HPV-associated OSCC (HPV-OSCC) has been determined to be a distinct disease entity from non-HPV associated OSCC. Patients affected by HPV-OSCC generally have a more favourable prognosis, with improved rates of locoregional control and survival compared with their non-HPV counterparts. Despite this, HPV-OSCC has a similar rate of distant metastases. Interestingly, recent evidence has emerged that demonstrates more frequent atypical metastasis patterns when compared to non-HPV associated disease. To the best of our knowledge, this report describes the first case of a confirmed HPV-OSCC with distant metastasis to the cavernous sinus.A 62-year-old non-smoking male presented to the head and neck oncology clinic with a five-month history of enlarging right neck mass causing neck pain, dysphagia, and dysphonia. HPV-associated base of tongue squamous cell carcinoma (cT4aN2c) was diagnosed, and he was treated with primary chemoradiation. Shortly after treatment, he presented with progressive bilateral cranial nerve palsies including left cranial nerve III and right cranial nerve VI involvement. Imaging identified masses in the left cavernous sinus with extension of tumor into the sella and in the right cavernous sinus at the level of Dorello’s canal. Endoscopic Image Guided Transsphenoidal biopsy of the left sellar mass confirmed distant metastases from the previously treated HPV-OSCC primary to the cavernous sinus. The patient was palliated with carboplatin and paclitaxel.The presented report is the first documented case of confirmed HPV-associated oropharyngeal squamous cell carcinoma metastasizing to the cavernous sinus, and the only HPV confirmed head and neck cancer case to present with metastasis to the cavernous sinus and limited extracranial disease. This case demonstrates the importance of recognizing presentations of atypical metastasis that are possible in HPV-associated oropharyngeal cancer. Given the rarity of metastasis to this region, vigilance in follow up is instrumental in early identification and treatment for these patients. Oropharyngeal squamous cell carcinoma (OSCC) has traditionally been associated with tobacco and alcohol use. In the past, these exposures accounted for up to 75% of cases . Public HPV-associated OSCC (HPV-OSCC) has recently been recognized to be a distinct disease entity from OSCC occurring in the absence of HPV infection. Human papillomavirus association is most commonly identified with p16 immunohistochemistry, whereby HPV-OSCC cells display p16-positivity. In general, HPV-OSCC is associated with a more favourable prognosis when compared to OSCC occurring in the absence of HPV infection, with improved rates of locoregional control and patient survival following curative-intent treatment of localized disease , 8. In aTo the best of our knowledge, this report describes the first case of a confirmed HPV-OSCC with distant metastasis to the cavernous sinus. We intend for this report to highlight the importance of recognizing presentations of atypical metastasis that are possible in HPV-associated oropharyngeal cancer.2 one week prior to the initiation of radiotherapy, followed by seven weekly doses of 250 mg/m2 concurrent with radiotherapy. During and soon after completing chemoradiotherapy, the patient experienced the expected toxicities and had evidence of favourable cancer response to treatment. A contrast enhanced CT scan of the neck performed five weeks after completion documented good anatomic response to treatment with resolution of all disease with the exception of a lymph node within right level II, which was much decreased in size, measuring 2 cm in greatest dimension.A 62-year-old male lifelong non-smoker was referred to the head and neck oncology clinic with a five-month history of enlarging right neck mass associated with neck pain, dysphagia, and dysphonia. He was otherwise healthy, aside from type II diabetes mellitus and a distant history of sarcoidosis. Physical examination, as well as contrast-enhanced CT scan of the neck, showed a large base of tongue mass that crossed the midline and projected into the suprahyoid epiglottis. There was a 4.6 × 3.4 cm mass representing a necrotic confluence of lymph nodes in right level IIA and IIB. Biopsy confirmed non-keratinizing, HPV-OSCC. Immunohistochemistry p16 testing was used for HPV diagnosis. PET/CT scan demonstrated bilateral level III lymphadenopathy, but did not show evidence of distant metastases. The patient was diagnosed with a cT4aN2c HPV-OSCC and referred to the institutional tumor board where a recommendation for definitive chemoradiation was made. Staging was according to the American Joint Committee on Cancer Seventh Edition. The patient received 70 Gy in 35 fractions to the primary site and involved nodes, with 56 Gy to the uninvolved regional nodes bilaterally, including the bilateral lateral retropharyngeal nodes. Due to the patient’s comorbidities the concurrent chemotherapy consisted of intravenous cetuximab given as a loading dose of 400 mg/mTwo months after completing chemoradiotherapy, the patient presented to medical attention with onset of binocular vertical diplopia. He was found to have subtle evidence of a new pupil-sparing, left-sided cranial nerve III palsy. His extraocular movement deficits progressed rapidly over one and a half weeks and upon repeat examination he was found to have a new right-sided cranial nerve VI palsy. Differential diagnosis included ischemia, neuromuscular disorder, metastasis, unrelated compressive lesion, and bilateral aneurysm. He was admitted to hospital for investigations. Electromyography showed no evidence of myasthenia or myopathy. Magnetic Resonance Imaging of the sella revealed two separate enhancing cavernous sinus lesions; one tumor in the left cavernous sinus that encased the internal carotid artery with extension into the sella Fig.  and anotThe patient underwent Endoscopic Image Guided Transsphenoidal biopsy of the sellar tumor which showed a malignant basaloid tumor consistent with non-keratinizing p16-positive squamous cell carcinoma, likely of oropharyngeal origin Fig.  and consMetastases involving the cavernous sinus are rare and have previously been described in several solid tumors. Cancers with primary tumors outside of the head and neck have been shown to occasionally metastasize to the cavernous sinus, including carcinomas of the lung , colon , 18, andTwo cases of oropharyngeal squamous cell carcinoma with metastasis to the cavernous sinus have been recently described. In one, disease progression was advanced on initial presentation, with a relatively small primary tumor but substantial nodal involvement and lung metastases (cT1N2bM1). In the other, nodal involvement was also extensive on the patient’s initial presentation, with N2c disease. There were no additional sites of metastasis in that case. However, no HPV testing or smoking histories were reported in either case, calling into question the nature of the cancer , 25. WirHPV-associated oropharyngeal cancer is considered to be a distinct disease entity and therefore has a different natural history and prognosis compared to non-HPV associated oropharyngeal cancer. Locoregional control and overall survival of HPV patients is better, with long term survival approaching an improvement of over 40%. However, a subset of patients develop early, widespread metastases and fair very poorly . As wellAlthough outcomes differ, treatment of HPV-OSCC currently aligns with that of non-HPV OSCC and may include surgical resection, radiation therapy alone, or chemoradiation. Among head and neck cancer populations unselected for HPV status, the evidence supporting the addition of concurrent chemotherapy using either cisplatin or cetuximab has shown definitive improvements in overall survival and locoregional control rates compared with definitive radiation alone , 31. How1, V2, and VI, as well as the internal carotid artery. Due to these anatomic relationships, lesions of the cavernous sinus may present with headache, chemosis, ophthalmoplegia, proptosis, miosis, diplopia, or sensory deficits in the ophthalmic and maxillary nerve distributions. Indeed, cavernous sinus metastases are capable of presenting with these signs and symptoms, and recognition of this pattern is necessary in HPV-OSCC patients as this disease has shown a propensity for atypical metastases. Metastasis to this region is rare, and having two separate metastatic tumors of the cavernous sinuses become simultaneously symptomatic was indeed extraordinary. Vigilance in close follow up of newly symptomatic patients is instrumental for early identification of tumor metastasis and for treating these patients.The cavernous sinuses are paired dural venous sinuses that contain, either running directly through or within the lateral wall, cranial nerves III, IV, VThe presented report is the first documented case of confirmed HPV-associated oropharyngeal squamous cell carcinoma metastasizing to the cavernous sinus, and the only HPV confirmed head and neck cancer case to present with cavernous sinus involvement in the absence of widespread metastases. This case demonstrates the importance of recognizing presentations of atypical metastasis that are possible in HPV-associated oropharyngeal cancer.


The Advisory Committee on Immunization Practices (ACIP) recommends that adolescents routinely receive tetanus, diphtheria, and acellular pertussis vaccine (Tdap), meningococcal conjugate vaccine (MenACWY), and human papillomavirus (HPV) vaccine (https://www.cdc.gov/vaccines/imz-managers/nis/downloads/NIS-PUF15-DUG.pdf). T-tests were used for statistical comparison of weighted data to account for the complex survey design. Weighted linear regression by survey year was used to estimate annual percentage point increases. Differences were considered statistically significant for p-values <0.05.NIS-Teen is an annual survey that collects data on vaccines received by adolescents aged 13–17 years in the 50 states, the District of Columbia, selected local areas, and territories.In 2016, ≥1-dose HPV vaccination coverage among teens was 60.4% , and 43.4% were up to date with the recommended HPV vaccination series  . During Tdap and MenACWY coverage was similar for each age group. For HPV vaccination  coverage was higher overall and by sex, for persons aged 17 years , except for HPV up-to-date status among males, which was highest among males aged 16 years . Among ahttps://stacks.cdc.gov/view/cdc/47510) (https://stacks.cdc.gov/view/cdc/47510).Differences in vaccination coverage by race/ethnicity in 2016 were similar to patterns observed in previous years  ; howeverc/47510) . Coveraghttps://stacks.cdc.gov/view/cdc/47510).Vaccination coverage varied by state . For exaIn 2016, adolescent vaccination coverage in the United States was sustained and continued to improve in several areas: compared with 2015, coverage with Tdap, ≥2 doses of varicella vaccine, ≥2 doses of MenACWY, and each dose of HPV vaccine increased. Since HPV vaccine was introduced for females in 2006 and for males in 2011, coverage has increased gradually among females and more rapidly among males. During 2015–2016, increases in coverage with each HPV dose, ranging from 3.4 to 6.2 percentage points occurred among males, whereas only a 2.8 percentage point increase in ≥2-dose HPV coverage occurred among females. Coverage with ≥1-dose HPV vaccine among males continues to approach that among females, particularly for adolescents aged 13 years, suggesting that HPV vaccination of both female and male adolescents has been integrated into vaccination practices. Although HPV vaccination initiation (receipt of ≥1 HPV vaccine dose) continues to increase, coverage remains 22–28 percentage points lower than those for Tdap and ≥1-dose MenACWY. These gaps indicate substantial opportunity for improving HPV vaccination practices.https://www.cdc.gov/ruralhealth/caseforruralhealth.html).Disparities in adolescent vaccination coverage were found by MSA status: HPV vaccination initiation among adolescents living outside MSA central cities was 16 percentage points lower than among those living in MSA central cities. Although adolescents living in non-MSA areas had substantially lower HPV and MenACWY vaccination coverage compared with those living in MSA central cities, Tdap coverage in these groups was similar. Reasons for these disparities are not well understood. Potential contributing factors might include differences in parental acceptance of certain vaccines and provider participation in, and adolescents’ eligibility for, the Vaccines for Children program.Variation in adolescent vaccination coverage among state and local areas might reflect differences in adolescent health care delivery, the prevalence of factors associated with lower vaccination coverage, and immunization program emphasis on, and effectiveness of, adolescent vaccination activities. Immunization programs in several state and local jurisdictions , although not necessarily having the highest HPV vaccination coverage in the nation, have experienced annual increases in coverage that exceed the national average over a 4-year period. Activities contributing to this success, as reported by these immunization programs, include enhancing provider education, assessing vaccination coverage levels in health care provider offices and providing feedback to the practices, conducting media campaigns, engaging community partners, and experiencing a “spillover” effect from middle school vaccination requirements for Tdap and MenACWY vaccines.At the end of 2016, the recommended HPV vaccination schedule was changed from a 3-dose to a 2-dose series for immunocompetent adolescents initiating the series before their 15th birthday. Three doses are recommended for persons initiating the series at ages 15 through 26 years and for immunocompromised persons (https://www.cdc.gov/cancer/hpv/statistics/cases.htm). Although it is too early to observe the impact of HPV vaccination on HPV-associated cancers, impact on infection with HPV types targeted by the vaccine and other endpoints have been reported , and only 53.9% of landline-completed and 47.4% of cell phone–completed interviews had adequate provider data. Second, bias in estimates might remain even after adjustment for household and provider nonresponse and phoneless households.https://www.thecommunityguide.org/topic/vaccination). Resources for clinicians to facilitate effective communication with parents and adolescents regarding HPV and other recommended vaccines are available at https://www.cdc.gov/hpv/hcp/index.html. Provider-based performance measurement could also facilitate increased adolescent vaccination coverage, including the Assessment, Feedback, Incentives, and eXchange program implemented by state and local immunization programs with individual providers (https://www.cdc.gov/vaccines/programs/afix/index.html), and the 2018 updated Healthcare Effectiveness Data and Information Set composite measure for health plans, assessing receipt of Tdap, MenACWY, and HPV vaccines by age 13 years . Protection against vaccine-preventable diseases will be increased if clinicians consistently recommend and simultaneously administer Tdap, MenACWY, and HPV vaccines at age 11–12 years.Adolescent vaccination coverage can be increased, and the gap between HPV vaccination coverage and coverage with Tdap and ≥1-dose MenACWY can be closed with increased implementation of effective strategies. Providers should use every visit to review vaccination histories, provide strong clinical recommendations for HPV and other recommended vaccines, and implement systems to eliminate or minimize missed opportunities  –associated cancers, diphtheria, pertussis, tetanus, and meningococcal disease, routine immunization of adolescents aged 11–12 years is recommended by the Advisory Committee on Immunization Practices (ACIP). Since HPV vaccine introduction in 2006 for females and 2011 for males, coverage has increased gradually for females and more rapidly for males, although coverage has not reached the tetanus, diphtheria and acellular pertussis vaccine (Tdap) and quadrivalent meningococcal conjugate vaccine (MenACWY) coverage.In December 2016, ACIP updated HPV vaccination recommendations to include a 2-dose schedule for immunocompetent adolescents initiating the vaccine series before their 15th birthday; 3 doses are recommended for persons who initiate the series at age 15–26 years and for immunocompromised persons. A new HPV up-to-date measure was added to the 2016 National Immunization Survey–Teen to account for the revised HPV vaccination schedule. HPV up-to-date estimates were 49.5% for females and 37.5% for males and 6.0–6.5 percentage points higher than ≥3-dose adolescent HPV coverage. HPV up-to-date vaccination coverage was 15 percentage points lower among adolescents living in nonmetropolitan statistical areas (MSAs) than among adolescents living in MSA central cities.Adolescent vaccination coverage continues to improve, but opportunity remains to increase HPV-associated cancer prevention. A better understanding of reasons for differences in HPV vaccination by MSA status might identify appropriate strategies to improve coverage. Protection against vaccine-preventable diseases will be increased if clinicians consistently recommend and simultaneously administer Tdap, MenACWY, and HPV vaccines at age 11–12 years.


The classification of human papillomavirus (HPV) intratypic lineages by complete genome sequencing is a determinant in understanding biological differences in association with this disease. In this work, we have characterised complete HPV genomes from southern Brazil. Fifteen cervicovaginal Pap smear negative samples previously categorised as HPV-positive were sequenced using ultradeep sequencing, and 18 complete genomes from 13 different HPV types were assembled. Phylogenetic and genetic distance analyses were performed to classify the HPV genomes into lineages and sublineages. This is the first report describing the distribution of HPV intratype lineages of high and low oncogenic risk in asymptomatic women from southern Brazil. Human papillomavirus (HPV) is the most prevalent sexually transmitted infection worldwide and is a major factor in the development of cervical cancer zur . With ovDistinct HPV types differ by more than 10% in their L1 sequences, whereas the classification of intratype lineages and sublineages requires complete genome information. Nucleotide distances between 1 and 10% distinguish intratype lineages, whereas differences between 0.5 and 1% define distinct sublineages .Biological differences have been proposed for HPV lineages and sublineages . StudiesFifteen samples of cervicovaginal smears originated from HPV-positive women previously typed by ® XT DNA Sample Preparation kit  and tagmentation and polymerase chain reaction (PCR) according to the protocol established by the manufacturer. Libraries were purified according to the manufacturer’s protocol and quantified by real-time PCR in an ECO System (Illumina Inc.) using the Kapa Library Quantification kit . Pooled libraries were subjected to clustering in a cluster C-bot station and sequenced in an Illumina® HiSeq 2500 system. After sequencing, reads were filtered in the Sickle-Master programme (available from: https://github.com/najoshi/sickle) to select reads with a quality ≥ 28 in the Phred scale and with a length ≥ 20 bp. Reads were evaluated with FastQC software  for their amount and mean quality.HPV DNA was first enriched by rolling circle amplification (RCA) using the Illustra TempliPhi DNA Amplification kit . Libraries were prepared for each sample using a NexteraBWA  was usedp-distance method in MEGA5  in 12 out of the 15 samples analysed. The remaining three samples did not provide enough information that enabled HPV complete genome assembly.The 18 complete genomes, spanning 13 different HPV types of the alpha genus (HPV6 (n = 3), HPV18 (n = 1), HPV31 (n = 2), HPV35 (n = 1), HPV39 (n = 1), HPV51 (n = 1), HPV56 (n = 1), HPV58 (n = 1), HPV61 (n = 1), HPV67 (n = 1), HPV68 (n = 2), HPV82 (n = 2) and HPV85 (n = 1)), were aligned with reference sequences from different lineages and sublineages of each HPV type . Newly aThis report is the first describing the distribution of HPV lineages/sublineages of high and low oncogenic risk in asymptomatic (Pap smear-negative) women from southern Brazil. We showed herein that the use of NGS enabled the detection of co-infections with multiple HPV genotypes due to its greater sensitivity when compared to Sanger sequencing, as shown previously by our group and others da .Interestingly, in one of the samples (07) the HPV type identified by Sanger sequencing was not detected by NGS, even though the latter is more sensitive. One possible explanation for this observation may be related to the use of the rolling circle amplification (RCA) enrichment during the preparation of samples for NGS, a technique that enriches episomal (unintegrated) HPV genomes. Thus, we hypothesize that HPV33, which was previously detected by Sanger sequencing in sample 07, is likely integrated, and its detection was disfavoured by the RCA protocol, while HPV68 may be episomal, and may have been enriched during RCA and hence detected. This may be considered an important limitation to our study and to the use of NGS for HPV typing. While amplicon-based NGS approaches could circumvent this bias , they doIn this study, the lineages identified that HPV6, the aetiological agent of anogenital warts and laryngeal papillomas, belonged to the A lineage and B1 and B3 sublineages. These results are in agreement with the multicentric study by With respect to HPVs with high oncogenic potential, HPV18 is the second most common type associated with cervical cancer. Our study has identified an European sublineage of HPV-18 (A3) in a sample of a woman with no cervical intraepithelial lesions. Previous work has also identified European HPV-18 lineages as predominant among asymptomatic women in southeastern Brazil . A high HPV58 is the second highest oncogenic risk type in prevalence in the city of Rio Grande, Brazil . In thisHPV is the most prevalent sexually transmitted infection worldwide and a major factor for the development of cervical cancer. Infection by multiple HPV types or lineages can be pivotal to the virus’s oncogenic potential in infected women. Next-generation sequencing becomes a powerful research tool to identify co-infections by different HPV types and/or lineages worldwide.


Recent studies have suggested that immune cells as part of tumor's microenvironment could partly explain the better outcome in HPV-associated oropharyngeal carcinoma. We performed a systematic review of the literature focused on differences in immune-infiltrate in HPV+ versus HPV- oropharyngeal cancers. This comprehensive search yielded 4308 original papers, of which 20 satisfied our eligibility criteria. Increase in both circulating and tumor infiltrating CD8+ lymphocytes is mainly seen in HPV+ oropharyngeal carcinoma. Interestingly, the survival benefit associated with increase in immune cells is equal both in HPV+ and HPV- oropharyngeal cancer. Based on these results, our review underscores the role of the immune system in the biological and clinical behavior of oropharyngeal squamous cell carcinomas (OPSCC) and might open doors to further investigate immune modulatory treatment options in OPSCC patients. Lay Abstract: In the last few years it has become clear that oropharyngeal carcinomas can be caused by an HPV infection. Overall, HPV+ carcinomas have a better outcome. Recent studies suggest that could be partly explained by immune cells in the tumor's microenvironment of HPV+ carcinomas. Following a systematic review of the literature focusing on immunological difference between HPV+ and HPV- oropharyngeal cancers, we found a survival benefit associated with increase in immune cells in both HPV+ and HPV-. We found an increase in CD8+ lymphocytes primarily in HPV+ carcinomas. Our review underscores the role of the immune system in the biological and clinical behavior of these cancers. Head and neck squamous cell carcinoma (HNSCC) is the sixth common cancer worldwide with about 550,000–600,000 new patients per year ,2. DespiHNSCC is a heterogeneous disease originating from multiple different anatomical subsites and depending on the origin varies in biological behavior resulting in varying survival rates and incidences. Traditional risk factors for HNSCC are excessive tobacco and alcohol use . In the Supplementary File 1). After removing duplicates, two authors (PMWK and SEM) independently screened the titles and abstracts of all retrieved records for inclusion, using predefined inclusion and exclusion criteria (see below). Subsequently, full texts of relevant articles were screened for a more detailed selection. A cross-reference search was performed, checking for articles not identified by the original search. Differences in opinion were settled by discussion. Subsequently, the full texts were screened by both authors, which resulted in 20 articles used in this review.A systematic literature search was conducted in EMBASE and PubMed databases of articles published until September 2015. The research terms were ‘T lymphocytes’ and ‘oropharynx cancer’ with all their synonyms in title and abstracts  extracted descriptive data of study population, HPV detection, HPV prevalence and T-lymphocyte infiltration. The following data of included authors were extracted: first author, year of publication, country, tumor location and percentage OPSCC, percentage HPV-positive tumors, HPV detection method, method of T-lymphocyte extraction . To compare the influx or circulation of CD3+, CD4+, CD8+ and FoxP3+ T lymphocytes between patients with HPV-positive OPSCC and HPV-negative OPSCC, p-values were extracted or calculated if sufficient data were provided. Additionally, if reported, the association between T-lymphocyte infiltration or circulation and survival was extracted.The search retrieved 4308 unique articles . After set al. correlated HPV DNA to HPV mRNA expression. Of the included studies, seven articles investigated the relation between HPV status and circulating T cells, using flow cytometry. Four of these studies also studied the HPV-specific T-cell response and HPV status [Supplementary Table 1). Rittà et al. also described the association between the viral load and the density of the TILs [Supplementary Table 1 describes the used antibodies for IHC and flow cytometry in the included studies.The study characteristics of the included studies are summarized in V status ,14,18,19V status ,15–17,2011–20 epitope as the CD8+ cells target. Also the ratio of CD4/CD8 is significantly lower in HPV+ OPSCC, compared with HPV- OPSCC. Only Heusinkveld et al. measured circulating FOXP3 cells and found no statistically significant difference between both groups [The extracted outcome data of included six studies are shown in A total of 13 articles investigated the CD3+ tumor infiltrating lymphocytes . Two artOnly two of six articles investigating the circulating T cells in OPSCC, described the influence of circulating T cells on the survival ,19. The et al. concludes that HPV positivity in itself is associated with a significantly better overall survival [et al. described in his article that TILs were no prognosticators in HPV- OPSCC, but in HPV+ OPSCC a high number of TILs were related to a significantly better disease-specific survival [et al. showed a strong HPV+ signal alone that is associated with a significantly better DSS, but that a more intense CD3+ staining has no prognostic value in HPV+ tumors [et al., Badoual et al., Oguejiofor et al. and Nordfors et al. [Seventeen studies investigated the relationship between tumor infiltrating lymphocytes and survival . Oguejiosurvival . Ward etsurvival . In cont+ tumors . This sas et al. ,10,17. Bs et al. ,13. But s et al. ,13,15.Looking at the different T-cell subtypes, CD4+ cells were not significantly related to survival, according to Balermpas, Oguejiofor and Badoual ,10,13. IIn this review, we have examined the differences in T-cell repertoire between HPV+ and HPV- OPSCC. To our knowledge this is the first systematic review investigating the relationship between the prognostic value of immune cells in HPV+ and HPV- OPSCC.The results of this systematic review should be viewed within the constraints of several limitations. First, studies were performed in various geographical areas with probably variable HPV incidence. Second, the techniques of HPV detection also varied, and due to different sensitivity and specificity, possibly also introduced heterogeneity in HPV status and subsequent correlations. Third, although we included only studies that investigated OPSCC, some also contained tumors from additional subsites. As the effect of immune cells might vary per subsite, it is not certain whether this influenced survival rates. Fourth, antibodies and  methods used for detection of immune cells were variable, again introducing (pre)-analytical heterogeneity. Because of this heterogeneity in methods and analyses, statistical pooling was not permitted. Hence, no overall disease-specific survival could be calculated. This warrants the introduction of useful criteria for quality assessment for molecular studies. This is particularly important due to the increase in biomarker studies in the field of personalized cancer care, because these specific molecular biomarkers of tumors including their microenvironment are used for clinical decision-making in the treatment of oncologic patients. We advise caution in interpreting these results because of the small number of candidate genes with overlap and the methodological differences between studies. Comparison and validation of these results was thus impaired. For future research, we recommend adequately designed studies with study populations of explicitly OPSCC, uniform protocols for interpretation of promoter methylation and validation in independent cohorts to evaluate these promising results on a larger scale. Despite this heterogeneity however, most studies point toward an increase in circulating and tumor infiltrating CD8+ lymphocytes in HPV+ OPSCC, but only in half of the cases, this effect is statistically significant. Moreover, this increase in both circulating and tumor infiltrating CD8+ lymphocytes in OPSCC is associated with increased survival. Interestingly this is not only seen in HPV+ OPSCC but also in HPV- OPSCC. The latter might be caused by and increase immunogenicity due to higher mutational load in  HPV- tumors. In general however, this review shows that there is a clear increase in CD8+ circulating and tumor infiltrating T lymphocytes in HPV+ tumors, which is associated with a better survival, independent of HPV status. There is not enough research done in the general increase of circulating lymphocytes (CD3-positive T cells) in OPSCC. This is probably the reason that this effect is not ready statistically significant. In contrast, we do see a significant increase of general tumor infiltrating T cells in HPV+ OPSCC and this has an association with a significantly better survival, although (as in CD8), this effect is independent of the HPV status. This increase in TILs in OPSCC strengthens the idea that circulating lymphocytes are also significantly increased in OPSCC, when the sample size is large enough. The same problem arises in the investigation of circulating CD4- and FOXP3-positive T cells. Although two articles describe a minimal increase of circulating CD4 T cells in HPV- OPSCC compared with HPV+ T cells, this sample size is quite small. Tumor infiltrating T cells have been shown to be increased in HPV-positive OPSCC. In most cases it has no significant role in the survival, regardless of the HPV status. Based on these results, our review underscores the role of the immune system in the biological and clinical behavior of OPSCC and might open doors to further investigate immune modulatory treatment options in OPSCC patients.Prognosis of head and neck cancer is still poor and targeted treatment options for oropharyngeal cancer are still limited. Recent studies have shown breakthrough results of immune modulatory therapy in many cancers. As the role of immune cells in HPV-positive and -negative oropharyngeal cancer is being elucidated, immune modulatory therapy could play an important role in the treatment of oropharyngeal cancer.The therapeutic options of oropharyngeal carcinoma are fairly limited.Major causes are smoking, alcohol and HPV.This makes oropharyngeal cancer highly immunogenic tumors.The difference in prognostic relevance of CD3+, CD4+, CD8+ and FoxP3+ T lymphocytes between patients with HPV-positive OPSCC and HPV-negative oropharyngeal carcinoma.Reporting of this review was done systematically.We conducted a systematic search in the databases of PubMed and EMBASE.Results were evaluated by two reviewers (SEM and PK) independently.Review manager 5.3, IBM SPSS 20.0 statistical software was used for the statistical analysis.Significant heterogeneity was explored through subgroup analysis.Increase in both circulating and tumor infiltrating CD8+ lymphocytes in oropharyngeal cancer is associated with increased survival both in HPV+ OPSCC and also in HPV- OPSCC.There is a clear increase in CD8+ circulating and tumor infiltrating T lymphocytes in HPV+ tumors, which is associated with a better survival, independent of HPV status.Immune cells play an important role in the biological and clinical behavior of OPSCC.This opens doors to further investigate immune modulatory treatment in both HPV+ and HPV- oropharyngeal cancer.Click here for additional data file.


P = 1.000), but the specificity of the HPV-Risk assay was significantly higher than that of HC2 . For the detection of CIN2+, similar results were obtained, with the relative sensitivity being 0.98  and the relative specificity being 1.02 . The performance of the HPV-Risk assay for the detection of CIN3+ and CIN2+ was noninferior to that of HC2, with all P values being ≤0.006. In conclusion, the HPV-Risk assay demonstrated noninferiority to the clinically validated HC2 by the use of samples from the VALGENT-3 panel for test validation and comparison.Human papillomavirus (HPV) testing is increasingly being incorporated into cervical cancer screening. The Validation of HPV Genotyping Tests (VALGENT) is a framework designed to evaluate the clinical performance of various HPV tests relative to that of the validated and accepted comparator test in a formalized and uniform manner. The aim of this study was to evaluate the clinical performance of the HPV-Risk assay with samples from the VALGENT-3 panel and to compare its performance to that of the clinically validated Hybrid Capture 2 assay (HC2). The VALGENT-3 panel comprises 1,300 consecutive samples from women participating in routine cervical cancer screening and is enriched with 300 samples from women with abnormal cytology. DNA was extracted from original ThinPrep PreservCyt medium aliquots, and HPV testing was performed using the HPV-Risk assay by investigators blind to the clinical data. HPV prevalence was analyzed, and the clinical performance of the HPV-Risk assay for the detection of cervical intraepithelial neoplasia grade 3 or worse (CIN3+) and CIN2 or worse (CIN2+) relative to the performance of HC2 was assessed. The sensitivity of the HPV-Risk assay for the detection of CIN3+ was similar to that of HC2 (relative sensitivity, 1.00; 95% confidence interval [CI], 0.95 to 1.05;  Screening with human papillomavirus (HPV) testing provides better protection against cervical cancer and high-grade cervical intraepithelial neoplasia (CIN) than cytology-based screening , 2. ConsThe international validation guidelines described by Meijer et al.  have bee5–The HPV-Risk assay is a real-time PCR-based assay that targets the E7 region of 15 (probable) high-risk HPV types and enables partial genotyping for HPV16 and HPV18. A previous evaluation of the HPV-Risk assay relative to the clinically validated comparator GP5+/6+-PCR showed that its clinical performance and reproducibility met the international validation criteria for HPV tests for use in cervical cancer screening .This study was performed to evaluate the clinical performance of the HPV-Risk assay for detection of high-grade CIN using the VALGENT-3 panel and to compare its performance to that of the clinically validated Hybrid Capture 2 assay (HC2) as a comparator test , 11.Of the 1,600 samples tested by the HPV-Risk assay, 30 (1.9%) samples were considered invalid for PCR amplification and were excluded from further analysis. HPV-Risk assay and HC2 results are shown in n = 1,316) and for women ≥30 years old . Within the total study group, the sensitivity of the HPV-Risk assay for the detection of CIN3+ was 97.6%  and the specificity was 89.0% . For the detection of CIN2+, the sensitivity and specificity were 93.7%  and 91.8% , respectively. For women aged ≥30 years , the sensitivity of the HPV-Risk assay for the detection of CIN3+ was 97.0%  and the specificity was 91.8% . For the detection of CIN2+ among women aged ≥30 years, the sensitivity and specificity of the HPV-Risk assay were 92.9%  and 94.2% , respectively.The absolute sensitivity and specificity of the HPV-Risk assay for the detection of CIN3+ and CIN grade 2 or worse (CIN2+) are shown in P value by the McNemar test [PMcN] = 1.000), but its specificity was significantly higher . Similar results were obtained for the detection of CIN2+ . The performance of the HPV-Risk assay was clinically noninferior to that of HC2 with respect to sensitivity and specificity for the detection of CIN3+  and also for CIN2+ .The absolute sensitivities and specificities of HC2 for the detection of CIN3 and CIN2+ are shown in PMcN = 1.000) and 1.02 , respectively. Relative sensitivity and specificity for the detection of CIN2+ were 0.97  and 1.02 , respectively. In women aged ≥30 years, the performance of the HPV-Risk assay was clinically noninferior to that of HC2 with respect to the sensitivity and specificity for the detection of CIN3+  and CIN2+ .When we performed the analyses for women aged ≥30 years, results similar to those obtained for the total study group were obtained . RelativIn this study, we evaluated the clinical performance of the HPV-Risk assay for the detection of high-grade CIN and compared its performance to that of a clinically validated test (HC2) using samples from the VALGENT-3 panel. The results of this study show that the sensitivity of the HPV-Risk assay for the detection of CIN3+ and CIN2+ was comparable to that of HC2 and that it had a significantly higher specificity, both in the total study group and in women aged ≥30 years. The HPV-Risk assay showed a noninferior clinical performance compared to that of HC2.Clinical validation studies of HPV tests are crucial because the HPV assays that are used for primary cervical cancer screening must ensure an optimal distinction between HPV infections associated with CIN2+/3+ and clinically irrelevant transient HPV infections . The curAn advantage of the HPV-Risk assay, in comparison with other commercially available HPV assays, is that no specific laboratory equipment is needed since the HPV-Risk assay can run on different real-time PCR platforms and is compatible with various collection media and different DNA extraction procedures. Furthermore, the HPV-Risk assay can be performed reliably on self-sampled material, both lavage-based and brush-based material, in addition to physician-taken cervical scrape samples , 12, 13.The strength of this study is the large sample set from the VALGENT-3 framework, which makes it possible to evaluate the clinical performance of various HPV assays compared to that of the validated and accepted comparator test (HC2) in a formalized and uniform manner. In addition, a network of test comparisons, which can later be pooled in multiple-testing meta-analysis, can be developed , 5, 11. 6–In conclusion, the HPV-Risk assay has a high sensitivity and a high specificity for the detection of CIN3+ and CIN2+ and has a demonstrated noninferiority to the clinically validated HC2. Results from this study provide additional evidence that the HPV-Risk assay can be considered clinically validated and therefore can be safely used for primary cervical cancer screening.The VALGENT-3 panel was collated in Slovenia . EthicalCervical scrape samples from the screening population were collected from December 2009 to August 2010, and samples from the enrichment population were collected from January 2014 to May 2015. All samples were collected in ThinPrep PreservCyt medium  according to European Union guidelines . HC2 tes17–Management of the women was based on the results of cytology and HPV testing , as previously described in detail . Women wTC) values for HPV16, HPV18, and/or other HPV types were ≤36. If no HPV signals were obtained and the TC value for the β-globin target was ≤33, samples were considered HPV negative. Samples were considered invalid when the TC value for HPV was >36 and that for β-globin was >33.For testing with the HPV-Risk assay, ThinPrep aliquots were transported to the VU University Medical Center  in June 2016. Extraction of DNA from the ThinPrep aliquots was performed using an automated extraction system  during the period from August to December 2016, with subsequent HPV-Risk assay testing being performed according to the manufacturer's instructions . Accordingly, the time between sample collection and HPV-Risk assay testing was approximately 7 years. The HPV-risk assay targets the E7 region of 15 (probably) high-risk HPV types. Detection is based on hydrolysis probes with 3 spectrally unique fluorescent dyes, providing genotype information for HPV16 and HPV18 and a pooled detection of the 13 other HPV types  . The fouAll HPV testing was performed by investigators blind to the clinical data and cytology findings. Results obtained by testing by both HPV assays were sent to the Unit of Cancer Epidemiology, Scientific Institute of Public Health , where analysis and evaluation of the data were performed.On the basis of the HPV-Risk assay results, the overall prevalence of high-risk HPV infection and the type-specific prevalence of HPV16 and HPV18 infection were assessed in the screening population of 1,300 women. For clinical validation, women with histologically confirmed CIN2, CIN3, and cervical cancer within 48 months after sample collection were classified as having high-grade disease (disease group). Women with two consecutive negative cytology results (at enrollment and at 12 to 48 months of follow-up) were classified as having no evidence of disease (control group). The clinical performance of the HPV-Risk assay for the detection of both CIN3+ (primary endpoint) and CIN2+ (secondary endpoint) was assessed in the total study group and in women aged ≥30 years, and the clinical performance of the HPV-Risk assay was compared to the clinical performance of HC2 as the standard comparator assay. Sensitivity and specificity were calculated from cross-tabulation of the test results with exact 95% confidence intervals (CIs).PMcN value of >0.05 indicated that the sensitivity or specificity of the HPV-Risk assay was not significantly different from that of HC2. Finally, the noninferiority (ni) of the HPV-Risk assay to HC2 was assessed, according to the international validation criteria  and relative specificities (ratios of the specificity of one test to the sensitivity of another test) with 95% CIs were calculated. The McNemar (McN) test was applied to assess differences between matched proportions. A criteria , 23. In


Squamous cell carcinoma of anus (SCCA) is one of the most frequent non-AIDS-defining diseases in HIV patients, mainly in men who have sex with men (MSM), and it is associated with human papillomavirus (HPV) infection.To determine the prevalence of high-risk HPV (HR-HPV) genotypes, premalignant lesions (HSIL) and SCCA in a cohort of HIV-positive MSM; to study the distribution of HPV genotypes according to anal histology results; and to analyze risk factors for this infection.©) was carried out. Logistic regression analysis was performed to identify risk factors for HR-HPV infection.This prospective single-center study was conducted between May 2010 and September 2016. At the study visit, cotton swabs were used to collect anal samples for cytology study in ThinPrep Pap Test liquid medium , and for HPV PCR (Linear Array HPV Genotyping Test). After, high-resolution anoscopy (HRA) , in LSILs (36.9%), and in HSILs (38%). In multivariate analysis, CD4 nadir < 200 cells/μL was the factor associated with infection by HR-HPV .HIV-positive MSM showed a high prevalence of HSIL+ lesions and of infection by oncogenic HPV, which appears to be favored by a deficient immune system. HPV 16 was the most frequently isolated genotype in anal mucosa, regardless of lesion type. SCCA is rare in the general population, with an incidence of around 1.8 cases per 100,000 persons/year. However, it is one of the most frequent non-AIDS-defining neoplasms in HIV patients, especially in MSM, with a very high incidence of up to 144 cases per 100,000 persons/year, although survival rates are similar to those in the general population. For this reason, it is of major interest to establish the prevalence of HPV infection in these patients as well as to study the distribution of HPV genotypes in relation to histological lesion type and identify predictive factors for this infection.Human papillomavirus (HPV) infection is the most prevalent sexually transmitted disease worldwide and is more frequent among females, men who have sex with men (MSM), and immunodepressed patients . Moreover, the sensitivity of anal cytology to screen for high-grade squamous intraepithelial lesion (HSIL)+ lesions  is variable and limited, resulting in an appreciable percentage of under-diagnosed patients, as demonstrated by some studies.–10Data on the distribution of HIV genotypes related to anal mucosa histology have only been published on patients with abnormal cytology and with high-resolution anoscopy (HRA) findings suggestive of dysplastic lesion.The objectives of this study were: to establish the prevalence and distribution of HPV genotypes in a population of HIV-positive MSM according to anal mucosa biopsy results; to determine the prevalence of HSIL+ anal lesions; and to identify the risk factors for HPV infection.22.1An observational, prospective, single-center study was conducted in a cohort of consecutive HIV-positive MSM enrolled between May 2010 and September 2016 in a screening, diagnosis, treatment, and follow-up program for dysplastic anal mucosa lesions in the Infectious Diseases Department of a 3rd-level hospital in Southern Spain. This study was approved by the ethics committee of the hospital. Epidemiological, clinical, and analytical data were gathered and treated following the data protection law in force .Inclusion criteria were: age ≥18 years, self-reported MSM, HIV positivity, and signing of informed consent to study participation. Exclusion criteria were: female sex, self-reported heterosexuality, and history of anal canal neoplasm.At the visit, the variables collected were: age, history of perianal-genital condyloma, number of sexual partners as anal receptor during previous 12 months, utilization of condoms, smoking habit, alcohol consumption in standard drinking units (SDUs), parenteral drug addiction, nationality, educational level, months since HIV diagnosis, and HIV stage according to the CDC classification; months receiving antiretroviral therapy (ART), virological failure ; and receipt of any concomitant treatment, and the presence of other infections, including chronic liver disease from hepatitis B (HBV) or hepatitis C (HCV) virus, syphilis, other sexually transmitted diseases, and latent, treated, or active tuberculosis infection. Analytical data were also gathered on CD4 nadir cell count, CD4 lymphocyte count and viral load at the HIV diagnosis, and on CD4, CD8, and viral load at the study visit. Between 4 and 12 weeks after the study visit, patients underwent HRA with a Carl Zeiss 150 fc© colposcope . After digital rectal exam, a disposable transparent anoscope was introduced for the installation of 5 mL acetic acid and left in place for around 3 minutes, examining the mucosa after its removal; next, 5% Lugol iodine was instilled and left for 1 minute, followed by a 2nd anoscopy. Samples were taken from apparently normal mucosa in the 4 quadrants and from areas with Lugol-negative acetowhite lesions. Biopsies were conducted using an endoscopic retrograde cholangiopancreatography (ERCP) catheter.During the study visit, 2 anal canal samples were taken using 2 cotton swaps impregnated in physiological saline: one for HPV detection and genotyping by qualitative polymerase chain reaction (PCR) (Linear Array HPV Genotyping Test) using a “GeneAmp PCR System 9700” thermocycler ; and the other for the cytology study; both samples were immersed in thin layer liquid medium for application of the thin-layer cytology technique using a ThinPrep Pap Test liquid medium . Both samples were sent to the pathology laboratory, and the results of both techniques were evaluated by a single pathologist (JE). Genotypes 16, 18, 26, 31, 33, 35, 39, 45, 51 to 53, 56, 58, 59, 66, 68, 73, and 82 were considered HR-HPVs, classifying genotypes 39, 45, 59, and 68 as subspecies of HPV 18, and genotypes 31, 33, 35, 52, 58, and 67 as subspecies of HPV 16. Genotypes 6, 11, 34, 40, 42 to 44, 54, 55, 57, 61, 70 to 72, 81, 83, 84, and 89 were considered low-risk HPVs (LR-HPVs). which divides lesions into atypical squamous cells (ASC), ASC-high, low-grade squamous intraepithelial lesion (LSIL), and HSIL. The histology study used the classification of the Lower Anogenital Squamous Terminology (LAST) Standardization Project for HPV, which divides lesions into LSIL (AIN1/condyloma), HSIL , and SCCA. These studies were always conducted by the same pathologist (JE).The cytology study used Bethesda classification,Definition of variables:Abnormal cytology was considered to include ASC of unknown significance (ASCUS), LSIL, or HSIL.In the histology study, “HSIL+ anal lesions” were considered to range from HSIL to SCCA, and “LSIL+ anal lesions” were from LSIL to SCCA.Patients were considered late-presenters when the CD4 cell count was <200 cells/μL at HIV diagnosis.2.2Means, standard deviations, medians, and percentiles were calculated for quantitative variables and absolute frequencies with 95% confidence interval for qualitative variables. Prevalence of HPV, anal mucosa cytology, and histology findings were calculated with 95% confidence interval.t test for independent samples was applied for quantitative variables that were normally distributed and the Mann–Whitney U test for those that were not, while the Pearson chi-square test was used for qualitative variables that were normally distributed and Fisher test for those that were not. The Kappa index was used to analyze the concordance between HR-HPV PCR and biopsy results, considering k < 0.20 poor, 0.21 to 0.40 weak, 0.41 to 0.60 moderate, 0.61 to 0.80 good, and 0.81 to 1.00 very good agreement, following Landis and Koch test.Bivariate tests were conducted to study the relationship between possible risk factors and HR-HPV infection. After evaluation of the distribution of variables using the Kolmogorov–Smirnov test, the Student  The model included variables that were statistically significant in bivariate analyses and those considered clinically relevant, including: age, age >50 years, employment status, number of partners during the previous 12 months, age at first sexual relationship, use of condom, the presence of perianal or genital warts, smoking habit, history of syphilis or other sexually transmitted infection, CD4 nadir, CD4 nadir <200 cells/μL, CD4 count at HIV diagnosis, CD4 count and viral load at the time of the study, receipt of ART, time with ART, and time since HIV diagnosis. A stepwise method was used, with an entry P-value of .05 and exit P-value of .10 at each step. The Hosmer–Lemeshow test was applied to analyze the goodness of fit of the model. SPSS 20.0  was used for the statistical analyses. P < .05 was considered significant in all tests.Multivariate logistic regression analysis was performed, based on Freeman formula [n = 10∗(k + 1)].33.1A total of 319 HIV-positive MSM were recruited between May 2010 and September 2016. The mean age was 36.7 years and the mean CD4 nadir was 361.1 cells/μL; CD4 nadir <200 cells/μL was recorded in 24% of participants. The median time with HIV was 31 months (interquartile range [IQR]: 9–91 months); 86.2% had been on ART for a median of 19 months (IQR: 6–65.5 months), with a mean of CD4 698.5 cells/μL, and only 1.8% were in virological failure. Table 3.2Anal cytology results were normal in 40.3% of participants and showed LSIL in 51.1%, HSIL in 3.1%, and ASCUS in 5.3% , with a median of 2 (1–3); LR-HPV genotypes were detected in 71.1% (95% CI 66–76), with a median of 1 (0–2); and simultaneous infection with low- and high-risk genotypes was detected in 59.5% (95% CI 54–65). The most frequently isolated LR-HPVs in anal mucosa were HPVs 6 (17.7%), 11 (18%), 42 (18%), 61 (9.4%), and 81 (11.3%); and the most frequently isolated HR-HPVs were HPVs 16 (32.9%), 18 (14.1%), 31 (14.5%), 45 (13.5%), 51 (16.4%), 55 (15.7%), 66 (12.9%), and 68 (13.2%)  of the patients with normal cytology were HSIL+ (10 HSIL and 1 SCCA), whereas no HSILs+ were found in the 25 (7.8%) patients with normal cytology and negative HR-HPV PCR Table . Among t3.3The most frequently isolated HPV genotypes were HPVs 6 (16%), 16 27%), 45 (18%), and 55 (16%) in normal biopsies; HPVs 16 (37%), 11 (20%), 42 (19%), and 55 (18%) in LSIL biopsies; and HPVs 6 (24%), 16 (38%), 18 (24%), and 68 (31%) in HSIL biopsies. Figure 7%, 45 . A significant association with HSIL+ lesions was found for genotype 68 , and genotype 53 .Infection by genotype 16 was significantly associated with LSIL+ lesions , retirement , higher CD4 cell count , and viral load <50 copies/mL . Remaining findings are listed in Table In bivariate analyses, the following factors emerged as protective factors against infection by oncogenic virus: older age   in comparison to the former study , or to the different techniques used to detect HPV in anal mucosa.A high prevalence of HPV infection in anal mucosa was observed in this cohort of MSM patients with HIV. HR-HPV genotypes were detected in 81.9% of the patients and LR-HPV genotypes in 71.1%, while both HR-HPV and LR-HPV genotypes were identified in 59.5%. The most frequently isolated genotype was HPV 16, followed by the low-risk genotypes 42, 11, and 6. An elevated prevalence of anal infection by HR- and LR-HPV was previously reported in MSM patients with HIV,20 and this genotype has also been associated with high-grade lesions in seropositive MSM individuals and with other oncogenic viruses, for example, HPV 51.With respect to the distribution and prevalence of genotypes according to histological findings, the most frequent low-risk genotypes were: HPV 6 in patients with histologically normal mucosa, HPVs 11 and 42 in LSIL, and HPV 6 in HSIL. The most frequent high-risk genotypes were HPVs 16, 45, and 55 in normal mucosa, HPVs 16 and 55 in LSIL, and HPVs 16, 18, and 68 in HSIL. The most prevalent genotype in all mucosa types was HPV 16, which was significantly associated with LSIL+ lesions. HPVs 18 and 53, less prevalent than HPV 16, were associated with the presence of HSIL+ lesions. In previous studies of seropositive and seronegative patients, the most prevalent HR-HPV in anal mucosa was genotype 16, regardless of sex or sexual orientation, In addition, the previous study was limited by an important selection bias, given that the distribution of HPV genotypes was only analyzed in individuals with abnormal cytology and pathological HRA findings, with biopsies only being ordered for 94 (19.4%) of the 483 participants.These results are similar to published data on the prevalence of HPV genotypes in HIV-positive MSM according to histological findings, although these showed some differences, even including the absence of LR-HPV 11 and HR-HPV 45, among others.,22 Around one-quarter of HSIL+ lesions were in patients whose anal mucosa cytology had been normal. Hence, if the screening for dysplastic lesions had been based on cytology alone, as recommended by the scientific societies, 10 of the patients with HSIL and the patient with SCCA would have been undiagnosed and untreated. On the other hand, we highlight that no HSIL+ lesions were detected in patients with normal cytology and negative HR-HPV PCR. Anal cytology showed only moderate sensitivity in the present series. The cytology was previously observed to be more useful in cases with high-grade lesions, a larger number of involved quadrants, HIV positivity and CD4 cell count <200 cells/μL. In contrast, the majority of our patients had an excellent immune status, which predicts low sensitivity for cytology results. It therefore appears essential to optimize the screening of precursor and anal cancer lesions in HIV-infected MSM. We therefore propose that HPV PCR is performed in patients with normal cytology, because a negative result would rule out HSL+ lesions in all such cases. Other authors have proposed the utilization of HRA in screening for these lesions, based on the high frequency of false-negative cytology results in screening for intraepithelial lesions.In the present study of HIV-positive MSM with, HSILs were detected in 13.5% and SCCA in 0.3%, that is, 1 out of 7 patients had these lesions, similar to a recent report on seropositive Spanish MSM in the ART era. their lower capacity to clear these viruses, with a higher incidence of anal mucosa infection,26 and faster progression in the severity of precursor lesions. It has been reported that the presence of AIDS is associated with an increased risk of HPV infection-related cancer, finding standardized incidence ratios of 68.6% (95% CI 59.7–78.4) for anal cancer in situ and of 34.6% (95% CI 30.8–38.8) for SSCA. The early diagnosis of HIV patients and minimization of late presentation is therefore vital, because HIV immunosuppression increases the likelihood of acquiring new HPV genotypes and may favor the persistence and reactivation of latent infection by this virus, increasing the risk of these lesions in severely immunodepressed patients.Finally, the sole predictive factor for HR-HPV anal infection in the multiple logistic regression analysis was a CD4 nadir <200 cells/μL. This factor may also explain the higher rate of HPV infection reported in seropositive versus seronegative MSM patients,Study limitations include its single-center design, although it proved possible to enroll all patients in the program, to which all HIV-positive MSM patients in the hospital catchment area were referred. In addition, the results cannot be extrapolated to other types of patient with HIV. The fact that genotyping and histological findings were obtained for all of the patients is a major strength of the present investigation, contrasting with studies in which biopsy was only performed in those with abnormal cytology and with HRA findings of lesions suggestive of dysplasia.In conclusion, there is a high prevalence of oncogenic HPV infection and HSIL+ lesions in the anal mucosa of HIV-positive MSM. This infection appears to be favored by a deficient immune system, underscoring the need to minimize late diagnoses of HIV. Although HPV 16 was the most prevalent genotype in the anal canal of these patients, the presence of HSIL+ lesions was associated with genotypes 53 and 68.The authors thank Mercedes Álvarez Romero of the Department of Infectious Diseases for helping to coordinate the patients, assisting with HRA examinations, and drawing blood samples, and to Marina Gutiérrez and Rodrigo López of the Pathology Department for processing samples.


Diabetes has been shown to increase HPV pathogenicity in different pathological context. Herein, with this newly-established cell line, we uncovered that diabetes promoted CUP-1 xenograft growth in syngeneic db/db mice. In sum, we successfully established a HPV-16 E7 transformed mouse epithelial cell line, which allowed subsequent studies of co-factors in multistep HPV carcinogenesis in an immunocompetent host. More importantly, this study is the very first to demonstrate the promoting effect of diabetes on HPV-associated carcinogenesis in vivo, implicating the importance of cancer surveillance in diabetic environment.Epidemiological evidence supports that infection with high-risk types of human papillomavirus (HPV) can interact with host and environmental risk factors to contribute to the development of cervical, oropharyngeal, and other anogenital cancers. In this study, we established a mouse epithelial cancer cell line, designated as Chinese University Papillomavirus-1 (CUP-1), from C57BL/KsJ mice through persistent expression of HPV-16 E7 oncogene. After continuous culturing of up to 200 days with over 60 passages, we showed that CUP-1 became an immortalized and transformed epithelial cell line with continuous E7 expression and persistent reduction of retinoblastoma protein (a known target of E7). This model allowed  Persistent infection with high-risk types of human papillomavirus (HPV) is strongly associated with the development of a wide range of malignancies, including cervical, oropharyngeal, anal, vaginal, vulvar and penile cancers. The pathogenic mechanism of HPV has been extensively studied and it is well established that E6 and E7 are its major oncogenes. Interestingly, though infection of high risk HPV types is common, only a small proportion of infected subjects eventually develop cancer . Undoubtin vivo.Diabetes has been shown to increase HPV pathogenicity in different diseases. For instance, patients with type-2 diabetes (T2D) are at increased risk for HPV-associated cancers, including cervical cancer and head and neck cancer . In addiCell lines represent a valuable tool for studying cellular and molecular responses to a defined condition. They are also essential in establishing xenograft and autograft models to delineate the influence of different host factors (e.g. diabetes) on tumorigenesis in animals. Though ample of HPV-associated human cell lines are available, there is currently very few HPV-related murine epithelial cell lines in market . The mosdb/+Leprdb diabetic (db/db) mice and non-diabetic control littermates (m+/db) were purchased from Jackson laboratory . All animal experimental protocols were reviewed and approved by the Animal Experimentation Ethics Committee of The Chinese University of Hong Kong. The animal study was approved by the Animal Research Ethics Committee of Hong Kong and performed in accordance to the Animals (Control of Experiments) Ordinance . Ketamine/xylazine combinations were administered intraperitoneally for anesthesia. Mice were sacrificed using carbon dioxide overdose. Animals were housed under specific pathogen-free conditions at the Laboratory Animal Service Centre (LASEC) according to the approved protocols and guidelines of the Animal Experimentation Ethics Committee (AEEC) of the Chinese University of Hong Kong.C57BL/KsJ mice, male C57BL/KsJ-+Lepr2. Cells were allowed to grow for 48–72 hours to approximately 50% confluency. The HPV-16 E7-expressing construct pJ4Ω-HPV16E7 was transfected into BMK cells by calcium phosphate precipitation. H-ras was co-transfected to augment the immortalization and transforming process as previously described  + S, where S is the initial population doubling. Cumulative population doubling was then plotted against days in culture to study CUP-1 growth rate. Population doubling time was also calculated according to the equation: log2 .Upon subculturing, CUP-1 cells were plated at an initial density of 2 × 10™ DNase (Thermo Fisher Scientific) to remove genomic DNA carryover. RNA was reverse transcribed to cDNA by poly(dT) primers using Superscript III reverse transcriptase (Thermo Fisher Scientific). HPV-16 E7 levels was determined by qRT-PCR assays using 2x TaqMan universal mix (Thermo Fisher Scientific). The primers used for amplifying HPV-16 E7 were: 5’-ACAAGCAGAACCGGACAGAG-3’ (forward) and 5’-GCCCATYAACAGGTCTTCCA-3’ (reverse). Real-time monitoring of fluorescence was performed on Veriti™ 96-Well Thermal Cycler (Thermo Fisher Scientific) with cycling condition conducted at 50°C for 2 minutes, 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 sec and 55°C for 1 minute. Endogenous level of housekeeping β-actin was simultaneously quantified for normalization using 5’-GCACGGATCGTCACCAACT -3’ (forward) and 5’-CATCTTCTCGCGGTGGCCT-3’ (reverse) primers.Cell lysate were harvested for total RNA extraction at every 10 passages from passage 10 (P10) to passage 60 (P60) to ensure continuous E7 expression throughout the whole culturing period. Total RNA was extracted from cell lysate using TRIzol  and treated with DNA-FreeCell lysate were harvested for total protein extraction every 10 passages from P10 –P60. CUP-1 cells were lysed with ice-cold RIPA buffer supplemented with protease inhibitors  and lysates were quantitated using BCA protein assay kit (Thermo Fisher Scientific) according to the manufacturer’s procedures. Twenty microgram of total protein was resolved on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membrane. The membrane was then blocked with either 5% dry milk or 5% bovine serum albumin (BSA) in Tris buffered saline with Tween20 (TBST) and probed with specific antibodies as follows: E7 , pan-keratin , S-100A4  and β-actin . Antigen complexes were visualized by ECL chemiluminescence .CUP-1 cells plated onto glass coverslips were fixed with methanol and blocked with BSA. Cells were then probed with specific antibodies against pan-keratin  and S-100A4 , followed by Alexa Fluor-conjugated secondary antibodies (Thermo Fisher Scientific). Finally, cell nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI). Slides were examined using fluorescent microscopy  and representative images were captured.2) × 0.5236, where “a” refers to the largest dimension of the tumor and “b” is the perpendicular diameter [3. The tumors were paraffin sectioned for hematoxylin and eosin (H&E) staining.Ten million CUP-1 cells were resuspended in 150 μL PBS and injected subcutaneously over the flank of 10-week-old nude mice, diabetic db/db mice and their non-diabetic (m+/db) littermates (10 mice per group). Mice body weight and tumor size were monitored and recorded every other day during the study period. Tumor volume was estimated using the formula  to late passages (Passage 60) were confirmed by qRT-PCR . ContinuProliferation profile of CUP-1 revealed that it did not show any sign of crisis upon transfection and grew continuously with a steady rate over the whole study period. CUP-1 cells did not show any sign of senescence up to 200 days of culture with over 60 passages , indicatMorphologically, CUP-1 cells displayed a spindle and dendritic-like shape in tissue culture vessel . They deTo avoid fibroblast contamination, only colonies presented with epithelial morphology were selected for subculture under phase contrast microscopy. The cell type of CUP-1 was further affirmed by Western blotting and immunofluorescence using pan-keratin as a specific epithelial marker and S100A4 as a fibroblast marker. Results showed that CUP-1 cells expressed high levels of pan-keratin in cytoplasm , whilst The most well-characterized E7 properties which contributes to its oncogenic action is its ability to degrade pRb and related pocket proteins. Consistent with E7 expression, Western blotting showed that pRB was degraded in CUP-1 cells both in P28 and P60 , indicat3). Mice were therefore sacrificed by carbon dioxide asphyxiation and tumors were excised. The dissected tumors appeared to be irregular in shape and had blood vessels on the surface .To study the effects of diabetes on HPV-16 E7-mediated tumorigenesis, CUP-1 cells were injected subcutaneously over the scruff of diabetic db/db mice and non-diabetic m+/db littermates. Similar with nude mice, visible tumor mass appeared on day 3 upon subcutaneous inoculation of CUP-1 cells. Interestingly, growth kinetics of CUP-1 xenograft differed significantly between the two groups. From day 3 to 7 after CUP-1 cell inoculation, there was a 6-fold increase in tumor volume in db/db mice. In contrast, tumor volume increased only 1.5-fold in non-diabetic m+/db mice from day 3 to 7 and started to regress on day 7 . CUP-1 tFurther histological examination of excised tumours  showed tIn this study, we successfully established a murine epithelial cell line, designated as CUP-1 by introduction of HPV-16 E7. CUP-1 have been continuously cultured for up to 200 days with over 60 passages, indicating that it gained infinite lifespan. Stable and continuous E7 expression from early to late passages was confirmed by qRT-PCR and Western blot. Unlike human keratinocytes transformed with HPV-16 E6/E7 , CUP-1 cAfter reaching full confluency, CUP-1 cells lost cell-cell contact inhibition and were able to form foci. This is in agreement with previous reports, in which the same phenomenon was also observed and is likely to be contributed by E7 expression in association with p600 to resist anoikis –15. Funcet al [Though the pathogenic mechanism of HPV has been extensively studied, questions remain over its interaction with environmental and host risk factors in tumorigenesis. Several studies have suggested that type 2 diabetes is associated with increased risk of cervical cancer and head and neck cancer which are related to HPV infection [et al  reportedet al  and Mexiet al .Using CUP-1, we successfully delineated the promoting effects of diabetes to HPV-16 E7-mediated tumorigenesis in db/db mice. Our results implicated the importance of cancer surveillance in this patient subgroup. More importantly, this implies the possibility of anti-diabetic drug as a therapeutic option for HPV-related cancers in diabetes patients. The underlying mechanism of this observation, however, awaits further investigation.in vivo examination of the involvements of these co-factors on HPV, in particular E7, -mediated tumorigenesis. The advantage of using CUP-1 xenograft model instead of traditional nude mice xenograft model is that it allows in vivo studies in an immunocompetent host and can take into account the effect of the host immune system.Similarly, CUP-1 could be used to study the effects of other host or environment co-factors to HPV-mediated tumorigenesis in any host with the same C57 genetic background. For instances, several epidemiologic studies have pinpointed the effects of environmental pollutants generated by cigarette smoking , heavy tOur present study successfully established a murine epithelial cell line, CUP-1 transformed with HPV-16 E7 and with functional E7 properties. With CUP-1, we successfully uncovered the promoting effects of diabetes in HPV-mediated tumorigenesis. This cell line represents an important tool for studying the effects of co-factors in HPV-mediated carcinogenesis, and also provides a translational platform for preclinical screening of novel therapeutics.


Carcinoma of the vulva is usually regarded as a disease of older women, with the typical age of 65–85 years. There are a limited number of reports of vulvar cancer cases younger than 30 years. These patients have usually risk factors such as human papillomavirus (HPV) infection and immunosuppression. Herein, we present a case of invasive squamous vulvar cancer in an 18-year-old patient without any risk factor. Vulvar radical local excision and bilateral inguinal sentinel lymph node biopsies were performed. The clitoris was preserved during the surgery. Patient did not receive adjuvant therapy. Follow-up after 12 months of the disease showed no evidence of disease. Vulvar carcinoma in very young women may develop without any predisposing factor. Early detection will result in better survival. So, there should be a high index of suspicion when a vulvar lesion is seen, even if the patient falls below the typical age range and does not carry any well-known risk factors such as HPV infection and immunodeficiency. Invasive squamous cell carcinoma (ISCC) of the vulva is a rare disease and reflects 3–5% of all cancers of the female genital tract. Characteristically, it occurs in older women and is rarely seen in women younger than 35 years. However, in recent years, the incidence has been increasing in women younger than 50 years of age . Vulvar Some studies have shown that hypertension, obesity, diabetes mellitus, and nulliparity are risk factors for vulvar cancer . Immune A persistent, itchy, ulcerous lesion may suggest vulvar cancer, but the only way to be certain that cancer is present is to do a vulvar biopsy.Herein, we present a very rare case of HPV-negative ISSC of the vulva in an adolescent without any risk factors.An 18-year-old patient presented with a pruritic, painful lesion on the vulva. There was no history of systemic disease, operation, or tobacco-alcohol use. She reported that she had been sexually active for one year. Physical examination revealed a hyperpigmented and erosive lesion, approximately 2 cm in diameter, centrally localized in the vulva . InguinaWritten informed consent was obtained from the patient for publication of the case.The ISCC of the vulva is very rarely seen in patients under 35. The typical patients are 65–90 years old . Recent HPV-positive tumors are expected to occur more often in younger patients than HPV-negative tumors . On the A SEER data study comparing vulvar cancer in younger versus older women found that, in younger women, disease is diagnosed at early stage and more likely to have surgical treatment without any adjuvant treatment . In our Patients younger than 45 years with vulvar cancer tend to have early stage disease and a better prognosis . Our patIn conclusion, there should be a high index of suspicion when a vulvar lesion is seen, even if the patient falls below the typical age range and does not carry any well-known risk factors such as HPV infection and immunodeficiency. A growing number of reports and studies on the ISCC of the vulva in young women are required.


Inadequate facilities and hygiene at slaughterhouses can result in contamination of meat and occupational hazards to workers. The objectives of this study were to assess current conditions in slaughterhouses in western Kenya and the knowledge, and practices of the slaughterhouse workers toward hygiene and sanitation.Between February and October 2012 all consenting slaughterhouses in the study area were recruited. A standardised questionnaire relating to facilities and practices in the slaughterhouse was administered to the foreperson at each site. A second questionnaire was used to capture individual slaughterhouse workers’ knowledge, practices and recent health events.A total of 738 slaughterhouse workers from 142 slaughterhouses completed questionnaires. Many slaughterhouses had poor infrastructure, 65% (95% CI 63–67%) had a roof, cement floor and walls, 60% (95% CI 57–62%) had a toilet and 20% (95% CI 18–22%) had hand-washing facilities. The meat inspector visited 90% (95% CI 92–95%) of slaughterhouses but antemortem inspection was practiced at only 7% (95% CI 6–8%). Nine percent (95% CI 7–10%) of slaughterhouses slaughtered sick animals. Only half of workers wore personal protective clothing - 53% (95% CI 51–55%) wore protective coats and 49% (95% CI 46–51%) wore rubber boots. Knowledge of zoonotic disease was low with only 31% (95% CI 29–33%) of workers aware that disease could be transmitted from animals.The current working conditions in slaughterhouses in western Kenya are not in line with the recommendations of the Meat Control Act of Kenya. Current facilities and practices may increase occupational exposure to disease or injury and contaminated meat may enter the consumer market. The findings of this study could enable the development of appropriate interventions to minimise public health risks. Initially, improvements need to be made to facilities and practices to improve worker safety and reduce the risk of food contamination. Simultaneously, training programmes should target workers and inspectors to improve awareness of the risks. In addition, education of health care workers should highlight the increased risks of injury and disease in slaughterhouse workers. Finally, enhanced surveillance, targeting slaughterhouse workers could be used to detect disease outbreaks. This “One Health” approach to disease surveillance is likely to benefit workers, producers and consumers.The online version of this article (doi:10.1186/s12889-016-3923-y) contains supplementary material, which is available to authorized users. Slaughterhouses are defined as places where animals are slaughtered for food . The devIn developing countries slaughter facilities vary from large industrial meat processing facilities in cities to small unregulated facilities in rural areas . This vaRegulation of the slaughter industry aims to improve hygiene and reduce the contamination of meat and spread of disease, as well as protecting workers from occupational health hazards . The meaThere are three types of slaughterhouses in Kenya depending on the size and whether the meat is for local consumption or transport out of the community. Slaughterhouses are further subdivided into ruminant or pig slaughterhouses, out of respect for the Muslim community. Category A slaughterhouses process over 40 bovines or greater than 8 pigs per day and are permitted to supply products all over Kenya. Category B slaughterhouses process 6–39 bovines or 1–7 pigs per day and are permitted to supply products up to 50 km from the slaughterhouse. Category C slaughterhouses process less than 5 bovines per day or less than 6 pigs and can only supply products to the local population centres.Changes to slaughterhouses are now being implemented across Kenya to varying degrees. Introduction of the new regulations is slow in rural areas because abrupt enforcement may result in an increase in the informal market as local meat handlers are unwilling to meet the costs of the improved facilities , 8.The majority of slaughterhouses in rural areas are classified under the new Meat Control Act as Category C, more commonly referred to as slaughter slabs. These facilities are privately owned and rented to butchers who employ their own team of slaughter workers , 8. TherThere are multiple failings in the slaughter process that result in meat contamination and allow the transmission of pathogens: inadequate infrastructure, poor hygiene, lack of ante and post mortem inspection, and inadequate training , 11. PreThere are no published reports describing the standards in slaughterhouses in western Kenya. This information is required to assess the potential risks to workers and consumers from these facilities and can be used to assess the impact of improvements. The present study reports on the facilities and hygiene practices in slaughterhouses in western Kenya as documented in 2012.The study was conducted in the Lake Victoria Basin region of western Kenya on the border with Uganda. The study area was a 45 km radius from Busia town, where the project laboratory is located Fig. . It is aA census of slaughterhouses was performed between May 2011 and January 2012. The location of slaughterhouses in the study area was obtained from the former District Veterinary Officers (now County Directors of Veterinary Services) who had oversight over meat inspection. Data collection was conducted between February and October 2012.All slaughterhouses in the study area were visited 3–6 days before data collection. The purpose of this visit was to explain the project objectives and give the necessary information regarding the study so that participants could give informed consent. Participants were informed as a group by a project enumerator that they would be asked questions about their work and their health and samples would be tested for a range of diseases; it was emphasised that participation was voluntary. The biological sampling and data collection process was explained verbally. Workers were informed that they would receive a confidential report of any diagnoses and free treatment for any diagnosed condition.On the day of data collection, informed consent was obtained from all participants individually. The enumerator outlined the objectives of the study as well as the questionnaire and biological sampling procedures. Participants were required to sign or apply a thumbprint to duplicate consent forms—one was retained and the other given to the participant. Inclusion criterion specified all workers, aged over 18 years and present at the slaughterhouse on the day of sampling. In slaughterhouses with 12 workers or less all willing participants were recruited. In slaughterhouses with greater than 12 workers a random selection of 12 willing participants from the workers present on the day were sampled. This restriction was necessary due to the time required to collect data each day, and also took into account that the slaughterhouses were professional environments where income earned by workers related to time worked.Exclusion criteria included third trimester pregnancy (self reported), severe anaemia , being under the age of eighteen (self-reported), severe inebriation (determined if the participant was unable to converse clearly without slurred speech or confusion), aggression toward the project enumerator, and being over eighty-five years (self-reported).A 114-item individual questionnaire  Personal digital assistant (PDA) using Pendragon Forms 5.1 . Microsoft® Access databases were used to manage data.http://www.ilri.org/gis).Slaughterhouses were georeferenced using a handheld Global Positioning System (GPS) device (Garmin eTrex®). The locations of slaughterhouses were mapped using ArcGIS™ version 9.1 and version 10.2.2 . Base layers were provided by the ILRI geographical information systems unit  was used.Variables were analysed for independence using the There were 180 slaughterhouses in the study area when the study began in May 2011. Twenty-four slaughterhouses were closed by the former District Veterinary Officers (now County Directors of Veterinary Services) between May 2011 and January 2012 for non-compliance with regulations. From the remaining 156, 142 slaughterhouses (91%) agreed to participate in the study with fourteen (9%) slaughterhouses refusing to participate. This included 4/57 (7%) cattle and 10/68 (15%) pig slaughterhouses. Although no specific reason was given for refusal, the study team surmised that fear of recriminations from the Department of Veterinary Services was an important factor.Figure Of the 142 slaughterhouses recruited in the study, 31 were mixed ruminant , 53 were cattle only and 58 were pig only. The total employment at these slaughterhouses was 1005 workers. Questionnaires were administered to 738 (73.4%) workers at all 142 slaughterhouses. Slaughtering was observed at 84 slaughterhouses whilst interviews were being conducted.The results are presented in two parts. The questionnaires completed by the foreperson together with the observations by the study team form the results regarding the slaughterhouse infrastructure and practices. The second questionnaires administered to the workers and the clinical assessment of the health of those workers form the results regarding worker practices, knowledge, and health.Only two slaughterhouses were Category B slaughterhouses. The remainder were Category C or informal. It was not possible to determine which slaughterhouses were working without authority and would therefore be classified as informal slaughterhouses, as forepersons were unwilling to admit to working outside the regulations.Despite the vast majority of slaughterhouses being classed as Category C, only 26% (95% CI 24–28) restricted meat selling within the local village, with the remainder transporting meat outside the immediate area. Figures Table 2 = 21.53, df = 2, p < 0.001).Both mixed ruminant slaughterhouses and cattle only slaughterhouses had better infrastructure than pig slaughterhouses. Ninety percent of mixed ruminant slaughterhouses and 75% (95% CI 72–79%) of cattle only slaughterhouses had a roof, cement floor and solid walls compared with 45% (95% CI 40–50%) of pig slaughterhouses . The viscera were washed outside the slaughterhouse on a concrete slab in all but one slaughterhouse, where there was a specific room inside the slaughterhouse. Only one mixed ruminant slaughterhouse stunned cattle before slaughter. The remaining 141 slaughterhouses cut the throat without prior stunning.Less than half of slaughterhouses reported that workers wore personal protective clothing. The forepersons in 32% (95% CI 29–34%) of slaughterhouses reported that workers wore protective coats; and in 34% (95% CI 31–36%) that workers wore rubber boots. This report was supported by the observational data that workers in 27% (95% CI 21–34%) of slaughterhouses wore lab coats and workers in 22% (95% CI 17–28%) of slaughterhouses wore rubber boots.2 = 13.38, df = 2 p = 0.001). Workers in mixed ruminant slaughterhouses (52%) and cattle slaughterhouses  were more likely to wear boots than workers in pig slaughterhouses  .Very few slaughterhouses provided protective equipment for workers, with workers providing their own protective coats in 78% (95% CI 73–84%) of slaughterhouses and workers providing their own rubber boots in 84% (95% CI 78–89%) of slaughterhouses. No workers were observed wearing gloves. Workers in mixed ruminant (55%) and cattle slaughterhouses  were more likely to wear protective coats than workers in pig slaughterhouses  . Eating was observed in 18% (95% CI 12–24%) of slaughterhouses.Soap was reported to be provided at 64% (95% CI 62–67%) of slaughterhouses but was only observed in 21% (95% CI 15–27%). Soap was observed in 16% (95% CI 7–25%) of cattle only and 12% (95% CI 2–22%) of pig only slaughterhouses. This was significantly less than in mixed ruminant slaughterhouses where soap was observed 50% (95% CI 44–56%) of the time (XNinety percent (95% CI 92–95%) of slaughterhouses reported that the meat inspector visited every day. However the meat inspector was seen at only 53% (95% CI 45–61%) of slaughterhouses during the course of data collection. Workers explained that the meat inspector may visit the butchery to inspect the meat if he/she was too late arriving and did not see the meat at the slaughterhouse. Antemortem inspection was reported at 7% (95% CI 6–8%) and observed at 6% (95% CI 3–10%) of slaughterhouses. Nine percent (95% CI 7–10%) of slaughterhouses reported slaughtering animals they identified as being sick/ill/unhealthy.The slaughterhouse workers ranged in age from 18–82 years with a mean age of 39 (95% CI 39–40). The mean time of employment as a slaughterhouse worker was 9.35 years (95% CI 9–10) with a range of 1 month to 59 years. The mean days worked per week were 4.9 with a mean work day of 2.5 h.The different jobs in the slaughterhouses included slaughtermen ; flayers ; cleaners ; the person who cleaned the offal ; and foreperson/owner . The slaughterman is responsible for cutting the animals throats in mixed ruminant and cattle slaughterhouses. The slaughterman is a practicing Muslim so that all meat products are Halal. Flayers are responsible for skinning and sectioning the carcass. There is not an official slaughterman in pig slaughterhouses. The same worker that cuts the throat also sections the carcass and in this study these people are classified as flayers. In ruminant and cattle slaughterhouses a specific worker is responsible for washing the offal to remove gross faecal contamination as offals are sold for consumption. Pig offals are not typically consumed in the study area, and these were not observed to be processed.Ninety-seven percent (95% CI 96–97%) of slaughterhouse workers were men. Seventy-four percent (95% CI 73–76%) of workers had primary level education. Eighty-two percent (95% CI 80–83%) of workers had a second occupation, predominantly as butchers  or farmers . Seventy-two percent (95% CI 70–74%) of workers had contact with livestock outside of work. The majority of workers had contact with poultry  and cattle . Contact with other animals included goats ; sheep ; and pigs .2 = 79.82, df = 2 p < 0.001). Forty-nine percent (95% CI 46–51%) of workers reported wearing rubber boots. Workers at ruminant slaughterhouses  and cattle slaughterhouses  were more likely to wear rubber boots compared with pig slaughterhouse workers   (Table Table Almost one quarter of workers smoked daily (23% 95% CI 21–25%) and 32% (95% CI 30–34%) workers reported consuming alcohol daily. The study team observed that 11% (95% CI 10–12%) of workers appeared to be intoxicated during work. Twenty-one percent (95% CI 21–23%) of workers ate at work. At pig slaughterhouses, workers were observed to consume pieces of the carcass that were cooked over an open fire. At large mixed ruminant slaughterhouses, there was someone preparing and selling tea to workers. Twenty-four percent (95% CI 22–26%) of workers reported defecating in the open regularly.Ninety-six percent (95% CI 95–96%) of slaughterhouse workers reported seeing the meat inspector every day Table . HoweverThirty-one percent (95% CI 29–33%) of the 738 slaughterhouse workers knew that disease could be transmitted from animals (Table Eighteen percent (95% CI 16–19%) of workers reported being unwell in the past 3 months. Commonly reported symptoms included: fever , backache , headache , joint pain , cough , and skin infections .Workers reported previous diagnoses of tuberculosis . Workers reported the following illnesses in the past 12 months: malaria ; typhoid ; respiratory illness ; and gastrointestinal illness . Twenty-five percent (95% CI 23–27%) of workers reported being injured at work at least once a month and 8% (95% CI 7–9%) had a wound at the time of interview.This study reports the conditions in slaughterhouses in western Kenya with respect to infrastructure, hygiene, meat inspection, and the knowledge and health of workers. The most notable findings were the lack of facilities to ensure adequate meat hygiene. Ideally the floor of the slaughterhouse should be hard concrete and impervious, to reduce dirt in the slaughterhouse and allow drainage and ease of cleaning . SimilarIdeally, there should be a division in the slaughterhouse between the dirty  and clean (eviscerating and splitting) operations to prevent carcass contamination . All slaSalmonella sp [International guidelines specify that hot and cold water should be readily accessible for cleaning, and that equipment and workers’ hands should be washed with soap and hot water , 30. Thinella sp –33. The Only 60% of slaughterhouses had access to a toilet, with 24% of workers admitting to regularly defecating in the open. This behaviour may promote the persistence of zoonotic diseases such as cysticercosis . The preThe purpose of protective clothing within the slaughterhouse is primarily to protect the meat product from contamination but has also been shown to be protect meat handlers against directly transmitted zoonoses including leptospirosis and brucellosis , 30, 31.Many of the activities in the slaughterhouses occurred without a meat inspector present, which is in violation of the stipulations in the Meat Control Act . In deveA lack of knowledge regarding the process of meat contamination is the biggest hindrance to improving conditions in the meat industry . This stSlaughterhouse workers have been identified in occupational health studies for elevated risk of injury, particularly to the upper extremities (mostly due to lacerations) and back injuries , 40. BotSeveral potential risk factors that have been associated with zoonotic disease exposure in slaughterhouse workers in previous studies were observed in the study population. These included cutting animals throats which has been associated with RVF and brucellosis exposure , 43 and Ill workers are a risk to meat contamination and should self report . HoweverThe majority of workers own livestock and a number of workers have a second occupation, predominantly in other aspects of the meat production industry, including as farmers or butchers, and are therefore exposed to animals and meat products outside the slaughterhouse. This increased exposure may act as a source of infection or a potential for dissemination since these activities are independently associated with disease exposure.Streptococcus suis, Methicillin-resistant Staphylococcus aureus and Hepatitis E [During the course of the study in 2012, some changes were noticed in slaughterhouses as the new Act was brought into effect. The changes were initially focused on mixed ruminant and cattle slaughterhouses, which may explain the significant difference between mixed ruminant/cattle and pig slaughterhouses documented in this study through the chi squared analysis. The lack of regulation of the pig slaughterhouses is likely to have significant public health impacts in this region particularly regarding food-borne zoonotic diseases such as cysticercosis. The prevalence of cysticercosis in pigs at slaughter in this region has been reported to be 37.6%, of which none were detected by regular meat inspection . Potential emerging zoonotic diseases risks to pig slaughterhouse workers include atitis E –46.Meat inspectors and Sub-county Veterinary Officers informed the study team that each slaughterhouse was urged to adopt one change in the first year or face closure. They were concerned that strictly enforcing the new standards would lead to a deficit in the meat industry in the region. A number of facilities, which did not make efforts to adopt the changes were closed through 2012. The informal meat industry was very difficult to quantify as slaughterhouse owners and butchers were unwilling to admit to slaughtering without authority as they feared prosecution from the public health department. This fear may explain the number of slaughterhouses that declined to participate. Despite only 2 slaughterhouses being classified as category B, a large number of slaughterhouses were trading meat beyond the local village where the slaughter was conducted which contravenes the regulations of the Meat Control Act and may allow the dissemination of disease.Fear of stigma associated with certain disease diagnoses such as HIV may have influenced participation in the study. The incentive of free treatment for diagnosed conditions and the high response rate (73.4%) should reduce response bias. In addition the questionnaires were pretested to identify any problems with the data collection tools before the study and observational data regarding facilities at the slaughterhouses was collected to confirm that responses to the foreman questionnaire were accurate.The findings of this study are similar to reports from other countries in East Africa regarding lack of facilities, hygiene, and inadequate meat inspection , 18. TheSlaughterhouse workers may act as sentinels for disease outbreaks in animals and people , 47. ThiThis study contributes to understanding the current situation in the meat industry in western Kenya. The study documents the conditions in slaughterhouses in western Kenya during the implementation of the revised Meat Control Act, 2012 and gives an indication where improvements need to be made. None of the slaughterhouses visited complied with the published regulations at the time of the study, with many falling far below a minimum standard. The infrastructure at the majority of slaughterhouses did not meet the guidelines with many slaughterhouses lacking basic structural requirements such as a roof and sanitation facilities such as a toilet or running water.The results of this study are an important contribution to understanding and regulating the meat industry in Kenya and it is likely that they can be generalised to rural areas in other resource limited settings. Improvements need to be made to facilities and practices in all slaughterhouses. In the initial stages, training is recommended to improve awareness for workers, managers, and inspectors of the risks of meat contamination and methods to reduce it. Secondly, improvement of facilities must be implemented with closure of sub-standard facilities and focusing resources on fewer facilities to improve meat hygiene in this resource-limited setting. These enforcements would need to take into consideration the effects on the price of meat and nutrition in the region and the risk of pushing meat producers into the informal market.