value of 0.05 was the maximum considered to be significant throughout the study.Multivariate analysis of variance (MANOVA) using one variable selected as fixed versus the other remaining dependent variables collected  from 144 samples were pooled together using an epMotion M5073 liquid handler . Pooled libraries were sequenced using the Illumina MiSeq platform using the dual-index sequencing strategy outlined by Kozich et al. .https://github.com/FernandoLab). Briefly, initial quality filtering was carried out to remove sequences that had ambiguous bases, incorrect lengths, and inaccurate assemblies. Subsequently, the quality-filtered reads were run through the UPARSE pipeline (http://www.drive5.com/uparse/) and subjected to chimera filtering and OTU clustering (at a similarity threshold of 97%), followed by the generation of an OTU table. Taxonomy was assigned to the OTUs using the assign_taxonomy.py command available in QIIME using the Greengenes database (May 2013). The OTU table was rarefied across samples to the lowest sample depth (1000 reads) using QIIME based on the Mersenne Twister pseudorandom number generator. All statistical analyses were performed with samples at an even depth. The sequencing data obtained from the sequencer were subsequently analyzed using the Illumina MiSeq data analysis pipeline developed by the Fernando lab (described in detail at All human subject protocols were approved by safety committees at the Ocean Road Cancer Institute (ORCI) and UNL in accordance with the Helsinki Declaration. Participation by patients was entirely voluntary, and written patient consent was required for inclusion in the study.Mycoplasmataceae was detected at the cervix at 66% of women tested. Among the Mycoplasmataceae family, Ureaplasma spp. was the most common, at about 51.4% of the cohort. Mycoplasma hominis was found in 34% of the cohort, while Mycoplasma genitalium was found in only 2.3% of the cohort. We found that HIV+ women had increased odds for all Mycoplasmataceae and L. iners. Mycoplasma hominis and genitalium infections were especially prevalent among HIV+ women compared HIV−. Those women who were HPV+ were more likely to have M. hominis . Co-infection with multiple HPV genotypes was associated with higher a prevalence for M. hominis and M. genitalium. Multiple HPV genotype infections were more common amongst HIV+ women (In this study, we analyzed the cervical microbiota of 1160 Tanzanian women. We found that the bacterial family of V+ women .Mycoplasmataceae was not associated with cervical dysplasia. However, we found that the abundance of Mycoplasmataceae bacteria, such as M. hominis, increased with more severe cervical lesions, LSIL and HSIL. Ureaplasma spp. abundance was also associated with HSIL women (M. hominis and M. Ureaplasma are associated with cervical dysplasia. We suggest that M. hominis infection of the cervix could lead to chronic inflammation, which favors HPV-related neoplasias.The prevalence of IL women . These d


The diagnostic efficacy of IHC-p16 varied among countries. In conclusion, IHC-p16 has high sensitivity and specificity for diagnosing HPV infection in HNSCC. The consistency of IHC-p16 findings with those of ISH indicate that their combination can be used to improve the specificity of diagnosis.Human papillomavirus (HPV) is a risk factor for squamous cell carcinoma of the head and neck (HNSCC). This study aimed to investigate the feasibility of IHC- p16INK4a (p16) as an alternative modality for diagnosing HPV infection. We searched PubMed, EMBASE, Web of Science, and Cochrane library for studies that evaluated the diagnostic accuracy of IHC-p16 staining. A total of 30 studies involving 2,963 patients were included from 2007 to 2019. The combined sensitivity was 0.94 (95% CI: 0.92–0.95); specificity, 0.90 (95% CI: 0.89–0.91); positive likelihood ratio (LR), 6.80 (95% CI: 5.63–8.21); negative LR, 0.10 (95% CI: 0.07–0.16); diagnostic odds ratio, 85.98 (95% CI: 55.57–133.03); and area under the curve value, 0.9550. Subgroup analysis showed that the IHC-p16 test was more consistent with the  Head and neck squamous cell carcinoma (HNSCC) is the sixth most common malignancy worldwide, with ~830,000 incident cases annually . Smokingin situ hybridization (ISH). PCR-based detection is more sensitive, while ISH-based detection is more specific  p16INK4a(p16) staining as an alternative modality for diagnosing HPV infection , 28. In inplasy.com (https://doi.org/10.37766/inplasy2020.7.0068).The protocol for this systematic review was registered on INPLASY (202070068) and is available in full on the This study was conducted according to the PRISMA guidelines and the Cochrane diagnostic test manual , 34. TheWe reviewed the full text of all observational studies, both retrospective and prospective, and randomized controlled clinical trials that compared the diagnostic efficacy of IHC-p16 positivity with the gold standard modality for HPV diagnosis. The inclusion criteria were: (i) the included patients had HNSCC; (ii) the samples tested were biopsy or puncture specimens; (iii) HPV E6/E7mRNA detection was used as the gold standard for the diagnosis of HPV infection; (iv) p16 expression was detected using IHC; (v) the total sample size was >10. All case reports, preclinical studies, case series, animal studies, and conference summaries were excluded. In addition, papers were also excluded if the specific location of HNSCC was not clearly defined. Further, the included studies must present the specific true positive (TP), false positive (FP), false negative (FN), and true negatives (TN) values or have adequate data so these can be calculated. If data were lacking, we contacted the author by email to ask for the data, and the study was excluded if the author did not respond. Study selection was divided into two parts. First, the authors (JW and BW) screened all the articles independently by browsing the titles and abstracts. Second, the same two authors independently evaluated the full text of the initially included articles. Any disagreements were resolved by the third author (XJ), and the study was finalized for inclusion.Two authors (JW and BW) independently assessed the methodological quality of the included studies using the QUADAS-2 tool . BrieflyData extraction was carried out in two parts. First, a researcher (HHW) used a pre-designed data extraction table to extract basic elements from the study, such as author, publication year, and patient source. Then, two other authors (YYZ and WB) independently extracted the specific values of TP, FP, FN, and TN from the text and cross-checked them according to the pre-set standards to ensure the accuracy of the original values extracted. Any differences in the data extraction were resolved through discussion and negotiation. The extracted data were verified by the third author (WB).I2 and Cochrane Q-tests. An I2 of >50% indicated heterogeneity, and the source of heterogeneity was further explored. After obtaining the sensitivity and specificity values, we further used the receiver operating characteristic curve (ROC) model to obtain the positive likelihood ratio (LR), negative LR, and the diagnostic odds ratio (OR) and their 95% confidence intervals (CI). Positive LR was defined as the ratio of sensitivity to 1-specificity. Negative LR was defined as the ratio of 1-sensitivity to specificity. The larger the positive LR and the smaller the negative LR, the better the diagnostic experiment. The diagnostic OR was defined as the ratio of positive LR to negative LR. The greater the diagnostic OR, the better the capability of p16 to distinguish between HPV infection and non-HPV infection. The ROC curve was also drawn to obtain the area under the curve (AUC) value to comprehensively evaluate the efficacy of p16 positivity in diagnosing HPV infection. In addition, a funnel plot was used to further evaluate the presence of publication bias.Because the head and neck are divided into many regions, and there are two methods for HPVE6/E7mRNA detection, we expected that the data included in the meta-analysis might be uneven. Therefore, we divided the study into several different subgroups based on factors such as tumor location and the detection methods for HPVE6/E7mRNA set as the gold standard. Given that the accuracy of p16 positivity in diagnosing HPV infection is related to the positive threshold, differences in thresholds between studies may have an impact on the sensitivity and specificity. Thus, we further evaluated whether there was a threshold effect using Spearman correlation coefficient. If there was no threshold effect, the sensitivity, specificity, and other indicators were further combined. Sensitivity was defined as the percentage of TP for diagnosing HPV infection in the total number of p16-positive cases (TP+FN). Specificity was defined as the percentage of TN for diagnosis of no-HPV infection in the total number of p16-negative cases (FP+TN). All data were combined using Meta Disc and STATA 15.0 software. We developed a forest map that graphically displays estimates of sensitivity and specificity and visualized heterogeneity between studies. Moreover, heterogeneity was examined using n = 7) and detailed data were not available (n = 22). Finally, 30 studies  and specificity was 0.90 (95% CI: 0.89–0.91)  . We foun89–0.91) ; and diasistency , and thesistency , showingI2-value, 12.1%; and AUC value, 0.9627. For PCR HPVE6/E7mRNA, the combined sensitivity was 0.93 (95% CI: 0.91–0.95); specificity, 0.89 (95% CI: 0.87–0.91); positive LR, 6.17 (95% CI: 4.70–8.09); negative LR, 0.09 (95% CI: 0.05–0.17); diagnostic OR, 75.25 (95% CI: 38.28–147.91); I2-value, 48.9%; and AUC value, 0.9424. After all the studies were grouped according to the test methods, the possibility of heterogeneity in each group was reduced. When ISH was used as the gold standard, the combined sensitivity, specificity, and diagnostic OR were higher, and the AUC value was larger than those in the PCR. This indicates that when ISH is used as the gold standard, p16 has higher diagnostic efficacy for HPV infection. The combined results are shown in In addition, we investigated whether the diagnostic efficacy of p16 positivity is consistent when different testing methods are used as the gold standard. PCR and ISH were analyzed separately. For ISH HPVE6/E7mRNA, the combined sensitivity was 0.94 (95% CI: 0.92–0.96); specificity, 0.91 (95% CI: 0.89–0.93); positive LR, 7.53 (95% CI: 5.77–9.83); and negative LR, 0.12 (95% CI: 0.06–0.22). The diagnostic OR was 101.09 (95% CI: 59.93–170.54); I2-value, 31.9%; and AUC value, 0.9598. In non-OPSCC patients, the combined sensitivity was 0.79 (95% CI: 0.77–0.88); specificity, 0.93 (95% CI: 0.91–0.94); positive LR, 8.43 (95% CI: 5.46–13.01); negative LR, 0.33 (95% CI: 0.17–0.66); diagnostic OR, 40.38 (95% CI: 14.36–113.49); I2-value, 33.9%; and AUC value, 0.9455. Compared with non-OPSCC patients, the sensitivity, positive LR, diagnostic OR, and AUC value of HPV infection diagnosed according to p16 positivity is higher in OPSCC patients, indicating that p16 positivity has higher diagnostic efficacy for HPV infection in OPSCC ; specificity, 0.88 (95% CI: 0.85–0.90); positive LR, 6.33 (95% CI: 5.12–7.83); negative LR, 0.08 (95% CI: 0.05–0.11); diagnostic OR, 104.49 (95% CI: 65.14–167.61); in OPSCC .In subgroup analysis by country to investigate whether the diagnostic efficacy of p16 positivity for HPV infection is consistent across countries, we grouped studies according to their origin: European, north American, and non-Western. The results showed higher diagnostic efficacy of p16 positivity in European and American countries. In contrast, in non-western countries, the combined diagnostic OR was only 68.96, and heterogeneity was observed, indicating that p16 positivity had no significant diagnostic value. The above results are shown in p = 0.61), indicating that there was no publication bias (The funnel plot for publication bias showed no statistically significant difference (ion bias .This systematic review and meta-analysis investigated the diagnostic accuracy of p16 for HPV infection. We found that p16 expression has high sensitivity and moderate specificity as an alternative biomarker for the diagnosis of HPV infection. The findings of this meta-analysis are consistent with those of previous studies where p16 expression for diagnosing HPV infection had 90% sensitivity and >80% specificity , 28, 65.The studies in this meta-analysis used different modalities as the gold standard for diagnosing HPV infection. The results showed that the diagnostic efficacy of IHC-p16 for detecting HPVE6/E7mRNA differed between PCR and ISH, with IHC-p16 being more consistent with ISH for diagnosing HPV infection. PCR is widely used because of its high sensitivity and specificity , but PCRIn subgroup analysis according to tumor location, p16 positivity for HPV diagnosis had higher sensitivity and specificity in OPSCC. The diagnostic OR was also two times higher than that of non-OPSCC. This may be related to the different infection rates of HPV in different tumor sites . AccordiIn subgroup analysis according to country, we found that the diagnostic efficacy of p16 expression varied between countries. This difference may be related to the different infection rates of HPV, which is influenced by alcohol and tobacco smoking and sexual behavior. Collectively, these results suggested the optimal diagnostic biomarker for HPV infection may different by country or region.This meta-analysis was conducted according to stringent guidelines. Relevant studies were identified from four databases using a pre-defined search strategy, and data were extracted according to pre-set tables. Further, the risk of bias for each study was analyzed, and the data were analyzed statistically using two software. However, this study also has some limitations, including the lack of prospective data and multivariate analysis.IHC-p16 staining is a highly effective modality for diagnosing HPV infection, particularly for OPSCC patients. However, the diagnostic efficacy varies between countries, and misdiagnosis could not be eliminated. When selecting patients for treatment de-escalation, HPVE6/E7mRNA should be detected using ISH based on p16 positivity to ensure accurate treatment.All datasets generated for this study are included in the article/XJ and LD: conceptualization and validation. BW: software. WB: formal analysis. HW: investigation. YZ: resources. HW, BW, and YZ: writing-original draft preparation. RJ, YX, and XJ: writing-review and editing. XJ: funding acquisition. All authors read and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Introduction: Persistent human papillomavirus (HPV) semen infection is increasingly associated with male infertility. Adjuvant HPV vaccination is suggested to reduce the time to clearance and the disease relapse in males with persistent HPV semen infection. However, only a sub-population of patients show a clinical benefit from adjuvant vaccination. Here, we aimed to address the effectiveness rate of HPV adjuvant vaccination in males with genital tract infection and the possible prognostic markers of healing. Methods: Clinical records from 379 patients with persistent seminal HPV detection, all receiving HPV adjuvant vaccination, were considered. Clinical data, including genital HPV-DNA assessment by INNO-LiPA genotyping, semen HPV-DNA analysis by FISH analysis and serum antibody titer, were collected at basal (T0) and after 6 months (T1) since the vaccination cycle ended. Results: Clearance of genital HPV-DNA was recorded in 326 (86%) patients. Serum HPV-antibody titer at T1 was the most important prognostic factor associated with HPV-DNA clearance. A serum antibody titer equal to or greater than the threshold value 1:125, obtained by ROC curve analysis, was prognostic of healing. Conclusions: Anti-HPV antibody represents a suitable marker of adequate immune response to HPV vaccination in patients with genital infection. Human Papillomavirus (HPV) is the etiological agent of the most common sexually transmitted infection worldwide, with an estimated 6.2 million new cases annually . The repHPV vaccination is usually administered before the beginning of sexual activity in a three-dose schedule at 0, 2 and 6 months. However, recent evidence showed the efficiency of a single dose of vaccine administered to females younger than 18 years old ,10. MoreCurrently, there is no validated treatment for asymptomatic patients with HPV infection. However, it is widely acknowledged that the development of a specific humoral immune response, whether natural or obtained by vaccination, is the only condition able to counteract reinfection and disease recurrences . Recent Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma spp.), Human Immunodeficiency Virus (HIV), Hepatitis B Virus (HBV), Hepatitis C Virus (HCV) and Treponema pallidum active infections, CFTR gene mutations and any alteration of the major endocrine axes.The study was conducted at the Unit of Andrology and Reproductive Medicine of the University Hospital of Padova, according to the principles of the Declaration of Helsinki, and approved by the Institutional Ethics Committee of the Padua General Hospital by Protocol No. 2336P and subsequent amendments. Among males attending our unit for andrological issues , we retrospectively considered the clinical and demographic records from 700 patients with genital and/or semen HPV detection and genotyping. Inclusion criteria were: acceptance of written informed consent for clinical data access, persistent HPV infection (at least 6 months) from genotypes included in the quadrivalent vaccine, such us 6, 11, 16, 18, or those with evidence of cross-protection, such as 31, 33, 45, 52 and 58 . ExclusiThe analysis was performed on data collected at study initiation (T0) and after 6 months from the end of the vaccination cycle (T1), thus one year since the study initiation, in a final sample size of 379 patients fulfilling inclusion criteria. Vaccine responding was defined as the absence of HPV-DNA in seminal fluid and cells coming from balano-preputial sulcus swab, detected by INNO-LiPA Genotyping assay, at T1.Semen analysis and anti-sperm antibodies were assessed according to the World Health Organization (WHO) Criteria. HPV-DNA bound to sperm was detected by FISH analysis (FISH-HPV) on whole semen, as previously described .HPV-DNA detection and genotyping were performed on balano-preputial sulcus swab  and in semen samples by INNO-LiPA Genotyping Extra assay, as previously described . This anThe detection and titer of serum total anti-HPV immunoglobulin G (IgG) were assessed on serum samples from peripheral blood obtained from all patients, at T0 and T1, by enzyme linked immuno-sorbent assay (ELISA) using a commercial kit supplied by DRG Diagnostic GmbH , as previously described . An intep-values < 0.05 were considered as statistically significant. The possible relevance of prognostic factors was assessed by calculating the odds ratio (OR) and related 95% confidence intervals (95% CI).Statistical analysis was performed using SPSS version 23.0 . Data are presented as means ± standard error of the mean (SEM) for continuous variables and as percentages for categorical variables. Comparison of continuous variables between two groups, particularly demographic data, semen parameters and antibody titers between healed and not-healed patients, was performed by Student’s t-test for unpaired data, previously assessed for normal data distribution by the Kolmogorov–Smirnov test. Proportions and discrete variables, when the number of groups was <5, were compared by the Fisher’s chi-squared exact test. The receiver operating characteristic (ROC) curve was calculated to correlate the antibody titer recorded at T1 with the healing rate after vaccine administration. The area under the curve (AUC) thus determined was assessed through the classification of Swets. The threshold value of the anti-HPV antibody titer for healing was calculated with Youden’s S statistic. For the considered comparison, 0.9 < AUC < 1.0 was found. The multivariate analysis through logistic-stepwise regression was also applied to continuous and non-continuous variables in order to evaluate the association with vaccine responding as a dichotomous outcome.Clinical and demographic data of the study group are reported in p = 0.0001 for FISH-HPV and 23.1% vs. 14.2%, p = 0.0021 for ASA). In particular, this evidence was essentially associated with the significant reduction of these two parameters in responding patients (p < 0.001 FISH-HPV) and p = 0.0015 ASA, respectively. In fact, such a significance for FISH-HPV and ASA was not observed in non-responding patients (both p > 0.05).p = 0.0001). The significant increase of serum-antibody titer at T1 vs. T0 was also maintained when patients were distinguished into responding and non-responding. Importantly, in responding patients, the serum-antibody titer at T1 was higher than that observed in non-responding patients at the follow-up .Vaccination was associated with seroconversion in all patients, with an overall increase of the mean serum-antibody titer from 1:72 ± 36.47 at T0 to 1:308 ± 42 at T1 .Of note, the serum HPV-antibody titer at T1 was the factor most significantly associated with the responding outcome in a multivariate analysis through the logistic-stepwise regression approach  to disclose the natural immune response associated with a previous exposure to HPV, (ii) to assess the immune response associated with vaccination in patients with accidental genital infection and (iii) to critically consider a further vaccine dose, in order to increase humoral immunity, in those patients showing persistence of HPV infection, in spite of a vaccination-related seroconversion. This approach certainly requires appropriate clinical validation but has valid precedents since, for example, it represents a shared procedure adopted in subjects with a low response to HBV vaccination. .Despite the fact that more studies including proper control arms are required to draw final conclusions, here, we provided evidence that HPV vaccination is able to improve the clearance of HPV from the genital tract. Moreover, the assessment of serum anti-HPV antibody titer represents a suitable approach to monitor the effectiveness of the anti-HPV immune response.


Human papillomavirus (HPV) E6 and E7 oncoproteins are critical for development and maintenance of the malignant phenotype in HPV-induced cancers. These two viral oncoproteins interfere with a plethora of cellular pathways, including the regulation of cell cycle and the control of apoptosis, which are critical in maintaining normal cellular functions. E6 and E7 bind directly with certain components of the Ubiquitin Proteasome System (UPS), enabling them to manipulate a number of important cellular pathways. These activities are the means by which HPV establishes an environment supporting the normal viral life cycle, however in some instances they can also lead to the development of malignancy. In this review, we have discussed how E6 and E7 oncoproteins from alpha and beta HPV types interact with the components of the UPS, and how this interplay contributes to the development of cancer. Papillomaviridae is a diverse family of small, non-enveloped DNA viruses, approximately 50–60 nm in diameter that infect all homoeothermic vertebrates including humans [Papillomaviridae in fish [Alphapapillomavirus species (α-HPVs) preferentially infect oral or anogenital mucosa in humans and primates [g humans ,2. Inter in fish . Current in fish . The Alpprimates . HPV infprimates . On the primates ,2,8,9. Hprimates ,8,10. HPprimates ,13,14,15Betapapillomavirus genus comprises approximately 50 HPV types and can be detected in normal and precancerous tissue, as well as in cancerous cutaneous epithelial tissue [The l tissue ,16. Althl tissue ,18. In al tissue .It is believed that HPVs infect cells in the basal layer of stratified squamous epithelia, exposed as a result of small abrasions and micro-wounds ,21. In nThe β-HPV types infect the basal layer of the squamous epithelium and hair follicle region, but the mechanism remains unknown ,26 althoThe Ubiquitin Proteasome System (UPS) is the pathway responsible for the majority of intracellular protein degradation in eukaryotic cells. It controls the turnover of thousands of short-lived, regulatory, damaged and misfolded proteins, in order to regulate and maintain various cellular functions and cellular homeostasis . It is aGaussia princeps luciferase protein complementation assay. HPV E6 and E7 proteins appear to have evolved various strategies to make use of the ubiquitin system to support the viral lifecycle [Many human pathologies, such as inflammatory and neurodegenerative diseases and certain cancers are, directly or indirectly, promoted by the deregulation of the UPS ,44. An iifecycle ,46,47. Iifecycle ,49,50. Eifecycle  and low-ifecycle ,53 HPVs ifecycle . Interesifecycle ,55. Furtifecycle . E6 and E7 oncoproteins inactivate the majority of these cellular substrates by interacting with components of the UPS, ultimately inducing their degradation at the proteasome. Among the diverse components of the UPS, the interplay between E6/E7 and ubiquitin ligases is one of the crucial aspects in this process. The best characterized ubiquitin ligases used by the two oncoproteins are E6-associated protein (E6AP) and the cullin-2 ubiquitin ligase complex . E6AP coThe HPV E6 oncoprotein contains around 150 amino acids and has two zinc fingers created by four CXXC motifs ,66,67. THPV E6s interact with a number of cellular ubiquitin ligases. E6AP or UBE3A E3 ubiquitin-protein ligase is the principal ubiquitin ligase that associates with α-HPV E6 proteins. The interaction occurs via an LXXLL binding motif on E6 and leads to stimulation of E6AP ubiquitin ligase activity ,82,83. THPV-16 E6 oncoprotein was also shown to interact with HERC2, another putative HECT domain-containing E3 ubiquitin ligase ,89. InteUBR5/EDD is another HECT domain-containing E3 ubiquitin ligase, alterations in which have been linked to carcinogenesis ,93. StudAnother E3 ubiquitin ligase that has been shown to interact with HPV E6 oncoproteins is TRIM25, which is involved in immune system regulation ,97. DiffGaussia princeps luciferase protein complementation assay (GPCA), identified a number of cellular ubiquitin ligases or substrates with ubiquitin ligase activity as interacting partners of HPV E6 [The BARD1 protein is a RING heterodimer that interacts with BRCA1, providing the E3 ubiquitin ligase activity that is required for BRCA1’s tumor suppressor function, as well as coordination of ubiquitination to maintain genomic stability ,100. BARf HPV E6 . These sf HPV E6 ,103. MGRf HPV E6 . These uf HPV E6 ,104. Interestingly, there are also ubiquitin ligases that interact exclusively with β-cutaneous E6 oncoproteins, for example, HPV-17a and HPV-3 E6 which were reported to interact with 10 subunits  of the Ccr4-Not multiprotein complex, which has been shown to be associated with various enzymatic activities including a ubiquitin ligase function ,105. WhiApart from ubiquitin ligases, there are also other elements of the UPS, such as DUBs, that have been shown to interact with E6 oncoproteins from α- and β-HPV types. One of these is CYLD lysine 63 (K63) deubiquitinase, a negative regulator of the NF-κB pathway, which was shown to form complexes with HR HPV-16 and -18 E6, although there was no evidence for direct interaction . NonetheHPV E6 oncoproteins were further reported to interact with two other DUBs: ubiquitin-specific protease (USP) enzymes, USP15 and USP46 . USP15 wHPV E6 oncoproteins from α- and β-types were also shown to bind directly to the proteasome. In vitro studies using GST-pull down assays have demonstrated that HR HPV-18 E6 interacts with several proteasomal subunits, including PSMC1, PSMC5, PSMD2, PSMC3, PSMC4, and PSMC2, while under the same conditions HR HPV-16 and LR HPV-11 E6 interacts with both subunits PSMD2 and PSMC1 . IntriguHPV E7 oncoprotein is a small acidic protein of approximately 100 amino acid residues, having no significant sequence similarities to any cellular protein, except for the LXCXE motif. The amino terminus contains two regions similar to the E1A adenovirus proteins: the conserved region 2 (CR2) and part of CR1 . The E7 The main function of the HPV E7 oncoprotein is to maintain the infected differentiating cell in a DNA replication-competent state, which it does in part, by targeting the retinoblastoma tumor suppressor (pRb), a critical regulator of cell cycle progression, which controls the G1 to S-phase transition . E7 alsoA large number of ubiquitin ligases have been reported to be HPV E7 interactors, a principal one being cullin 2 (CUL2), a core component of the cullin-RING-based ESC (ElonginBC–Cullin–SOCS-box) E3 ubiquitin-protein ligase complex ,127. ThiE7 oncoprotein, similarly to E6, generally has a short half-life, approximately one hour . It has The HPV-16 E7 protein binds to eleven POZ/BTB (Pox virus and Zinc finger/Bric-a-brac Tramtrack Broad complex) domain-containing proteins  . The funAmong the binding partners that interact with the E7 CR1 is p600 (ubiquitin N-recognin domain-containing E3 ligase 4) ,62, a unMoreover, HPV E7 was found to interact with TNF receptor-associated factors (TRAFs), a family of E3 ubiquitin ligases associating with the TNF receptor superfamily, and contributing to the activation of NF-κB and MAP kinases . TRAF2, Some other ubiquitin ligases that are involved in pathogen recognition and which associate with E7 are members of the TRIM family . This coAdditional interactions have been identified between the E7 oncoproteins of LR HPV-6, HR HPV-16, -18, -33, and β-HPV-8 and -38 and RING ubiquitin ligase NEURL1, while only HR  and LR (HPV-6) E7 binds to another RING ubiquitin ligase RNF135 [E7 oncoprotein also interacts with some DUBs; amongst these are USP26 and USP33, which are ubiquitin carboxyl-terminal hydrolases known to be involved in the ubiquitin-dependent proteolytic degradation of proteins via the 26S proteasome. Both USP26 and USP33 were identified as interactors of HR HPVs , but not of β-HPVs (HPV-8 and -38), and have not so far been investigated in detail [USP11, which belongs to a class of DUB that cleaves polyubiquitin chains, and thus inhibits the proteasome-mediated degradation of target proteins, is on the other hand, the only USP component so far shown to be bound by E7. USP11 increases the steady state levels of HPV-16 E7 by attenuating E7 ubiquitination and degradation, thus protecting E7 as well as influencing its effects on cell proliferation . This inRecent studies have shown that the levels of E7 protein in cancer cells are regulated by ubiquitin-dependent proteolysis via the 26S proteasome ,135,157.The interplay between E6/E7 oncoproteins from various HPV types and the UPS has been shown to be one of the critical strategies used by these viruses to create an optimal environment in which they can successfully replicate. However, this process, which is generally highly regulated and controlled through the various stages of the viral life cycle, can in some instances be perturbed, initially causing an imbalance in cellular homeostasis, and ultimately resulting in the transformed cell phenotype. As can be seen from


Human papillomavirus (HPV) DNA has been detected in the testis tissue of 6.5% of 185 men with non‐obstructive azoospermia (NOA). Others have suggested that seminal HPV originates from contamination from the genital skin and mucosa. One hundred unselected azoospermic men and 43 normal men undergoing vasectomy were recruited. Testicular biopsies for HPV examination were collected from all the men. Additionally, the normal men undergoing vasectomy delivered a semen sample and had a swab for HPV examination taken from the genital skin before vasectomy. A piece of each Vas deferens obtained during the vasectomy was examined for the presence of HPV. Two of the primarily azoospermic men were shown to have cryptozoospermia. It was not possible to detect HPV in the testis tissue of any of the included 98 azoospermic men or the 43 proven fertile men. In the proven fertile men, HPV DNA was detected in the semen of 15 men (35%), on the genital skin of 28 men (65%), and in the Vas deferens in three cases (7%). In 13 (87%) men with HPV‐positive semen samples, HPV DNA was also detected in the skin swabs, and in 11 men (73%), identical HPV genotypes were found in the two locations. All participants provided their written informed consent.2.2, and the sample was sent for analysis.Before disinfection of the scrotal skin and vasectomy, a swab was taken from the entire penile surface and the anterior half of the scrotal surface using sterile gloves. A kit including a flocked nylon swab stick and a tube with 1 ml of transport medium was used. The swab was prewetted with the medium, and the swab stick was broken in the tube ; the screw cap was closed2.3When potential participants in a phone call accepted to consider participating, written information for participants and a plastic container for the ejaculate were sent by postal mail. On the day of vasectomy, the participants brought a relatively fresh ejaculate together with their informed consent. Since vasectomy might be stressful, some men preferred to produce the ejaculates in the evening before the operation. The ejaculates were in no cases older than 12 hr when delivered to the hospital, and they were all conserved in a plastic container in the temperature range between room temperature (20ºC) and body temperature (37ºC). However, because the interval between ejaculate production and analysis was often extended compared to intervals of <2 hr in routine infertility work‐up, only ejaculate volume and sperm concentration were read—using a Makler chamber . Sperm motility was not analysed due to the variable and often several hours long periods from ejaculation to the delivery of the ejaculate.2.43]) from all men were examined for HPV, and in addition, all azoospermic men were examined histologically for testicular pathology.All testicular biopsies were performed after giving a funicular blockade with 200 mg of lidocaine  using a 14G TruCut needle  as described in detail by Fedder . In men 2.5During the vasectomy, ½–1 cm of each Vas deferens was routinely removed and sent for histological examination. The tissue was formalin‐fixed and paraffin‐embedded, and from the tissue block, three sections of 15 µm from each Vas deferens were cut. The sections from the right and left Vas deferens sections (right and left) from each man were mixed, and DNA was extracted as described below.2.6DNA purification  was performed using the Maxwell 16 LEV Blood DNA kit . Pre‐treatment before DNA purification varied according to sample type. Three hundred microlitre of the swabs and the tissue slices of testis and Vas deferens samples were incubated with 330 µl Proteinase K suspension at 56°C for 1 hr and overnight, respectively, and thereafter used for DNA purification. From the ejaculates, 300 µl samples were incubated with 300 µl DTT (20 mM) for 30 min at 37°C. Thereafter, the samples were centrifuged, and the supernatant was discarded. The pellet was re‐suspended in 330 µl Proteinase K solution, incubated for 1 hr at 56°C and then used for DNA purification.All samples were analysed for the presence of HPV using the INNO‐LiPA HPV assay  according to the manufactures protocol. In short, PCR was performed using biotinylated primers amplifying part of the L1‐region of the HPV genome (if HPV is present). A primer pair for the human gene HLA‐DPB1 was included in the PCR to ensure the presence and quality of the human sample. The PCR amplicons were denatured and bound to complementary DNA probes on a membrane. The presence of bound sample was visualised by the addition of streptavidin‐conjugated alkaline phosphatase and the BCIP/NBT chromogen.The INNO‐LiPA HPV genotyping assay allowed us to identify the following HPV types: 6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 56, 58, 59, 66, 68, 69, 70, 71, 73, 74 and 82.2.7t test. The sperm concentrations, total sperm counts and ejaculate volumes in ejaculates from the HPV‐positive and HPV‐negative men undergoing vasectomy were compared using the Mann–Whitney test for comparison of unpaired data.After controlling for normal distribution of the data, the ages of the azoospermic men and the healthy men for vasectomy were compared using Student's 3p < 0.05).One hundred men referred for diagnostic testicular biopsy due to azoospermia had testicular biopsies examined for HPV DNA. During the diagnostic work‐up, including the examination of more than two ejaculates, two men (of the 100) were found to have cryptozoospermia with intermittent presence of spermatozoa in the ejaculates. The mean age of the azoospermic men was 33 years compared to 40 years for the healthy men undergoing vasectomy  syndrome in testicular biopsies from 38 men, hypoplasia/atrophia/maturation stop in 42 men and normal testicular tissue in 18 of the 98 men with permanent azoospermia : 11 had Congenital Bilateral Absence of Vas deferens (CBAVD) based on a CFTR mutation or a history of epididymitis. For the azoospermic men with hypoplasia/atrophia/maturation arrest or SCO, the aetiologies represented a great spectrum. Twenty‐six (~33%) of the 80 patients had a history of cryptorchidism, and four (~5%) had a Y microdeletion. Other probable causes of azoospermia detected in the men with abnormal testicular tissue included chromosomal abnormalities (2), intake of anabolic steroids (3), chemotherapy (2), history of orchitis (1) and hypogonadotrophic hypogonadism (1). In only four (~5%) of the men with histologically abnormal testicular tissue, a CFTR mutation was detected, and one of these men in addition had a history of cryptorchidism and one a varicocele. Three of the 80 men had a history of severe genital chlamydia infections.In the two men finally diagnosed as having cryptozoospermia, intratubular germinal cell neoplasia (IGCN) was detected in one case , and a benign Leydig cell tumour in the other one. In the last case, the tumour was removed completely.None of the 43 men recruited as proven fertile, healthy controls had a history of cryptorchidism or genital tract infections, and no abnormalities were noticed on the clinical routine screening before vasectomy. HPV DNA was detected in the semen of 15 men (35%), on the genital skin of 28 men (65%), in the Vas deferens in three cases (7%), and no testis tissue samples were HPV positive ., the semen, and the penile and scrotal skin; this design serving as an appropriate basis to evaluate the natural history of HPV infection in the male genital tract. All samples from each man were collected on the same day.It might be difficult to recruit a control group of healthy volunteers undergoing a testicular biopsy. An important strength of this study is that the azoospermic men were matched with a control group of healthy, proven fertile men of similar age (40 versus 33 years). In addition, carefully systematic examination of the azoospermic men made it possible to compare the HPV status with testicular histology and aetiology of the azoospermia. Finally, the men undergoing vasectomy were examined for the presence of HPV DNA in four locations: unilateral testicular biopsy, the scrotal parts of the Vas deferensThe possible presence of HPV DNA was examined in an unselected cohort of azoospermic men with the purpose of comparing the prevalence in patients representing different aetiological groups. However, the chance to detect HPV in testicular tissue might have been increased by only including men with unexplained azoospermia . It might also be considered a weakness that only one HPV test was included for the detection of HPV DNA. However, the INNO‐LiPA HPV assay is a very widely used assay that is considered sensitive and validated and detects 32 of the most important HPV types  in men from infertile couples and 10% (95% CI: 7%–14%) in men from other populations (Laprise et al., A slightly, but not significantly, lower sperm concentration/total sperm count observed in this study was in agreement with the literature (Gizzo et al., Human papillomavirus DNA in semen might originate from a male genital tract reservoir or could be a result of contamination of HPV from the skin/mucosa during ejaculation. In agreement with the results of Golob et al.  and LuttThese prevalence rates of more than 60% having HPV on the genital skin as found in this study are higher than the prevalence rates of ~37% found in infertile Slovenian men referred for fertility treatment (Golob et al., Similarly, the prevalence of ~35% fertile men having HPV in the semen in this study was higher than a prevalence of ~14% found in infertile men in Golob et al.  and ~16%Human papillomavirus DNA in the Vas deferens has been detected in at least two studies. Rintala, Pöllänen, Nikkanen, Grénman, and Syrjänen  found HPIn another study by Svec, Mikysková, Hes, and Tachezy , HPV DNASince HPV is known to cause malignancy in the female Cervix uteri and in the anus, penis and pharynx (Garolla et al., The majority of sexually active people will acquire at least one HPV infection during their lifetime (Dunne, Nielson, Stone, Markowitz, & Giuliano, Foresta et al.  found th4.1In the majority of men with azoospermia, a probable cause can be identified (Fedder et al., The authors declare no conflict of interest.JF designed the study in collaboration with the other authors. JF informed all participating and non‐participating potential study participants, collected the written informed consent, performed all genital skin swabs and testicular biopsies, and collected and analysed all ejaculates from the men undergoing vasectomy. DØ performed all HPV analyses, and BE and NE carried out histological evaluations of testicular tissues from all azoospermic and cryptozoospermic men. ML performed the vasectomies. All authors contributed to the final article based on a draft written by JF.


While the vast majority of the cervical lesions have been attributed to the HPVs, the role of EBV and HSV1/2 as co-factors in the progression of these abnormalities needs more investigation. In this study, we aimed to determine the co-existence of EBV or HSV in cervical lesions infected with high-risk HPVs. Totally, 102 formaline-fixed cervical lesions with different pathological grades  were enrolled in this study. DNA was extracted, and its integrity was examined by PCR assay. Two conventional PCRs were performed for the detection of EBV and HSV1/2 genomes in the tissue specimens. Besides, an in-house Real-Time PCR, as well as a nested PCR assays following sequencing, was performed to detect HPV genotypes in EBV or HSV positive samples. The mean age of the participants was 42.8±13 years. Out of 102 samples, 32% (n=33) were confirmed to be LSIL, 42.2% (n=43) were HSIL, 22.5% (n=23) were SCC and 2.9% (n=3) were adenocarcinoma. EBV genome was detected in 13(12.7%) samples including 2 of LSIL, 8 of HSIL and 3 of SCC. All EBV positive samples harbored high risk HPV types 16,18 and/or 31 co-infections. However, the HSV genome was not found in any of the samples. Our result revealed that the frequency of EBV infection is higher in HISL than LSIL. Moreover, the amount of HPV load showed an elevated level among co-infected patients, which indicates that EBV might be an enhancing factor of disease progression. In contrast, HSV may not has a role as a co-factor in cervical lesions pathogenesis. Cervical cancer is the fourth most prevalent, and the fifth lethal cancer amongst women worldwide . The association between high-risk oncogenic papillomaviruses (HPVs) and cervical lesions is well known . The persistence of viral infections is attributed to a significant part of human cancers worldwide (~12–15%) . It was also suggested that viral co-existence might also make the progression of cervical lesions faster into cervical cancer. The coexisting of sexually transmitted viruses such as Herpes simplex viruses , Cytomegalovirus, Epstein- Barr virus (EBV) and, Human immunodeficiency virus are supposed to enhance the infection or disease progress during HPVs persistence . EBV and HSV belong to the Herpesviruses that persistently infect the majority of the adult population. While HSV has not been suggested as a direct tumorigenic agent, HPV, along with EBV is associated with 38% of all infection-related cancers . EBV was the first recognized human oncovirus which is responsible for different lymphoid and epithelial malignancies . Oncogenesis potency of EBV in the cervix may be associated with an increased expression of the latency proteins, LMP-1, and EBNA-2 . Studies showed that EBV could transform the cells via EBV/C3d  receptor interaction which in turn makes them more permissive/sensitive to other oncogenic viruses infection. Biopsies of the uterine cervix showed that the EBV receptors are mostly expressed on ecto- and endo-cervix regions, therefore, it might be involved in the development of cervical cancer as a “helper” factor . Regarding new reports, the presence of EBV has been documented in other cancers including breast, prostate, oral, and salivary gland carcinomas . The presence of EBV in cervical cancer and its precursor lesions  was firstly reported in the early 90s . Earlier studies have proposed that EBV is associated with the development of cervical cancer. In this regard, Marinho-Dias et al., (2013) reported a 10.1 % prevalence of EBV among cervical lesions. Another study by Santos et al., (2009) showed 21.21% and 64.29% of High Grade Squamous Intraepithelial Lesion (HSIL) and cervical cancer were infected with EBV, respectively.EBV gene expression levels gradually increased from 27% in CIN1 to 35% in CIN2/3 . Szostek et al., (2009b) suggested that the co-infection of HPV with EBV could increase the integration possibility of the HPV-16 genome that might contribute to the development of cervical cancer. Besides, Khenchouche et al. found the highest co-infection of HR-HPV and EBV in squamous cell carcinoma cases (SCC) (67%) . Therefore, they concluded that the HR-HPV and EBV co-infection in SCC might accelerate cervical cancer progression. Also, Kienka et al., (2019) reported that 100% (4/4) of HSIL HPV positive samples were infected with EBV suggesting the importance of their co-infection in developing more advanced cervical disease .Moreover, it has been shown that the co-infection of EBV and high-risk HPVs in cervical tissues is more frequent in patients with HSIL in comparison with low-grade lesions . A recent study reported 43.63%. of EBV infection in cervical cancer. They also reported that HSV1/2 is a human pathogen, mainly associated with primary and recurrent genital infections with no transforming property . The co-factor role of HSV-2 in cervical lesions progression has been postulated to be due to the induction of mutations throughout the HPV genome . Yang et al., (2004) reported that HSV-2 was associated with cervical and oral tumors and their study suggests that HSV is a risk factor in the development of cervical and oral cancers. Also, several studies have shown the co-infection of HSV-2 and HPV-16/18 in various cancers such as bladder, oral and vulvar squamous cell carcinoma . However, the actual role of HSV-2 in the development of transformed cervical lesions remained a question.Considering the importance of cervical cancer, and the possible effect of persistent viruses in the progression of the tumor diseases, this study was designed to investigate the prevalence of EBV and HSV1/2 infections in patients with cervical lesions in the southwest of Iran, Shiraz. In this regard, the detection of EBV and HSV1/2 genome was performed in high-risk HPV infected lesions. Study design and samples In this cross-sectional study, 102 formalin-fixed paraffin-embedded biopsy (FFPE) samples including 33 ISIL cases with CIN I; 23 HSIL cases with CIN II; 20 HSIL cases with CIN III; 23 cases with SCC and 3 other cases with adenocarcinoma were collected. All the samples were selected from the archive of the department of pathology of Motahari Clinic affiliated to Shiraz University of Medical Sciences, 2014-15. The study was approved by the local Ethics Committee of Shiraz University of Medical Sciences Ethic No: 1398.181). The Hematoxylin and eosin-stained slides were reclassified into different histologic grade of cervix LSIL, HSIL and SCC by the pathologist according to the WHO classification of the tumors for the female genital tract classification system .DNA extraction and qualificationTotally, for all samples, 10 μm-thick sections of FFPE tissue blocks were cut and collected in autoclaved microcentrifuge tubes and then subjected to deparaffinization step as described in a previous study . Genomic DNA was extracted using the QIAamp FFPE Tissue Kit  according to the manufacturer’s instructions. The accuracy of the extraction method was qualified by OD metry using  and the extracted DNA was stored at -20°C until further analysis.Real-time PCR assay for β-globin geneIn order to assess the quality of the extracted DNA and also quantifying the number of cell copies in every sample; all the extracted DNAs were subjected to an in house Real-Time PCR assay to detect β-globin gene as a housekeeping gene to evaluate the integrity of DNA and reporting HPVgenomic copies in a defined and normalized number of cells. The mean values were expressed as HPV DNA copies/ng DNA. The sequences of primers, PCR reaction components as well as amplification conditions were described previously . EBV PCR amplification Specific primers for EBV genome detection were designed by AlleleID software, version 7.0 . The sequences of primers targeting the BHRF1 gene region of the EBV are provided in PCR detection of HSV1/2HSV detection was performed using specific primers . PCR reaQuantification of HPV 16 and/or18 genomic copies and determination of other HPV genotypesA nested PCR assay using MY09/11 consensus primers followed by GP5+/6+ primers was done for the detection of HPV DNA sequences in all samples. HPV negative samples were excluded from the study and HPV positive samples underwent HPV16 and 18 DNA detection and quantification assessments. In this regard, an in-house duplex TaqManTM based real-Time PCR was performed, as described before . The sequences of primers, PCR reaction components as well as amplification conditions were described previously . In order to determine the genotype of HPV detected in the samples that were tested negative for HPV16 and/or18 DNA, we subjected the 150 bp amplified PCR products of GP5+/6+ primers to a bidirectional Sanger sequencing assay. Statistical analysisSPSS 21 software was used for statistical analysis. Chi-Square, T-test and Mann-Whitney tests were used to compare different parameters. P values less than 0.05 were considered to be statistically significant.Demographic characteristics of the participantsThe mean age of the patients was 42.8±13 years and that of the LSIL group was 39±13; HSIL 38.6±11.85; SCC 50.7±11 and ADC 67±7. A statistically significant correlation was observed between older ages and severer lesion grade (P <0.001). Out of 102 samples, 32% (n=33) were confirmed to be LSIL, 42.2% (n=43) were HSIL, 22.5% (n=23) were SCC and 2.9% (n=3) were adenocarcinoma. The mean age of EBV positive patients was 45.9 ±14.8 years.EBV PCR resultOne sample with a negative β-globin PCR result was excluded from the study and EBV PCR assay was performed for 101 positive β-globin samples. EBV genome was detected in 13/101 samples (12.7%) including, 2 samples with LSIL, 8 with HSIL and 3 with SCC . The freHSV detection PCR resultIn the present study, none of the 101 samples was positive for the presence of HSV DNA in all grades of cervical lesions .HPV detection and viral genomic copies quantificationEBV genome. The HPV genomic copies results showed that the mean of HPV type 16 and 18 genomic copies in EBV positive samples were 6.4 with 95%CI (5.8-7.9) and 5 with 95%CI (4.58-5.4) copies of the virus per cell, respectively. These results also indicated that although the mean of HPV-16 genomic copies in EBV/HPV co-infected samples was higher than those HPV16 mono-infected samples (6.05) copies of HPV16 with 95%CI (5.7-6.3) per cell, it was not statistically significant . Similarly, no statistically significant difference  was found between genomic copies of HPV18 mono-infected [mean of 4.9 copies of HPV18 with 95%CI(4.7-5.1) per cell]patients in comparison to EBV/ HPV co-infected ones. The co-infection of EBV and HPV was detected significantly more frequently in HSIL lesions and SCC groups than in LSIL . Our results showed that all of the EBV positive samples had co-infection with high-risk HPVs including types 16, 18 and 31 . AnalysiThe latency period between HR-HPV infection and cervical cancer progression suggests the involvement of other etiologic agents in malignancy progression. In this regard, the frequent detection of EBV in high-grade cervical lesions, suggests that EBV could be a cofactor of HPV-associated cervical carcinogenesis . In this study, we attempted to find a correlation between the presence of EBV and HSV amongst the HPV-positive cervical lesions. The results of our study showed that the frequency of EBV in cervical samples was 12.7% (13 out of 101 samples). In the same line with our results, the prevalence of EBV infection in cervical samples reported by Zhang et al., (1994) was 15.6% (15/93). Similarly, a study from Poland reported an EBV co-existence among 14% HPV positive specimens . However, the study from Bulgaria showed that just 9.6% of all specimens were positive for EBV and HPV simultaneously . Also, a higher prevalence of EBV coexisting with HPV reported from India (20%) , Thailand (32%)  and Syria (34%) , demonstrating a more important role of EBV as a cofactor. Our results also showed that the EBV genome was mostly found in HSIL (CINII and CIN III) which may indicate the importance of EBV presence in the progression of lesions. In accordance, it has been reported that the EBV genome was more frequent in HSIL than LSIL lesions . Furthermore, Silver et al. showed that women with LSIL or higher-grade of cervical lesions were almost 4 times more likely to be EBV positive than those without the disease . Also, EBV infection in HSIL was significantly more prevalent compared to LSIL . Szostek et al., (2009a) reported that EBV DNA was more frequent in cervical cancer tissue specimens than LSIL and HSIL. Furthermore, simultaneous co-infection of HPV and EBV was associated with more advanced HSIL cervical lesions, as all HPV positive samples with HSIL lesion harbored EBV DNA . Khenchouche et al., (2013) showed that the highest co-infection of HR-HPV and EBV was found in squamous cell carcinoma cases (67%). Data from Szostek et al., (2009b) research showed that the presence of EBV DNA was more frequent in cervical cancer tissues than LSIL and HSIL. However, in our study in HSIL but not cancerous samples the prevalence of EBV was significant. There are several suggestions regarding the effect of EBV/HPV co-infection on the outcome of the cervical lesion, Al-Thawadi et al., (2018) reported the co-expression of LMP1 and E6 genes of EBV and high-risk HPVs to be an important factor associated with squamous cell carcinomas and overexpression of diffused Id-1, which is an important regulator of cell invasion and metastasis. These data indicated that EBV and HPVs co-infection in cervical cancer is associated with a more aggressive cancer phenotype. During HPV infection, inflammatory signals are detected in cervical lesions and EBV-infected B-cells attracted to such inflammatory lesion; which in turn perform as a co-factor in the progression of cervical lesions . EBV may also alter the immune response to HPV-transformed cells by producing the viral BCRF1 gene product, an interleukin-10 homolog which modulates the HPV related tumor environment . Moreover, data of Zoster et al., (2009b) suggested that co-infection with EBV may facilitate the integration of the HPV-16 genome and subsequently the development of cervical cancer. Also, two studies reported that HPV/EBV co-infection correlated with a 5 to 7-fold increased likelihood of HPV integration into the host genome and enhanced genomic instability of HPV-infected cervical epithelial cells . Furthermore, Aromseree et al. declared that HPV–EBV co-infection was more common in episomal HR- HPV and EBV may have a role in carcinogenesis even in the absence of integrated HPV-DNA . Another theory that supports cancer progression by EBV is the possible EBNA1 roles which can lead to the transformation of cells through down-regulation of apoptosis and triggering DNA repair . The result of HPV genomic quantification in tissue specimens infected with both HPV and EBV showed that the average HPV 16 genomic copies in mono-HPV infections were lower than those co-infected with EBV and HPV. This finding indicates that EBV might have an effect on cellular pathways and interferes with HPV replication. Higher copy numbers of HPV-16 in EBV/HPV co-infected lesions could be determined as a bad prognosis in cervical cancer progression. Regarding the frequency of HSV infection in cervical lesions, our results did not show the presence of HSV1/2 genome in cervical lesions and cancerous tissues. Similar to our results, another report from Iran also showed no prevalence of HSV in cervical cancer tissues using real-time PCR assay . Ahmadi et al., (2017) also showed that only one out of 45 patients (2.22%) was infected with HSV-2. In contrast, in a study carried out in Bulgaria, the prevalence of HSV infection was 30.8% in HPV infected lesions . Besides, Thanh et al. reported that HSV-2 could not be a cofactor to HPV in the etiology of cervical malignancies. The low prevalence of HSV-2 is probably due to the fact that cervical sampling is not appropriate for this type of study because of the characteristics of viral biology related to neurovirulence . In conclusion, the results of this study showed that the frequency of EBV infection is higher in HSIL (CINII and CIN III) grade than LSIL. Moreover, higher HPV genomic copies was found in samples infected with both HPV and EBV than those only infected with HPV. Therefore, the co-infection of EBV and HPV might influence the progression of cervical lesions towards higher pathological grades. Also, our findings showed that there was no association between HSV infection and different grades of cervical lesions. Further investigation is required to clarify the role of EBV and HSV infection in cervical cancer.


This survey study examines the prevalence of and risk factors associated with nonfatal drug or alcohol overdose among veterans who have experienced homelessness. How common is nonfatal overdose among veterans who have experienced homelessness, and what are the risk factors and substances involved in overdoses?In this survey study including 5766 veterans nationwide who have experienced homelessness, 7.4% of veterans reported an overdose in the previous 3 years. Among veterans reporting overdose, alcohol was the most commonly involved substance.These findings suggest that nonfatal overdose is a relatively common issue among veterans who have experienced homelessness and one that warrants additional attention. Individuals with a history of homelessness are at increased risk for drug or alcohol overdose, although the proportion who have had recent nonfatal overdose is unknown. Understanding risk factors associated with nonfatal overdose could guide efforts to prevent fatal overdose.To determine the prevalence of recent overdose and the individual contributions of drugs and alcohol to overdose and to identify characteristics associated with overdose among veterans who have experienced homelessness.This survey study was conducted from November 15, 2017, to October 1, 2018, via mailed surveys with telephone follow-up for nonrespondents. Eligible participants were selected from the records of 26 US Department of Veterans Affairs medical centers and included veterans who had received primary care at 1 of these Veterans Affairs medical centers and had a history of experiencing homelessness according to administrative data. Preliminary analyses were conducted in October 2018, and final analyses were conducted in January 2020.Self-report of overdose  in the previous 3 years and substances used during the most recent overdose. All percentages are weighted according to propensity to respond to the survey, modeled from clinical characteristics obtained in electronic health records.A total of 5766 veterans completed the survey , and data on overdose were available for 5694 veterans. After adjusting for the propensity to respond to the survey, the mean (SD) age was 56.4 (18.3) years; 5100 veterans (91.6%) were men, 2225 veterans (38.1%) were black, and 2345 veterans (40.7%) were white. A total of 379 veterans (7.4%) reported any overdose during the past 3 years; 228 veterans (4.6%) reported overdose involving drugs, including 83 veterans (1.7%) who reported overdose involving opioids. Overdose involving alcohol was reported by 192 veterans (3.7%). In multivariable analyses, white race , self-reporting a drug problem  or alcohol problem , and having witnessed someone else overdose  were associated with increased risk of overdose.These findings suggest that nonfatal overdose is relatively common among veterans who have experienced homelessness. While overdose involving alcohol was more common than any specific drug, 1.7% of veterans reported overdose involving opioids. Improving access to addiction treatment for veterans who are experiencing homelessness or who are recently housed, especially for those who have experienced or witnessed overdose, could help to protect this population. Individuals who have experienced homelessness, including approximately 4% of Americans,3 are at increased risk. Overdose is one of the most common causes of death for younger individuals experiencing homelessness, with opioids implicated in most cases.4 However, the prevalence of nonfatal overdose among this population is unknown. Understanding the prevalence of and risk factors associated with nonfatal overdose among individuals experiencing homelessness could inform risk mitigation interventions at approximately 300 federally funded Health Care for the Homeless programs in community health centers,5 approximately 55 patient-centered medical homes in the US Department of Veterans Affairs (VA) that are tailored for individuals experiencing homelessness (known as Homeless Patient Aligned Care Teams [H-PACTs]),6 and other clinical settings that care for individuals experiencing homelessness.Drug overdose accounts for approximately 70 000 deaths annually in the United States,7 do not include housing status. Additionally, studies from single institutions or geographic regions have limited generalizability owing to region-specific variables, including drug supply contamination.4 To our knowledge, there has not been a nationally representative study of overdose prevalence among individuals experiencing homelessness.Previous studies on risk factors associated with overdose in individuals experiencing homelessness are limited by several factors. One challenge is that overdose data reported to the National Center on Health StatisticsAs part of a study to examine the association of primary care service design with care experience among patients experiencing homelessness, we administered a survey that included questions about overdose to veterans who have experienced homelessness. This study describes the prevalence of self-reported overdose involving drugs or alcohol in the preceding 3 years, the reported contribution of various drugs and alcohol to overdose, and the demographic, social, and health characteristics associated with overdose in this population.This survey study provides analysis of responses to a national survey of veterans who have experienced homelessness who were receiving primary care at 26 VA medical centers across the United States. The primary aim of the overall study was to compare care experiences across primary care models among veterans who have experienced homelessness. This report represents a secondary aim of that study. This study was approved by the VA’s Central Institutional Review Board. Participants were informed that by answering the survey they were agreeing to participate in the research study, so signed informed consent was not obtained.International Classification of Diseases, Ninth Revision, Clinical Modification8 or International Statistical Classification of Diseases, Tenth Revision, Clinical Modification9 diagnosis of homelessness or VA-specific administrative indicators of receipt of VA homeless services,10 in the preceding 30 months. Eligibility also required use of the VA’s primary care services at 1 of 26 VA medical centers, including a single active panel assignment and 2 or more visits to a clinic with an administrative code indicating primary care at the same study site in the preceding 24 months. Included VA medical centers were those with the largest H-PACTs. We randomly selected a subsample of eligible veterans from each study site based on initial power calculations and expected survey response rate of 40%, with the goal of a final sample consisting of two-thirds from H-PACTs and one-third from nontailored mainstream primary care clinics. We later excluded participants if they had no contact information from VA or other records or had died prior to survey initiation. To compare survey respondents and nonrespondents, we retrieved demographic and clinical information from VA medical records in the 2 years preceding recruitment.Eligibility was determined from the VA’s national electronic medical records in November 15, 2017. Eligible veterans were those who had evidence of having experienced homelessness, based on at least 1 A professional survey organization, Strategic Research Group of Columbus, Ohio, was contracted for recruitment and survey collection. Contact information from VA records was verified, updated, and standardized through a commercial address verification and update product (MelissaData). For instances in which both the MelissaData- and VA-provided addresses resulted in returned mail, Strategic Research Group searched for individual addresses using Whitepages Premium, an online service for finding individuals and verifying addresses. Residential and institutional addresses, including homeless shelters or offices, were used. Participants were recruited in 4 successive 4- to 6-week waves of approximately 3658 individuals each starting in March 2018. Each wave included 4 mailings: an introductory letter, a survey packet with $1 incentive enclosed, a postcard reminder, and, for nonrespondents, a second survey packet. Strategic Research Group called all nonrespondents up to 5 times during a 4-week window with an option to complete the survey by telephone or to provide a new address to receive the survey by mail. A debit card of $10 value was issued after survey completion. We continued to accept returned surveys through October 1, 2018.13 and on consultation with national experts. Experiencing overdose was based on the question “In the last 3 years, have you had an overdose where you needed to go to the emergency room or get medical care right away?” with a follow-up question asking what substances were involved  , severe chronic pain (a binary indicator based on screening positive for chronic pain on the 2-item screen and rating current pain ≥7), and psychological distress symptoms in the past 2 weeks based on the Colorado Mental Health Symptoms Index19 as modified and validated for homeless populations .20 A 6-item indicator of social support was devised from combining 4 items from the National Institutes of Health Patient-Reported Outcomes Measurement Information Set emotional support scale21 with a single item on perceived isolation and a tangible support indicator based on ability to borrow $20 from others .In addition to self-reported demographic characteristics, including age, race, ethnicity, and current housing status, other measures obtained via survey included self-report of drug or alcohol problems, based on the validated 2-Item Conjoint Scale,t tests to test for group differences on continuous variables and χ2 tests for differences on categorical variables. Next, we constructed a variable for the propensity to respond using the administrative data that were available for each participant who was invited to participate, which included demographic variables , medical conditions , health care utilization information , and primary care type . Responses were weighted by the inverse of response propensity (1 / propensity) so that respondents with lower overall propensity to respond were given greater weight, and vice versa. We then compared individuals who experienced any overdose with those who did not.In the initial step of our analysis, we compared survey respondents and nonrespondents on sociodemographic and clinical variables. We used independent sample 24 We also included variables associated with homelessness and adverse health outcomes that we hypothesized would be associated with overdose in individuals experiencing homelessness: severe chronic pain26 and social support.28 The multivariable analyses excluded individuals with missing data on 1 or more study variables. These models controlled for the identity of the VA medical center through application of a random effects term. P values were 2-sided and considered statistically significant at less than .05. Analyses were conducted using SAS statistical software version 9.4 (SAS Institute). Preliminary analyses were conducted in October 2018, and final analyses were conducted in January 2020.Finally, we used multivariable logistic regression models to explore which factors were associated with reporting any overdose, overdose involving drugs, and overdose involving alcohol. The intent of these models was explanatory and illustrative rather than predictive. Covariates included sociodemographic and clinical variables shown to be associated with overdose in prior studies: age, race, psychological distress, current homelessness, and medical comorbidities.Among 85 719 eligible veterans who had experienced homelessness, we attempted to recruit 14 656 veterans. We excluded 22 veterans who had no contact information and 294 veterans who died prior to survey initiation. Therefore, 14 340 eligible veterans who had experienced homelessness were invited to participate in the survey, and 5766 veterans (40.2%) returned at least partially completed surveys. Characteristics of survey respondents and nonrespondents are presented in the eTable in the P < .001), were more likely to be experiencing homelessness at the time of the survey , were more likely to be receiving medication for mental health , had higher psychological distress scores , and were more likely to report a problem involving alcohol  or drugs . Veterans who reported an overdose were significantly more likely to have witnessed an overdose than those who did not report an overdose .Among 5694 veterans included in the overdose analyses, the mean (SD) age was 56.4 (18.3) years; 5100 veterans (91.6%) were men, 2225 veterans (38.1%) were black, and 2345 veterans (40.7%) were white. Overdose in the previous 3 years was reported by 379 veterans (7.4%), and witnessing someone else overdose was reported by 873 veterans (16.2%). Characteristics of veterans with and without overdose are presented in Alcohol was the most common substance involved in a recent overdose, reported by 192 veterans (3.7%) . OverdosResults of the multivariable models for experiencing any overdose, overdose involving alcohol, and overdose involving drugs are presented in In this large survey study of veterans who have experienced homelessness, 7.4% reported an overdose requiring urgent medical attention in the past 3 years, and 16.2% had witnessed someone else overdose. Witnessing overdose was significantly associated with personal experience of overdose. Alcohol was the predominant substance reported in overdose, although 1.7% of veterans reported overdose involving opioids.30 Alcohol overdose, or acute intoxication, is a frequent cause for emergency department visits.31 Although alcohol rarely appears as the sole toxin in fatal overdoses,32 heavy alcohol use has a substantial negative effect on long-term morbidity and mortality.33 Alcohol use disorder is also treatable with approved medications and interventions, such as motivational interviewing and cognitive behavioral therapy.34 Despite this, data show that most individuals with alcohol use disorder do not receive treatment.35One potentially actionable finding from this study is that alcohol was the most common substance reported with overdose, nearly as common as all drugs combined and more than 2-fold as common as opioids. Previous studies have shown that alcohol, cannabis, and cocaine are the most widely used substances among individuals experiencing homelessness.2 and comparable with an analysis of patients with Medicaid insurance who had received at least 1 opioid prescription, in which 0.5% of patients per year required medical services for overdose involving opioids.36 A 2018 study using Medicaid data37 found that individuals who survived nonfatal opioid overdose were at 24-fold higher risk of death compared with the general population, with drug overdose, respiratory illness, HIV, and suicide all contributing to mortality. For health systems that serve individuals experiencing homelessness, a patient’s report of recent overdose signals elevated medical risk and may even merit standardized screening or offer of additional services.The second most common overdose cause was opioids. Opioid overdose in this sample  was higher than for the general population38 However, since pharmacies are required to dispense naloxone to a named individual rather than a facility, this can be difficult to accomplish for homeless shelters. Finally, opioid overdose could also be prevented through medication therapy for opioid use disorder,39 yet focused efforts to assure delivery of treatment for opioid use disorder in this population are uncommon.40Additionally, maximizing access to the opioid overdose reversal agent naloxone is advisable.41 few studies explore social networks among veterans who have experienced homelessness, a unique population whose relationships may be shaped by history of military service and unpredictability of housing. Peer-based programs have been applied widely in VA medical centers,42 including with veterans who have experienced homelessness.43 Peer-based strategies that focus on homelessness may be an opportunity for tailored assistance, potentially tapping into the experience and wisdom of veterans who have experienced homelessness in reaching their peers.The findings of this study may suggest additional targets for clinical and policy intervention. The association of witnessing overdose with experiencing overdose suggests that efforts at risk mitigation should take into account patients’ social networks. Although relevant to substance use,sleeping rough), in homeless shelters, or newly in housing. This could be advanced through outreach, colocation of mental health and primary care services, or enhancing the capacity of primary care clinicians to deliver mental health care. Motivational interviewing to address substance use has been shown to be effective in homeless populations,47 provided there are viable addiction treatment options available.49 While federal grants have expanded care for opioid use disorder, block grants from the Substance Abuse and Mental Health Services Administration, such as those that would assist with alcohol use disorder, have received flat funding since 2014 and fallen relative to inflation.50Given the prevalence of high emotional distress in individuals who have experienced overdose, enhanced mental health services could mitigate some risk for individuals residing on the streets ,This study, the largest survey of veterans who have experienced homelessness to date, to our knowledge, found that recent nonfatal overdose was a relatively common issue among veterans who have experienced homelessness, occurring in 7.4% of survey respondents, with alcohol being involved more often than opioids. Improving access to mental health and addiction treatment for veterans who are experiencing homeless or who are recently housed and targeting health and social services for those at increased risk could enhance the safety of this population.