For poorly understood reasons, invasive cervical cancer (ICC) incidence and mortality rates are higher in women of African descent. Oncogenic human papillomavirus (HPV) genotypes distribution may vary between European American (EA) and African-American (AA) women and may contribute to differences in ICC incidence. The current study aimed at disentangling differences in HPV distribution among AA and EA women.Five-hundred and seventy-two women were enrolled at the time of colposcopic evaluation following an abnormal liquid-based cytology screen. HPV infections were detected using HPV linear array, and chi-squared tests and linear regression models were used to compare HPV genotypes across racial/ethnic groups by CIN status.p = 0.03) when all women were considered. This association remained unchanged when only women with CIN2/3 lesions were examined . The most frequent high-risk HPV genotypes detected among EAs were 16, 18, 56, 39, and 66, while HPV genotypes 33, 35, 45, 58, and 68 were the most frequent ones detected in AAs.Of the 572 participants, 494 (86 %) had detectable HPV; 245 (43 %) had no CIN lesion, 239 (42 %) had CIN1, and 88 (15 %) had CIN2/3. Seventy-three percent of all women were infected with multiple HPV genotypes. After adjusting for race, age, parity, income, oral contraception use, and current smoking, AAs were two times less likely to harbor HPV 16/18 (OR 0.48, 95 % CI 0.21–0.94, Our data suggest that while HPV 16/18 are the most common genotypes among EA women with CIN, AAs may harbor different genotypes. In 2009, there were an estimated 34,788 new cases of human papillomavirus (HPV)-associated cancers in the USA overall, of which 61 % were among women  and 39 % among men  .Although AAs and Hispanics have lower incidence rates for the more common cancer sites, they have the highest incidence of ICC of any ethnic group in the USA. Currently, low-grade squamous intraepithelial lesions (LSILs) and atypical squamous cells of undetermined significance (ASCUS) cases are classified in ‘high-risk’ HPV-positive or HPV-negative groups. Although the general consensus is that HR-HPV genotypes 16 and 18 causes 70 % of ICC cases and 50 % of CIN2–3, HPV-genotype distribution of high-risk HPV types may vary by age , which i®). Inclusion criteria were, a visit to one of 10 colposcopy clinic following an abnormal Pap test, of at least LSIL, age 18 years or older, and English or Spanish speaking. Questionnaires were written in English, and a Spanish-speaking coordinator assisted and interpreted the content to Spanish-speaking study participants. Also, both self- and interviewer-administered instruments were identical in content. Women who did not intend to receive follow-up care in one of the 10 DPC clinics or moved out of the area for other reasons were excluded. Of the 1,657 women with cytological abnormalities approached in the colposcopy clinic, 1,303 were enrolled, a response rate of 79 %. These analyses are restricted to the first 572 in whom data on race/ethnic, HPV infection, and CIN status were available. Participants with detected HPV genotypes were comparable to those of the entire cohort not included in this analysis with respect to age (p = 0.83), HPV infection (p = 0.87), yearly income (p = 0.36), marital status (p = 0.44), cigarette smoking (p = 0.62), and prior HPV vaccination (p = 0.57). This study was approved by Duke University School of Medicine Institutional Review Board, and all study participants signed an informed consent explaining the nature of the study.Study participants were recruited from all 10 Duke University and Duke Primary Care (DPC) clinics in Durham County, North Carolina, during 2010–2012. All clinics used the same study protocol and the Duke University Pathology Laboratory for cytology and histological evaluation. All study participants were initially screened for cervical abnormalities with the Thin-Prep liquid-based cytology test , was abstracted from the medical records.®, Malborough, MA, USA). Colposcopy-directed biopsies were also obtained from the lesions. All specimens were tested for adequacy (KG) using the 2002 ASCCP guidelines. The specimens were stored at 4 °C prior to HPV testing.At the enrollment visit, the colposcopist co-investigator or designee obtained a cervical specimen using a plastic spatula and cytobrush and suspended it in the Thin-Prep specimen vial containing a proprietary media with at least 50 % methanol  laboratory.Testing for 37 HPV-DNA subtypes was performed by Dr. Gravitt’s laboratory at Johns Hopkins University, as previously described . BrieflyWomen were classified as having either single or multiple infections. Of the first 691 women for whom HPV genotyping has been completed, 572 also had covariate data including race/ethnicity and age. Pearson’s chi-squared and Fisher’s exact tests were used to compare women with no evidence of CIN and those with CIN . We computed the proportion of single and multiple HPV infections and grouped them according to potential oncogenicity , 20 by Cp < 0.0001). Most women with CIN1 (70 %) and CIN2 (61 %) were younger (aged 20–29 years), whereas those with CIN3 were 30 years or older (p < 0.0001). Those with CIN were more likely to be infected with at least one of the 31 HPV genotypes detected, which included low-risk types (p = 0.06), and to be unmarried (p = 0.001). Only 73 (13 %) study participants reported being vaccinated against HPV, and most (63 %) completed the vaccine doses between the ages of 18–29 years old (p = 0.08). Most (92 %) women with CIN reported having their sexual intercourse at age 16 years or younger. Seventy-three percent of women with CIN reported being current cigarette smokers, compared with 15 % of women without visible CIN lesions (p = 0.25).Of 572 participants, 494 (72 %) were HPV-positive; 245 (43 %) had no visible CIN, 239 (42 %) had CIN1, and 88 (15 %) had CIN2/3 , 31, 33, 35, 51 and 52 (in single infections) and 39 and 52 (in multiple infections) were the most common. In CIN3 lesions, the prevalence of HPV genotype 16 was 36 %, followed by 31 (13 %), 33 (9 %), 35 (9 %), 51 (9 %), and 52 (9 %) in single infections, and HPV 16, 52, 39, 18, 35, 45, 59, and 66 in multiple infections  women harbored multiple HPV genotypes (Table p = 0.03). Adjusting for age, race, income, parity, oral contraceptive use, and current smoking did not materially change these associations , p = 0.03) (Table p = 0.01); an association that persisted in AA women with CIN1 , but not in those with CIN2/3 lesions   , 10. To Several global studies have reported variability in the prevalence of HPV genotypes associated with cervical lesions by geographic region and race/ethnicity, with obvious potential implications for vaccine development –13. Few Reasons why AA women are less likely to be infected with HPV 16 and 18 are not known. One possibility is that women of European descent are more susceptible to persistent infection with HR-HPV 16/18 genotypes. Alternatively, AA women may have lower exposure to HR-HPV 16/18 genotypes, or maybe more resistant to infection with 16/18, and possibly be carriers of other HR-HPV genotypes that would increase susceptibility to developing high-grade lesions and subsequently, ICC. Another possibility could be a differential ability to clear the HPV infection among racial/ethnic groups and/or the presence of different variants of HPV 16 and 18 . We alsoSeventy-three percent of all women infected with HPV harbored multiple HPV genotypes, supporting previous evidence –14 that Approximately 90 % of cervical HPV infections are cleared within 2 years by the host immune system. However, persistent infection with high-risk HPV genotypes predicts progression from HPV infection to high-grade cervical intraepithelial lesions (CIN2/3) and to ICC. It is possible that multiple infections with oncogenic HPV genotypes may influence the risk of CIN2+; however, co-infections were equally common in AAs and EAs.These findings should be interpreted in the context of the study limitations. The study participants did not include a sample size of Hispanic women large enough for analyses; therefore, the distribution of HPV genotypes in this ethnic group could not be determined. Future studies that include Latino women are required to elucidate the relative importance of high-risk HPV genotypes in this ethnic group. The study was not powered to histologically confirmed CIN2 and CIN3 lesions, and hence, CIN2 and CIN3 were combined, which while increasing the statistical power, makes discerning aspects of CIN2 not related to ICC difficult. However, CIN2/3 is the point of treatment in the USA, and the two CIN categories are sometimes difficult to distinguish. Two prior studies with a much larger sample size of CIN2/3 cases reported similar racial differences , 10. DesIn summary, we have examined HPV genotypes in all women with abnormal cytological findings, including CIN1 and 2/3 in an ethnically diverse US population and found high-risk HPV genotypes that vary by race/ethnicity, even before triage decisions are made. While small sample limits inference, the findings support the contention that there are racial/ethnic differences in the distribution of oncogenic HPV genotypes among women with and without CIN that require exploration in relation to progression to ICC. Larger studies are needed to confirm this evidence and to help guide cervical cancer risk stratification guidelines.


High risk (HR) human papilloma Virus (HPV) genotypes have been associated with cervical cancer. In Tanzania there is a limited data on the epidemiology of HPV and genotypes distribution among HIV infected women. Here we document varieties of HPV genotypes associated with cervical squamous intraepithelial lesions (SIL) among HIV- infected women at Bugando Medical Centre, Mwanza-Tanzania.A cross sectional hospital based study involving HIV infected women was conducted between August and October, 2014. Exfoliated cells from ectocervix and endocervix were collected using cytobrush. HPV genotypes were detected using polymerase chain reaction (PCR) followed by sequencing using specific primers targeting broad range of HPV types. Cytology was done to establish squamous intraepithelial lesions. Log binomial regression analysis was done to establish risk ratios (RR) associated with HPV infection using STATA version 11.P = 0.006) and women with SIL A total of 255 HIV infected women with mean age 39.2 ± 9.1 years were enrolled in the study. HPV DNA was detected in 138/255  of HIV infected women. Twenty six genotypes were detected in various combinations; of these 17(65.3 %) were of HR genotypes. HR genotypes were detected in 124(48.6 %) of HIV infected women. Common HR genotypes detected were HPV-52(26), HPV-58(21), HPV-35(20) and HPV-16(14). The risk of being HPV positive was significantly higher among women with CD4 counts <100 (RR: 1.20, 95 % CI: 1.05-1.35, Significant proportion of HIV infected women with low CD4 counts have various grades of cervical SIL associated with varieties of uncommon HR genotypes. There is a need to evaluate the effectiveness of the current vaccine in preventing cervical cancer in developing countries where HIV is endemic. Human papillomavirus (HPV) is a very common sexually transmitted infection which is acquired through body fluids and skin-to-skin contact . It has Using molecular techniques, different HPV genotypes have been identified and categorized as high risk (HR) genotypes that are considered to be carcinogenic. These include HPV- 16, 18, 31, 33, 35, 95, 45, 51, 52, 56, 58, 59, 68, 73 and 82; and are considered to cause approximately 95 % of cervical cancers . Human pAs reported earlier in Africa, women particularly those who are HIV infected are under-represented in the global estimate of HPV genotypes . This neA cross sectional hospital based study involving 255 HIV infected women was conducted between August and October, 2014 at Bugando Medical Centre (BMC) HIV care and treatment clinic (CTC). The clinic attends patients who are on antiretroviral therapy (ART) and those who have not started ART. The CTC provides routine Papanicolaou (PAP) smear and gynaecological services with no routine screening of HPV. An average of 80 revisiting HIV infected women are seen per day .The study included all HIV infected women aged >18 years attending the CTC during the study period while excluding women who underwent wedge resection of the cervix and those with missed CD4 counts within 3 months of specimen collection. Socio-demographic and relevant clinical information collected were number of sexual partners, parity, marital status, age at 1st sexual debut, history of STI, history of genital warts, contraceptive use, and CD4 counts.Cervical exfoliated cells from the ectocervix and endocervix were obtained using cytobrush; there after the tip of the cytobrush was placed into a transport medium and stored at -10 °C until processing. Papanicolaou (PAP) smear was also taken, processed and interpreted based on the revised 2001 Bethesda system .http://blast.ncbi.nlm.nih.gov/Blast.cgi) to identify the HPV genotypes. The efficiency of nucleic acid extraction was controlled by a real-time PCR assay from our routine diagnostics simultaneously targeting human cytomegalovirus (HCMV) and the human albumin gene.After vigorous vortexing of transport media, cytobrush was removed and 200 μl of sample was used for nucleic acid extraction with the MagNaPure LC DNA Large Volume Kit on a MagNaPure LC instrument  with elution volume set at 100 μl. For the detection of HPV DNA, two different end-point PCR assays (GEN-1 and GEN-2) were done as previously described [P value of <0.2. Cross tabulation was done to detect variables with collinearity. Factors found with collinearity were not subjected into log multinomial regression analysis. The chi square test and fisher’s exact test were done to compare the distribution of HPV genotypes and SIL. Women with mixed genotypes were regarded as having HR genotype if HR genotype was present. Level of significance was measured by p-value whereby factors with p-values of less than 0.05 were considered statistically significant.The data were entered in the computer using Microsoft excel software and analysed using STATA version 11. Continuous variables were summarized as mean with standard deviation or as median with interquartile range whilst categorical variables were summarized as proportions. Socioeconomic status (SES) was defined using education, employment and business status of the participant. Log binomial regression for estimation of relative risk ratios was done followed by log multinomial regression analysis for the factors with The mean age of enrolled women was 39.2 ± 9.14 years. A total of 111(43.5 %) were married and 46 (18.03 %) and 209(81.7 %) had higher and lower education respectively. Majority of the enrolled women 138(54.1 %) had high socioeconomic status. A total of 158(61.9 %) were residing in rural areas  were found to have HPV DNA in their cervical cells. Out of 138 HPV infected women; 124 (48.6 %) were found to be infected with HR genotypes.p = 0.028). High risk genotypes detected were 16, 18, 31, 33, 34, 35, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73 and 82 whereas LR genotypes detected were 6, 40, 42, 47, 54, 74, 83 and 90. The total frequency of the genotypes detected was 160 (Table 4); of these common HR genotypes were HPV-52, HPV-58 , HPV-35 and HPV-16.In 138 HPV infected women; 26 genotypes were detected in various combinations. A total of 17/138(12.3 %) of HIV infected women had multiple HR genotypes. Of 26 genotypes, 17 were HR while 9  were LR genotypes (P = 0.003) Fig. P = 0.264) was observed. Of 17 women with multiple high risk genotypes, 1(5.8 %) had high grade SIL, 9(52.9 %) had low grade SIL and 7(41.2 %) had no SIL.Out of the 255 women; 91(35.6 %) had SIL on cytological studies. Of 91 HIV infected women with SIL, 74 (81.3 %) had low grade SIL whilst 17(18.7 %) had high grade SIL. Eleven (64.7 %) of women with high grade SIL had positive HPV DNA in their cervical cells. Amongst these eleven women none-16 and non-18 genotypes were detected in 9(81.8 %) of them. Out of 124 women infected with HR genotypes, 55 (44.4 %) had any kind of SIL compared to only 31 (26.3 %) of 118 women with no HPV infection (P = 0.006) and presence of SIL  remained with significant higher risk of being infected with HPV on log multinomial regression analysis.On log binomial regression analysis; women with good socioeconomic status, low CD4 counts, use of ARV ≤6 months and presence of SIL significant high risk of HPV infection as shown in Table Infection with oncogenic or HR human papillomavirus (HPV) genotypes is the major cause of cervical cancer. With the introduction of the quadrivalent HPV vaccine which prevents four HPV types: HPV 16 and 18, as well as HPV 6 and 11, that cause 90 % of genital warts the knowledge concerning the distribution of HPV genotypes has become increasingly public health concern . This stVariation of HPV genotypes can occur even within the country which might have implication in vaccine efficacy in the same country. As evidenced by the fact that in the current study, HPV-16 was not the commonest genotype among women with high grade SIL while in the previous study in Tanzania among HIV and non HIV infected women HPV-16 was the commonest genotype among women with high grade SIL . There iThe current study confirms the clear association between HPV infection and SIL. As in previous studies –31, it wThe major limitation of this study is inability to perform HIV viral load which could be used to correlate virological failure and HPV infection. In addition some important information such as tobacco use and information for those who declined were not collected. However the study established HPV prevalence and genotypes distribution among HIV infected women.In present study, HIV seropositive women with low CD4 counts and various grades of cervical SIL are significantly infected with HR HPV genotypes. In countries where HIV is endemic the governments should consider addition of HPV vaccine in national immunization programme focusing on epidemiology of HPV genotypes. Data on the efficacy of the current vaccine in preventing SIL/cancer and warts are highly needed in developing countries for appropriate policy decision making.


In Asian countries, small-scale rural poultry meat production can face challenges due to food safety policies that limit economic growth and hinder improvement of sanitation and disease prevention. In this study, an integrative, participatory research approach was used to elucidate the sanitation and disease prevention practices in small-scale poultry slaughterhouses in rural northern Thailand.Salmonella contamination in meat processing facilities. The microbial risk assessment was combined with stakeholder perceptions to provide an overview of the existing situation, as well as to identify opportunities for upgrading slaughterhouses in order to more effectively address matters of food safety, processing, and government licensing.Initial steps included the identification of key stakeholders associated with the meat production chain, development of a research framework, and design of a methodology based on stakeholder consultations. The framework and methodology combine issues in five major areas: (1) public health, (2) socioeconomics, (3) policy, (4) veterinary medicine, and (5) communities and the environment. Methods used include questionnaires, direct observation, focus groups, and in-depth interviews. In addition, a microbiological risk assessment approach was employed to detect Salmonella contamination in both the meat and the surrounding environment.The conceptual framework developed elucidated the complex factors limiting small-scale slaughterhouse improvement including a lack of appropriate enabling policies and an apparent absence of feasible interventions for improvement. Unhygienic slaughterhouse management was reflected in the incidence of There is potential for the use of an integrative approach to address critical problems at the interface of rural development and public health. The findings of this study could serve as a model for transdisciplinary studies and interventions related to other similar complex challenges.The online version of this article (doi:10.1186/2049-9957-3-46) contains supplementary material, which is available to authorized users. Please see Additional file Interest in an integrative approach has been increasing, especially in the area of ecosystem health, in response to growing evidence that many important ecosystem changes are the result of human activity . The curFood safety is an issue which is related to ecosystem health that concerns all human beings, making it an important focus of public health strategies around the world, including Thailand. Cases of foodborne diseases are still increasing, especially in developing countries, so food safety remains a serious challenge.Salmonella is one of the most common organisms causing foodborne diseases worldwide. In the US, Salmonella is the second largest cause of food poisoning, where it spread widely between 2009 and 2010 [Salmonella in 2010. In Thailand, Salmonella was found to be the second largest cause of food poisoning, following rotavirus in 2008 [Salmonella[Salmonella contamination of chicken meat, [and 2010 . In EU c in 2008 . The mailmonella. Processen meat,  especialen meat, .To enhance food safety and control foodborne diseases, efforts have been made to enforce regulations specifying standards for poultry slaughterhouses in Thailand, however, many small-scale slaughterhouses have yet not been able to meet these standards . This stSince food safety, especially in rural areas of Thailand, is a complex challenge and involves many sectors, an integrated approach was applied in this study. The main stakeholders were identified in the early stages of the project, using participatory methods, e.g., researchers meeting with key stakeholders including slaughterhouse owners, as well as Department of Livestock Development (DLD) officers at the national and regional levels, to identify problems. The information obtained from discussions with stakeholders was then reviewed with experts in veterinary science, socioeconomics, and public health before being used as the basis for developing a conceptual framework.Royal Thai Government Gazette, the Eleventh National Economic and Social Development Plan of Thailand (2012–3016), the DLD strategic plan, as well as domestic and international research reports on poultry slaughterhouses.Policies, laws, and regulations which include the key phrases “food safety”, “slaughterhouse standards”, “current situation of poultry slaughterhouses”, or “foodborne diseases in Thailand” were collected from published and unpublished sources including the Perceptions regarding the implementation of existing regulations were obtained through focus group discussions (FGDs) with DLD regional officers. The principle investigator and co-principle investigator led the FGDs. Purposive sampling was used to identify participants using the criteria: (1) DLD provincial officers; (2) heads of DLD district offices; and (3) individuals having responsibly for slaughterhouse control. Two FGDs were conducted with a total of 22 participants between May and June 2012. Qualitative content analysis was used to analyze FDG data using the five-step process as described by Agus et al. : (1) FolThe Chiang Mai province is located in the northern part of Thailand and is characterized as having dense areas of poultry production. In 2010, more than three million chickens were produced in this province . For thaSalmonella spp. was used to elucidate the public health and environmental hazards of poultry meat production. A study by Padungtod and Kaneene found a 9% incidence of Salmonella contamination in meat processed by slaughterhouses in northern Thailand [Evidence of the prevalence of Thailand . Based oSalmonella spp. In addition, environmental samples, including 500 grams of soil taken from around slaughterhouse buildings, were collected and stored in sterile plastic bags. In addition, one-liter samples of wastewater were collected using sterile bottles before the water was drained into the environment. Sample collection was conducted from July 2011 to May 2012.A preliminary survey found that, in 2010, Chiang Mai had 55 small-scale poultry slaughterhouses with approximately 25,000 birds being sent to these slaughterhouses each day. Samples were collected from slaughterhouses located within 100 kilometers of the laboratory at Chiang Mai University to insure that samples could arrive there within three hours. A total of 410 meat samples from 41 slaughterhouses were collected. Each carcass was placed in a large bag with 250 ml of sterile peptone water which was then shaken inside the bag for one minute, then the rinse water was poured into a sterile bottle and used for identification of Salmonella spp. The cold chain was not broken during sample collection and transport to the Diagnostic Center. Scientists used a standard Diagnostic Center form to record information on each ice chest including the number of the ice chest, the owner of the slaughterhouse, the sender of the sample, and individual sample identification information. Samples were then stored in a refrigerator at 4°C prior to individual sample testing which were conducted the following morning. After each use, each ice chest was washed with dishwashing liquid and water and then dried in a plate dryer. To further preclude possible contamination, each ice chest was withdrawn from use for between five and seven days after delivering the samples. The corresponding author  supervised sample collection in the field. The Diagnostic Center is certified by the Bureau of Laboratory Quality Standard (BLQS), Department of Medical Sciences, Ministry of Public Health.All samples were collected in the morning immediately after completion of the slaughtering process, put into single use zip lock plastic bags, kept on ice in an ice chest, and sent within three hours of collection to the Diagnostic Center, Faculty of Veterinary Medicine, Chiang Mai University, for testing for the presence of Salmonella identification was performed according to a modified version of the US Food and Drug Administration Salmonella culture method  [Salmonella contamination in carcasses, soil, and wastewater was calculated by dividing the number of samples positive for Salmonella by the total number of samples processed. Concurrent with the sample collection, data on the characteristics of the slaughterhouses, including slaughterhouse management and perceptions of the owners regarding slaughterhouse standard regulations, were collected by the principle investigator at the slaughterhouses using structured questionnaires and interviews. The questionnaire focused on processing capacity, biosecurity, disease control management, veterinary services, health status of workers, environmental management procedures, socioeconomic situation, and perception of zoonotic aspects. Manual) , and ISO Manual) . The preIn addition, a checklist of DLD regulations regarding the location of the facility, characteristics of the structures, the slaughtering process, and waste management was applied as a cross-check of the data. Descriptive statistical analysis was accomplished using Microsoft Excel 2010 (Microsoft Corp.).The main stakeholders in this study were the slaughterhouse owners, DLD officers at the national and regional levels, regional public health officers, and local administration officers. Brainstorming meetings and interviews confirmed the stakeholders’ views on the importance of food safety and food policies in Thailand. They realized that poultry slaughterhouses are an important link in the poultry meat production chain, that the standard regulations should be followed, and that there are many factors affecting the improvement of slaughterhouses. They concurred that the main problems to be addressed are the inability of most small-scale slaughterhouses to comply with the current standard regulations and a lack of appropriate strategies to motivate and assist small-scale slaughterhouses to comply with these regulations.The conceptual framework of this study  . This reIn addition, good manufacturing practices (GMPs) for poultry slaughterhouses were announced, and their adoption has been mandatory since 2006. However, in practice, the GMP guidelines, which were intended to further improve operations including hygiene standards, have been enforced only in slaughterhouses which have been issued a government license, most of which are larger operations.In the FGDs with DLD regional officers  on the implementation of laws and regulations, the officers accepted that they could not strictly enforce the ministerial regulatory criteria intended to promote the improvement of small-scale slaughterhouses. They acknowledged that the criteria are intensive and require high levels of investment, making them suitable for large- and medium-scale operations which generate sufficient profit, but not for small-scale facilities with low productivity and small profits. They agreed that if they attempted to strictly enforce the regulations, they would meet resistance from the slaughterhouse owners. They also acknowledged that during their regular visits to slaughterhouses every three to four months, they should focus on establishing a spirit of collaboration and cooperation in order to promote hygienic management and disease control in slaughterhouses rather than strictly enforce the regulations. They also indicated that the current regulations should be more flexible and practical. As one officer said, “It would be useful if there was a prototype or a blueprint of a good, hygienically managed slaughterhouse that owners could use as a model for investment”. The majority of the participants agreed with this comment.Department of Livestock Development officers indicated an awareness of the need to work integratively with officers from other agencies, including public health officers and local administrative officers, in order to improve food safety. However, they mentioned that there were obstacles to such joint efforts. For example, working with local administrative organizations was problematical because those organizations still had no official role in that area or any personnel specifically responsible for slaughterhouse control. In the case of Public Health agencies’ work with food safety control, their main focus is on meat products sold in the market rather than conditions at slaughterhouses. On a positive note, just over half the participants (54.5%) indicated that they were willing to work in an integrative manner with other agencies to address issues of food safety. Table Of the participating slaughterhouse owners  to firm up the skin, then stored in a small vessel containing ice. The wastewater from the slaughtering process was discharged directly onto the area around the slaughterhouses.The majority of the small-scale slaughterhouses, which processed fewer than 50 birds/day, did not satisfy all of the seven criteria described in the slaughterhouse law and regulation. The owners constructed simple facilities with only necessary equipment and located within their community. Birds were sold only in the local community the same day they were slaughtered, but quantities were small, just enough to meet local demand. Incomes were limited and not sufficient to invest in improvements to the slaughterhouses to meet the Ministerial criteria. Results of the slaughterhouse assessment and the hygiene management evaluation are presented in Tables Regarding slaughterhouse owners’ perceptions of relevant laws and regulations, 33 out of 41 owners (80.5%) stated that some of the criteria in the current standard regulation were impractical for small-scale slaughterhouses. For example, meat from most of the smaller slaughterhouses was not inspected because the limited slaughterhouse income was not sufficient to hire a meat inspector. Owners stated that they would have to stop operating their business if the DLD strictly enforced all the standard slaughterhouse regulation requirements. Although 25 out of 41 owners (61%) accepted that they did need to improve their slaughterhouses, they indicated a desire that the regulatory criteria be more practical.Salmonella spp. were isolated from 30 out of 410 carcass samples (7.3%) taken during this study. As for contamination in the environment, 12 out of 41 soil samples (29.3%) and nine out of 41 wastewater samples (21.9%) tested positive for Salmonella spp.Over the last 30 years, several successful attempts have been made to control various infectious diseases in countries all over the world, especially in developed nations. However, threats still exist such as antimicrobial resistant bacteria and unsafe farming and food production practices, as well as threats created by the impact of urbanization and agricultural intensification . In addiTo more effectively identify avenues for enhancing safe processing in small-scale poultry slaughterhouses, practitioners of veterinary and human medicine, social scientists, and economists cooperatively followed an integrative approach in the development of the conceptual framework and in participatory problem identification from the outset. That framework demonstrates the complexity of the problem and the linkages between the different disciplines. This study follows the successful integrative approach which was used to gain an understanding of and develop a suitable research agenda in the case of the emergence of leptospirosis in Hawaii . This stThis study confirms that good hygienic management is not widely practiced in small-scale slaughterhouses in northern Thailand. This finding is consistent with the report in Bangladesh by Rimi et al. in 2013, which reported commonly observed improper practices such as slaughtering sick poultry in rural communities . This stThe majority of slaughterhouse owners pointed out that they could not improve their operations in accordance with the current standard regulation. For instance, the regulation specifies that slaughterhouses must not be located in communities, a challenge to the small-scale slaughterhouse owners in this study currently located within a community. In fact, some of the slaughterhouses were in operation prior to the regulation. An authorized meat inspector is required to inspect the meat, but owners pointed out that they could not afford to hire a meat inspector because of the limited profitability of their operations. Owners stated further that if the DLD decided to strictly enforce the regulation, they would have to cease operation of their slaughterhouse and seek a new occupation, an outcome that would certainly adversely affect the socioeconomic status of the owners and their families.Department of Livestock Development regional officers agreed that the current laws and regulations are, in fact, more suitable for large- and medium-scale operations which can afford the necessary high investment. Nonetheless, the DLD is attempting to encourage even small-scale poultry slaughterhouses to meet the standard. The slaughterhouse blueprint developed by the DLD and distributed to officers and slaughterhouse owners, however, is designed for operations processing 200–300 birds/day which is four or more times the daily production of small-scale slaughterhouses. Thus, the DLD-proposed blueprint poses a considerable challenge to small-scale slaughterhouses.Salmonella as a public health hazard was clearly demonstrated in this study, which found a prevalence of 7.3% in the final product (chicken carcasses). That figure is close to the 9% prevalence of Salmonella in poultry carcasses after slaughtering and final products in Thai slaughterhouses reported in a study by Padungtod and Kaneene in 2006 [Salmonella of 43%. Reports from elsewhere in the world also indicate a higher prevalence of Salmonella. For example, Elgroud et al. reported in 2009 that the prevalence of Salmonella in chicken slaughterhouses in Algeria was over 53% [Salmonella in chicken carcasses from small-scale poultry slaughterhouses in Brazil [Salmonella in poultry slaughterhouses in Alberta City, Canada was 37% [Salmonella in chickens from slaughterhouses in Spain was 17.9% [The importance of  in 2006 , but mucover 53% , while Fn Brazil . Similaras 17.9% .Salmonella prevalence in these reports is significantly higher than that found in this study. One possible reason for the lower Salmonella prevalence found in the current study could be that the survey was conducted at small-scale facilities, the majority of which processed fewer than 50 birds/day. Processing fewer birds might result in a lower bacterial load in those facilities and thus a lower Salmonella prevalence in carcasses compared with other studies such as the one by Padungtod and Kaneene (2006), which was done in medium- and large-scale slaughterhouses. Moreover, the traditional slaughtering process commonly found in smaller operations includes the final processing step of immersing the carcass in hot water for a short time to firm the skin. Immersion makes the skin more attractive, an important factor for small-scale operations which sell the final product (carcasses) in the local community. That process also has the effect of decreasing pathogen contamination. However, this method could also have the negative effect of increasing the temperature of the carcasses, making them more suitable for bacterial growth and thus more susceptible to rotting. For that reason, it is not appropriate for carcasses treated this way to be stored overnight.Salmonella contamination found in soil collected around slaughterhouse buildings and the 21.9% contamination rate in wastewater drained onto the area around the slaughterhouse without treatment, however, is evidence that improper hygienic practices can affect not only end consumers, but also members of the local community and the surrounding environment. These results mirror findings in previous studies in other regions of the world. For example, 100.0% of sludge samples collected from eight pig and five poultry slaughterhouses in Belgium and the Netherlands were found to be contaminated with Salmonella[Salmonella spp. [Salmonella[The 29.3% prevalence of lmonella, and 7.4lla spp. . Seven olmonella. Thus, iSalmonella contamination found in carcasses was low compared with some previous studies, hygiene management should be improved to enhance food safety. Current laws and regulations are not suitable for most small-scale slaughterhouses in Thailand as few have the capacity to conform to the existing regulations. It is suggested that the potential health hazards to the community from Salmonella and other foodborne diseases emanating from small-scale poultry slaughterhouses should be studied further. In addition, specific regulatory criteria which meet appropriate food safety standards but which are specifically designed to be suitable and practical for small-scale slaughterhouses should be developed, pilot tested in the field, and then implemented.Improving small-scale poultry slaughterhouses to meet food safety standards and addressing the standard certification requirement are challenges that need to be addressed. This study, which found that unsatisfactory and inadequate management of hygiene was common in small-scale poultry slaughterhouses in Thailand, demonstrates that the use of an integrative approach for exploring a complex problem and developing a research conceptual framework can be an effective approach. Even though the level of Multilingual abstracts in the six official working languages of the United Nations.(PDF 239 KB)Additional file 1:Questionnaire for Slaughterhouses.(DOC 181 KB)Additional file 2:Check list for slaughterhouses.(DOCX 17 KB)Additional file 3:


Disease progression in multiple sclerosis (MS) is highly variable and predicting prognosis is notoriously challenging. Patients’ prognosis beliefs, responses to prognostic uncertainty and experiences of prognosis-related communication with healthcare professionals (HCPs) have received little study. These issues have implications for patients’ psychological adjustment and are important in the context of the recent development of personalised prognosis forecasting tools. This study explored patient perspectives on the experience of prognostic uncertainty, the formation of expectations about personal prognosis and the nature of received and desired prognosis communication.15 MS patients participated in in-depth semi-structured interviews which were analysed using inductive thematic analysis.Experiencing unsatisfactory communication with HCPs, Appreciating and accepting prognostic uncertainty, Trying to stay present-focused, Forming and editing personal prognosis beliefs, Ambivalence towards forecasting the future, and Prognosis information delivery. MS patients report having minimal communication with HCPs about prognosis. Over time MS patients appear to develop expectations about their disease trajectories, but do so with minimal HCP input. Provision of prognosis information by HCPs seems to run counter to patients’ attempts to remain present-focused. Patients are often ambivalent about prognosis forecasting and consider it emotionally dangerous and of circumscribed usefulness.Six themes captured key aspects of the data: HCPs must carefully consider whether, when and how to share prognosis information with patients; specific training may be beneficial. Future research should confirm findings about limited HCP-patient communication, distinguish predictors of patients’ attitudes towards prognostication and identify circumstances under which prognostic forecasting benefits patients. Multiple sclerosis (MS) is a chronic, degenerative disease of the central nervous system. It is the leading cause of non-traumatic neurological disability in young adults , and canClinical guidelines  emphasiprognostic uncertainty in pwMS is lacking.It is not surprising that uncertainty, or a lack of clarity or predictability, is noted to be a common experience for pwMS and their families; the uncertain and ambiguous nature of the disease and the implications of this for patients and their families has received substantial research attention . AlthougThe current study explores how pwMS experience prognostic uncertainty and communication with HCPs about prognosis. It aims to understand how prognostic uncertainty affects pwMS, if and how they form expectations about their disease trajectory and what communication they receive and want. These topics are both interesting in their own right and timely to investigate given recent developments of an online analytical processing tool (OLAP) which supports individualised prognostic risk forecasting in MS ,15,16. TThis study was approved by the University of Southampton ethics committee. Data was collected between Spring 2014 and Summer 2015 via semi-structured telephone interviews with pwMS. Telephone interviews were chosen to allow participants from a wide geographical area (across the UK) to participate, to limit burden on pwMS and to ensure that mobility limitations, MS symptoms, work or family commitments did not preclude participation. Although some researchers consider face-to-face interviews superior for establishing a rapport with participants when researching emotive topics, we have previously successfully used telephone interviews to elicit deeply personal, rich and detailed accounts of illness experiences in pwMS ,18.ChoosFive of the interviews were conducted by LD  and 10 were conducted by EM . EM had practical training in qualitative interviewing and ongoing supervision from LD throughout data collection. A schedule of open-ended questions  encouragInclusion criteria were a diagnosis of MS and age ≥18 years. An overview of the study was disseminated via the UK MS Society website and social media and through emails to members of the UK MS Register. PwMS who were interested in participating responded to the researchers directly. Participants received information sheets and signed consent forms prior to enrolment. 23 people expressed interest in the study. We begun by interviewing all participants that consented but as data collection  progressed we deliberately sampled from the pool of available participants to ensure the final sample varied in terms of MS course , time since diagnosis, age and gender. Purposive sampling was possible because participants typically disclosed this information to the researcher at the point of expressing interest or of returning the consent form. At later stages of the data collection we also made deliberate efforts to attract interest from younger and more newly diagnosed pwMS by editing the recruitment adverts/information to make it clear that we were especially interested in hearing from people with these characteristics. Once we began purposive sampling we thanked those that consented for their interest and willingness to be involved and advised them that we were nearing the end of the project and narrowing our focus in order to explore the experiences of participants with particular profiles. Where relevant we advised volunteers that their participation would not required and offered them the opportunity to receive a summary of the research results. Recruitment ceased after 15 interviews as by this point we had succeeded in collecting data from a diverse sample and analysis had reached the point where no new themes were occurring (i.e. saturation). Participants received a £15 retail voucher to thank them for their time.The data was analysed using thematic analysis , a systeThe analysis identified 6 key themes which incorporated 21 subthemes see . In the Participants’ accounts of interactions with HCPs about their prognosis frequently digressed to detailed emotional accounts of negative communication and support experiences around the time of diagnosis.I was left over the weekend with no knowledge, no ability to contact anyone and spent most of the weekend on the phone to the Samaritans. His lack of communication was just really upsetting. Distress relating to diagnosis experiences clearly persisted over time and appeared to create negative expectations and perceptions of future interactions with doctors (particularly neurologists). Neurologists tended to be viewed as ‘diagnosers’ with little time and interest in patients’ ongoing struggles. In contrast, MS nurses tended to be perceived as ideally situated for discussing emotional and practical issues relating to MS. Participants tended to be positive about the communication and support they received from nurses although they did not all have access to a nurse and were conscious that the valued supportive role nurses could provide was limited by their workload.MS nurses are the ones who generally are the best ones to talk to you face-to-face. All participants perceived that opportunities to discuss their prognosis had been limited. In some cases this appeared to reflect infrequency of contact with HCPs, the brief nature of consultations and abrupt clinician  communication style. In other cases, the participant deliberately held back questions because they did not feel ready to hear the answers. This sense of not being ready and wanting to ‘hold back’ appears to relate to the ‘Trying to stay present-focused ‘ theme described in more detail below.I didn’t want to know about it that much anyway, because I was just trying to absorb it all. Participants clearly comprehended MS to be a variable disease with inherent uncertainty. Participants were tolerant of the fact that HCPs did not have all the answers and could not confidently predict how MS would affect them over timeI know due to the nature of MS … that they probably couldn’t answer all of the questions…a lot of the questions that I have had. Along with theoretical ‘knowing’ that MS prognosis is uncertain, several participants described philosophical stances towards accepting an inability to know or influence the future. This perspective was described as originating from religious belief or as an outcome of undertaking psychological therapyI recognise open answers are a reality in the whole of life. l I just try to live one day at a time””, “take it as it comes”  and “get on with it [life]” , “try not to think about it too much” . This approach appeared to be based on the wisdom that “whatever will be will be”  and that “if the worst does happen, I’ll deal with that as and when” . This present-oriented approach appeared to inhabit a spectrum ranging from those who had extensively considered future scenarios then concluded that their time/energy is best spent dealing with day-to-day problems and pleasures, to those who (aware or not) avoided thinking about the future because they feel unable to face it.A ubiquitous strategy for living with an uncertain but threatening future was to stay in the present as much as possible. “It’s not wanting to realise what the, it’s realising that things could get worse or that things, that things will get worse but it’s hiding away, it’s more comforting. I refuse to think about what is going to happen, I don’t like that I’m afraid. Despite attempting to be present-focussed, participants inevitably experienced thoughts and worries about future disability and dependence. For instance, one participant  described a deliberate present-oriented approach to coping with MS but also revealed intrusive, distressing future-related thoughts about becoming dependent on others and spoke of how she had contemplated assisted suicide if such circumstances arose.Despite discussing the inherent uncertainty surrounding MS progression and declaring that they did not know their likely prognosis, most participants nevertheless described well-formed personal beliefs about their prognosis. Early beliefs seemed to be strongly influenced by salient, emotional images or memories of other pwMS they knew personally, or had seen in the media. Often these individuals had severe, debilitating disability due to MS and accordingly early personal prognosis beliefs tended to be fearful and extreme.And other people I know have got it are, absolutely, are stricken with it, I’ve got one on the verge of death. [I have] little experience of good stories, mainly bad, and all with the outcome of eventually death, which doesn’t really, reinforce your own situation very well. Early beliefs appeared to evolve over time and be subject to ongoing editing and revision as other sources of information about MS prognosis were encountered. Participants typically describe HCPs as only a minor influence in how their prognosis understanding came about. Importantly though, HCPs communication appeared to attenuate or moderate early fearful beliefs about prognosis by providing verbal explanations about the variability of outcomes and the non-necessity of progression and/or severe decline.I immediately thought it was a degenerative disease because I didn’t know anything about it. Umm… and he [the neurologist] said ‘not so, necessarily’. Some participants described how their neurologists had given indications that their MS was either progressing slowly or not highly active. This communication–tentatively positive yet vague- was frequently the only conversations about prognosis participants remembered having with HCPs. Others claimed to have never received information about their likely prognosis from HCPs.But the prognosis I think was quite vague. I don’t think I was told anything about how it would progress. Participants who had several years or decades of MS experience appeared to base their understandings of future likelihoods on their experiences to date (including looking back with hindsight at pre-diagnosis disease activity). This meant that pwMS who were many years post-diagnosis or post symptom onset expressed more confidence about how they expected their disease to affect them than those more recently diagnosed. People tended to believe that future experiences would mirror past experiences.Going on past time, past diagnosis, the amount of time that I’ve had the MS then it’s more likely now to stay on the same level now for an indefinite period, there shouldn’t be any rapid decline or rapid improvement. I’ve got experience of it in the last 5 years. It’s accelerating. I can clearly see there is a long slow deterioration. But it’s twenty years I am looking at. Participants were often unable to settle on a position about whether they wanted more insight into their likely long-term prognosis. Participants changed their minds backwards and forwards during the interview and demonstrated internal conflict.I kind of presumed that it wasn’t actually possible to … to tell. But God if it was possible I would really like to know. I think… Oh God, or do I? Actually yes I would, of course I would. Oh I don’t know. Maybe, I don’t know, Yeah I think so, I’d like to know. Would that be useful? You know this? That’s a real toughy, I’ve never even thought of that before. Would I want to know? Um, yeah I think I would, I think I would but I’m not 100%. It’s a toughy though isn’t it? Because participants were not pressed to arrive at a final, unambiguous answer during the interviews, reporting frequencies “for” and “against” more prognosis information is not straightforward. However, considering overall interview content, five participants were broadly classifiable as wanting more information, six as not wanting more information, and four as very unsure. No relationships between information preferences and disease type (progressive vs. relapsing-remitting), disease duration, gender or age were apparent.The hypothetical nature of accurately predicting MS progression appeared to compound the participants’ difficulties in articulating their views, as did the challenge that they would only be able to judge the likely impact once they had heard whether their prognosis was positive or negative.Often, participants appeared to assume that information about the future would be bad news. A dominant concern was that negative information would elicit hopelessness and demoralisation.If the future is really bad and really bleak then if I knew about that now then I suppose I could be tempted to, to just, give up now or something. If I had to think about what could happen, well then, you know, you might as well shoot me. Would I want to know? No I wouldn’t. Because I think it can make you cuckcoo, I think ignorance is bliss in some ways. Those that had already experienced moderate to severe disability believed that mental adaptation to a slow, creeping progression seemed easier to handle than knowing what one must face in advance.I find that the changes are so gradual and over time that you kind of, each little thing that happens you get used to it, before something else will happen. It’s not happening all at once, each change is gradual and easier to deal with that way. Perceived usefulness of prognosis information appeared pivotal in whether participants would want to receive it. Often the information was not considered useful as participants did not believed it would change anything for them.If somebody knew something was going to happen but they knew how to treat it, then I would want to know, then you’d want the treatment. But if there is no treatment and there is nothing you can do about it then you probably wouldn’t want to know. No, it’s almost better to just avoid it. I don’t think it makes any difference about knowing. What would you do with that information then? And you can’t change it. Why bother knowing it anyway? If they could tell me then it wouldn’t make any difference. Several participants did believe that receiving prognosis information would be useful. Anticipated impacts included prompting them to undertake personally valued activities whilst physically able, taking life easier to try to influence disease outcomes, financial and practical planning and preparing family members.I would be looking to see if there were any answers, solutions to the problem and if you don’t know then you can’t look for solutions can you. I might think of moving to a bungalow or something like that. I would like to know what I am dealing with…. But I would just want to prepare, I suppose the best I can, prepare my children. If I was told I would be in a wheelchair in ten years’ time, there is loads of things I would want to do, before that. Participants’ believed that HCPs must make challenging judgements about what prognosis information to share and when. Participants suggested that individual and personality differences influenced information preferences.Some people can understand and take in information a bit more than other people can so they need to be careful about how they tell people. I think it is just judging the patient. I think, it’s very hard to know whether someone wants to know, or not,… unless you actually ask the question but then if you say ‘would you like to know?’, I don’t know, it depends on the individual, on the person. If I’d have been given that info when I was diagnosed I would probably have jumped off a cliff.” P12: Female, 44, PP, 2yrs)). Beyond that, there was no consensus between participants  about what constituted appropriate timing.Some participants also described how timing affects receptiveness to prognosis information. Of those who mentioned timing, all felt the point of diagnosis was unsuitable Those that expressed an opinion about the mode of personalised prognosis information delivery favoured face-to-face communication from a HCP and were concerned with privacy and comfort of the setting.From a professional. A consultant. Not a letter, not an email. Relevant expertise was considered vital for prognosis information deliverers but support-giving skills were also considered pertinent and MS nurses were noted for having these.If I was going to be told about how in the future it will be really bad, I’d want someone who’s an expert who could answer any questions I had. Well I mean, I think that’s all. I wouldn’t mind what status they had but I’d just want them to be knowledgeable. An MS nurse, because they are very well trained and very empathetic with the situation, to have an in-depth consultation with the person, in a neutral environment, to try and sort of lighten the picture rather than darken it. Participants emphasised how they felt prognosis information should be communicated not as a one-off conversation but as part of joined-up care that prioritised their emotional wellbeing together with appropriate disease-modifying treatments and symptom management.They should have people that, other people around and place to support you after you have been told these things. This study represents the first investigation into the experiences of pwMS regarding prognostic uncertainty and prognosis communication. The analysis suggested that limited communication about prognosis takes place between pwMS and HCPs, a finding which, together with the global dissatisfaction expressed about interactions with HCPs, was not unexpected and concurs with previous research on communication in MS ,8. InterIn spite of minimal HCP communication and a realistic appreciation of the difficulties in accurate prognostication, we found that pwMS developed beliefs and expectations about their prognosis; particularly about the pace of worsening. The idea of personal beliefs about MS prognosis emerging over time is consistent with the Common-Sense Model of illness perception . This thA number of novel findings emerged which should inform improvement of HCP behaviour around discussing and/or delivering prognostic estimates. Firstly, this study highlights the emotive nature of MS prognosis as a topic and indicates that pwMS may require careful preparation and emotional support when this is discussed; referral to psychology services or to patient support groups may be helpful for some patients, particularly given the fact that several participants referred to self-harm, suicide or having mental health difficulties as consequences of getting negative prognosis predictions It also appears that clinicians would benefit from training in skills for discussing prognosis and that the role of MS nurses, as well as neurologists, should not be overlooked. Secondly, current findings demonstrated that although patients are unlikely to welcome detailed prognosis forecasts at diagnosis, early communication from neurologists, (even if brief and vague), about the variability of outcomes and the non-inevitability of wheelchair use is incredibly significant for pwMS, functioning to counter initial extreme and distressing illness perceptions. In early consultations clinicians could also acknowledge that prognosis-related discussions can be overwhelming whilst simultaneously conveying openness to revisiting dialogues when the pwMS feels ready.Finally, the ambivalence towards receiving forecasts of prognosis demonstrated in this study suggests that HCPs should not assume that all PwMS want to know their prognosis. It was clear that caution and sensitivity need to be exercised if prognosis forecasting tools are introduced into clinical practice and their predictions shared with pwMS. Prognostic calculators appear to be best suited to presentation as part of a clinical consultation or support programme, rather than stand-alone tools.A number of limitations to the current study should be noted. The first concerns sampling. We purposively sampled participants with varied clinical and sociodemographic characteristics in order to build up a picture of issues that are relevant for a range of pwMS. However, in accordance with the qualitative methodology, we did not seek to sample representatively. The recruitment approach may have attracted those with strong views about prognosis communication and may have failed to recruit those less engaged with actively managing their MS, and those who use more denial and avoidance based coping strategies. If this was the case we might expect a representative sample to have less appetite for information about their prognosis. This study may also have attracted patients who have had negative experiences and/or limited communication with HCPs and who wanted to share their frustrations about this. If this was the case, our findings might indicate a more negative picture of patient-doctor communication than is warranted.The suggestion that limited patient-HCP communication about personal prognosis takes place requires replication in a larger sample. It would also be valuable to establish whether the apparent paucity of prognosis communication is a UK phenomenon or more widespread; the limited existing research suggests scarcity might indeed be common ,6. ReseaThis study revealed substantial variability in responses to prognostic uncertainty and appetite for information. However, the study design was not suitable for detecting or quantifying associations between particular viewpoints or experiences and patient characteristics (e.g. progressive and relapsing remitting disease subtypes). More research is needed to delineate which pwMS want more prognosis information and to establish the best timing and clinical setting to discuss prognosis. Large questionnaire studies would permit investigation of associations between prognosis information preferences and clinical, sociodemographic and psychological variables.This study collected only very limited clinical data (MS type and time since diagnosis) and this was by participant self-report. Although, anecdotally participants appeared to have no difficulties in remembering or communicating this information, it is possible that there may have been inaccuracies. In the current study, objective clinical data on use of disease modifying therapies, relapse severity/frequency, neurological disability  with paS1 Fig(DOCX)Click here for additional data file.


Background: There is growing evidence that human immunodeficiency virus (HIV)-infected women might have a different human papillomavirus (HPV) type distribution in cervical dysplasia specimens as compared to the general population. This has implications for primary prevention.Objective: We aimed to obtain preliminary data on the HPV genotypes prevalent in histological samples of HIV-infected women with cervical intraepithelial neoplasia (CIN) 3/CIS of the cervix in Miami, FL, USA.Methods: Retrospective data were collected on HIV-infected women referred to the University of Miami-Jackson Memorial Hospital colposcopy clinic between years 2000 and 2008. The histology slides of CIN 3/CIS biopsies underwent pathological review and sections were cut from these archived specimens for HPV DNA extraction. HPV genotyping was then performed using the GeneSquare™ HPV genotyping assay. We report on our first set of 23 samples.Results: Eight high-risk HPV types were detected. Types in decreasing order of frequency were 16, 35, 45, 52, 59, 31, 58, and 56. Most cases had multiple infections. HPV type 16 was the most common (45%) followed by HPV-35 and -45 with equal frequency (40%). No samples contained HPV-18.Conclusion: Our preliminary results suggest that cervical dysplasia specimens of HIV-infected women more likely (55%) contain non-16 and -18 high-risk HPV types. We show that this held true for histologically confirmed severe dysplasia and carcinoma-in situ. Epidemiological studies guide vaccine development, therefore HPV type prevalence in CIS and invasive cervical cancer among HIV-infected women should be more rigorously explored to ensure that this highly vulnerable population receives appropriate primary prevention. This histological confirmation was not performed in previously reported studies on HPV type prevalence in HIV-infected women  genotypes are the necessary cause of 99.7% of cervical cancer. Seven of them  account for nearly 90% of all cervical cancer cases in the general population worldwide with little regional variation  and quadrivalent 6/11/16/18) vaccines against additional HR-HPV types –11. The /16/18 va/18 and qMeta-analyses have shown that, in the general population, many HR-HPV types are common in low grade cervical dysplasias however, as the grade of dysplasia increases HPV types 16 and 18 predominate. This follows a fairly linear upward trend. Similarly, as the grade of dysplasia increases, the other HR-HPV types become less common following a downward trend Figure . Our aimAfter obtaining IRB consent, retrospective de-identified clinical data were collected on 510 HIV-infected women with abnormal Pap smears seen at the University of Miami-Jackson Memorial Hospital (UM-JMH) colposcopy clinic between 2000 and 2008  by cervical biopsies. Twenty patients had samples that were considered probably too old for successful DNA extraction and were excluded. Samples were limited to one event per patient using the case with the most severe pathology. All slides underwent central Gynecologic Pathology review. There were 24 of 30 remaining histology slides that were confirmed CIN 3 while the others were down-graded to CIN 2. The corresponding paraffin-embedded blocks of the CIN 3 were pulled and two 20-micron sections were cut from each block. DNA was extracted from these sections using the Ambion™ Recover All kit as per manufacturer’s instructions. We were able to successfully extract DNA for HPV genotyping on 23 samples. Clinical data on the final 23 cases were also obtained including patient demographics, cytological/histological results, as well as CD4 counts and HIV-1 viral load. After DNA was extracted from paraffin sections, we performed Nanodrop analysis to check concentration/ratio of DNA. This was followed by analysis of DNA on an Agilent™ Bioanalyzer chip to assess quality. HPV genotyping of these samples was performed using the Genesquare™ kit developed by Kurabo Industries LTD , which tests for 23 HPV types including all high-risk types and some low risk types. This kit uses multiplex PCR assays with HPV type-specific primers. We chose this PCR-sequencing method because it offers sensitive detection of HPV types at a lower cost and with increased processing speed than the standard Roche Linear array. Furthermore, there is no cross-hybridization because HPV type-specific primers are used as compared to the Roche method, which uses the L1 consensus primer (high-homology region). However, the Genesquare assay has not yet been validated in a large trial.Fifty patients (74 events) were diagnosed with CIN 3  was used for statistical analysis. Descriptive statistics such as mean, standard deviation, median, first and third quantiles were reported for patients’ age and some of patients’ clinical characteristics, such as number of HPV infections, viral load, and CD4 count. Frequencies and percentages were calculated for characteristics such as race/ethnicity, grade of dysplasia, treatment, and recurrence of dysplasia. One-sample exact binomial test was used to test the differences in HPV genotype prevalence in cervical lesion of women between HIV-infected women and the general population reported. Type-I error rate is set to 5%.n = 23), 83% of women were African-American or Haitian-American and 50% were age 40 or younger (Table p < 0.05). However, the prevalence of HR-HPV types 16, 31, 56, and 58 were not significantly different than those in cervical lesions of women in the general population.In this sample n = 3, 83% ofIn conclusion, we found that certain HR-HPV types that are considered relatively rare in high-grade cervical lesions of women in the general population were actually more common in high-grade lesions of HIV-infected women at the UM-JMH colposcopy clinic. We also found that the most prevalent HPV types 16, 35, 45) in these high-grade lesions among HIV-infected women at our institution differed from those of other HIV-infected women in other geographic regions . In that same article, the most carcinogenic HPV types concentrated in species alpha-9 and alpha-7 were distinguished by elevated risk of progression given persistence, rather than persistence alone. Alpha-5 and alpha-6 species were also at elevated risk of progression given persistence  they are not able to evade the immune system as efficiently as HPV types 16 and 18 except in HIV-infected individuals who are immunocompromised. To test this hypothesis regarding immunocompromised status and link with HPV type prevalence, a simple genotyping study in immunocompromised women not due to HIV  could be evaluated.In our sample, 95% 19/20) of the HPV types detected belonged to either alpha-9 or alpha-7 species. The alpha species of HPV are those subgroups of the virus that infect the genito-anal epithelium (/20 of thn = 8) contained a single infection. HPV-16 accounted for 75% of these single infections , however, these preliminary results support the feasibility of retrospective HPV genotyping from archival paraffin-embedded blocks in our lab as well as the need for further research in this area. Because developing countries are known to have higher rates of HIV than the developed world and HPV is more aggressive in HIV-infected individuals, the highest potential for global public health impact by a HPV vaccine would be conferred by its effectiveness in the HIV-infected population or those at risk of acquiring the infection. We are now planning to better characterize differences between HIV-infected and HIV-negative women in terms of their HPV type prevalence and their immunity profiles with the goal of guiding the development of both therapeutic and prophylactic vaccines that would be effective not only in the general population but also in the HIV-infected population.In summary, our study design was based on the positive predictive value of CIN 3 lesions in predicting HPV genotypes in invasive cervical cancer from the same population. Strength of our study was the use of histological pathology review for all samples confirming the grade of the lesions. Our findings are consistent with that of others who found rare HR-HPV types to be more common in dysplasia of HIV-infected women. In this pilot study, the sample was small (The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Human papilloma virus (HPV) associated Head and Neck Cancers (HNCs) have generated significant amount of research interest in recent times. Due to high incidence of HNCs and lack of sufficient data on high-risk HPV (hr-HPV) infection from North -East region of India, this study was conceived to investigate hr-HPV infection, its types and its association with life style habits such as tobacco, alcohol consumption etc.A total of one hundred and six primary HNC tumor biopsy specimens were collected. These samples were analyzed for hr-HPV DNA (13 HPV types) using hybrid capture 2 (HC2) assay and genotyping was done by E6 nested multiplex PCR (NMPCR).The presence of hr-HPV was confirmed in 31.13% (n = 33) and 24.52% (n = 26) of the HNC patients by nested multiplex PCR (NMPCR) and HC2 assay respectively. Among hr-HPV positive cases, out of thirteen hr- HPV types analyzed, only two prevalent genotypes, HPV-16 (81.81%) followed by HPV-18 (18.18%) were found. Significant association was observed between hr-HPV infection with alcohol consumption (p <0.001) and tobacco chewing (p = 0.02) in HNC cases. Compared to HPV-18 infection the HPV-16 was found to be significantly associated with tobacco chewing (p = 0.02) habit.Our study demonstrated that tobacco chewing and alcohol consumption may act as risk factors for hr-HPV infection in HNCs from the North-East region of India. This was the first study from North-East India which also assessed the clinical applicability of HC2 assay in HNC patient specimens. We suggest that alcohol, tobacco and hr- HPV infection act synergistically or complement each other in the process of HNC development and progression in the present study population. The Head and Neck cancer (HNC) incidence accounts for approximately 25–30% of all cancer cases in India . HNC is The master cell cycle regulators, p53, pRB and p16 are important tumor suppressor genes having significant role in cell cycle regulatory pathway and cancer . These gThe North Eastern (NE) region of India has high age adjusted incidence rate (AAR) per 1, 00,000 persons for HNCs in India . Among aA total of 106 HNC patients were enrolled in this study . Tumor tissue specimens were obtained from the Head and Neck oncology surgery unit of Dr. Bhubaneswar Borooah Cancer Institute (BBCI), Regional Cancer Center, Guwahati, India. The histopathologically confirmed HNC cases were enrolled during the period of October, 2011 to September, 2013. The demographic and lifestyle information were collected using a pre-designed questionnaire through personal interview. The information collected about demographic variables included age, gender, ethnicity, education, socio-economic status, life style factors of alcohol, cigarette smoking, betel nut chewing and other food habits. The American Joint Committee on Cancer’s TNM staging was used for staging and diagnosis. The patients were informed about the study and written consent was obtained prior to collection of the specimens. This study was approved by the institutional ethics committee of Dr. Bhubaneswar Borooah Cancer Institute (BBCI), Guwahati, Assam (India).Tissue biopsy specimens were collected in 1mL volume of phosphate buffered saline (PBS) and the genomic DNA was extracted from the homogenized tissue by using QIAamp DNA mini kit  following the manufacturer’s instructions. The quantity and quality of the isolated genomic DNA was confirmed by Nano-spectrophotometer  and agarose gel electrophoresis respectively.™ kit  as per the instructions of the manufacturer. In the patient STM samples, chemiluminescent detection of the 13 most common hr-HPV types  was performed using automated DML 2000 Luminometer system . Light signals were measured as relative light units (RLUs) with light intensity indicating the presence or absence of target DNA in the tested specimen. The mean RLU value for high risk HPV calibrator of positive control samples was considered as positive cutoff (CO) value. Patients specimen with RLU/CO value ratio ≥ 1 with high-risk HPV probe were considered “positive” for any of the hr-HPV types . The absence of hr-HPV DNA below the detection limit was denoted in sample with RLU/ CO <1. When RLU = CO it confers presence of approximately 5000 virus copies in the specimen. Two confirmed cases of cervical cancer with hr-HPV positivity were also used in each test run and carrier DNA as negative control [TM software ver. 2.0. All the samples were tested twice and samples with hr- HPV positive outcome in both test run were only scored as hr- HPV positive.Genomic DNA isolated from biopsy samples of the patients were suspended in the specimen transport medium (STM)  and the presence of HPV was detected by HC2 High-Risk HPV DNA Test control . The tesst round was done in 20 μL volume using 25–100 ng genomic DNA, 1X Maxima Hot Start PCR buffer, 1.5–2.0 mM MgCl2 , 250 nM of each primers , 250 μM of each dNTPs and 0.5 U Maxima Hot Start Taq DNA Polymerase . The thermal cycling condition for 1st round of E6 PCR was as: Denaturation- 95°C- 5 min, 38 amplification cycles with Denaturation- 95°C- 30 sec, Annealing- 55°C- 45 sec, Extension-72°C- 90 sec, and final Extension- 72°C for 7 min. The 1st round E6 PCR product (630bp) was used as a template (1 μL) for the 2nd round of nested reaction for HPV genotyping using multiplex set of HPV type specific primers. The reaction volume and constituents were similar as mentioned above. The 2nd round nested PCR condition for HPV genotyping was similar to 1st round with changes only in Annealing- 55°C- 40 sec and Extension-72°C- 45 sec [™ system . The hr-HPV positive cervical squamous cell carcinoma samples DNA was used as positive control in each PCR run. The primer sequences are described in The samples tested positive for hr-HPV DNA in the HC2 assay were further analysed for HPV genotyping. The nested E6 PCR reaction 1- 45 sec . All PCRThe data was represented as mean ± SD or in frequencies (%). The χ² test and Fisher exact test were employed to identify the association between clinico-pathological characteristics and hr-HPV status. Univariate analysis was done by calculating the odds ratios (ORs) along with 95% confidence intervals (CIs) and P ≤0.05 was considered to be the significance level. Epi-info Version 6 software was employed for the statistical analysisIn the study population (n = 106), 68.86% of patients (n = 73) were males and 31.13% were (n = 33) females. Patients’ age ranged from 25 to 80 years (Mean = 53.41 years). More than three fourths (80.18%) of the patients were from rural areas, rest (19.56%) were from semi urban and urban areas. Non-vegetarian dietary habit was reported by >92% of the patients . The stuThe high risk HPV DNA was detected by nested multiplex PCR (NMPCR) in 31.13% (n = 33) and hybrid capture in 24.52% (n = 26) of the HNC patients. The sensitivity and negative positive predictively was found to be 100% each; the specificity and positive predictive value (PPV) was observed as 91.19% and 78.78% respectively. This test classified 93.29% of the cases correctly and the area under the curve was found as 89.50% (p<0.001) with 95%CI (81.30% - 97.80%). To confirm the biologically active HPVs, p16 expression was analyzed in both HPV positive and HPV negative cases, and over expression of p16 was found in HPV positive cases which is an indirect indicator for the presence of HPV (data not shown). In HC2 tested samples, mean RLU value was in the range of 99–3466, and the mean positive cut off (CO) value for hr-HPV was 347  as per the instruction. The High risk HPV positive cases were genotyped for HPV subtypes using E6 nested multiplex PCR. The primer details used for the E6 nested multiplex PCR [Among hr-HPV positive cases (n = 33), oral cavity 72.72% (n = 24), larynx 6.06% (n = 2), hypopharynx 6.06% (n = 2) and oropharynx 15.15% (n = 5) had hr-HPV DNA . There wAmong hr- HPV positive cases, HPV-16 was present in 81.81% (n = 27) and HPV-18 in 18.18% (n = 6) of the cases. The tobacco chewers had significantly increased risk of HPV-16 infection (p = 0.02) but not of HPV-18. The risk of HPV-16 being positive is 11.88 times more in chewers as compared to non-chewers with 95% CI (1.01–317.2). However the smoking habit does not show any significance compared to non-smokers (OR- 0.14) with 95% CI (0.01–1.28). The HPV-18 positivity was found in 5 cases (83.33%) of non-chewers compared to chewers. HPV-16 infection in smokers was found to be higher (p = 0.05) compared to HPV-18 , Though The Human papilloma virus (HPV) has been identified as an etiological factor in subset of head and neck cancer especially oropharyngeal cancer . Most ofWe used E6 nested multiplex PCR (NMPCR) for HPV genotyping, and found HPV-16 in 81.81% (n = 27) and HPV-18 in 18.18% (n = 6) of the hr-HPV positive (n = 33) cases, which is 31.13% of HNC patients (33/106). Our study data is supported by previous studies from India which had found HPV DNA in the range of 22% to 73% in oral cancer cases –45. AmonWe observed in our data that tobacco chewers have significantly higher hr-HPV infection, posing tobacco chewing as a risk factor for hr- HPV infection. The concentration of nicotine and its exposure duration to the cell may alter the antigen mediated signaling pathways . AlteratIn the present study hr-HPV infection was found to be significantly associated with alcohol consumption in HNC cases. Oh et al (2014) recently demonstrated that hr-HPV load and alcohol consumption may have synergistic effect on hr-HPV infection and its persistence in cervical cancer . AnotherEthanol, a major constituent of alcoholic beverages, is oxidized to acetaldehyde by alcohol dehydrogenases (ADH). Acetaldehyde is metabolized to acetate by aldehyde dehydrogenases (ALDH). Poor oral hygiene and smoking can change the oral bacterial flora which can lead to increased acetaldehyde production . In animAlcohol can also modify innate immune response in dose dependent manner which can result in altered inflammatory response to infection . Since iOur study provides evidence about increase in vulnerability for hr-HPV infection in tobacco chewers and alcohol drinkers mostly in oral cavity cancer patients. Our observation is well supported by previous HNC studies from India and worldwide . From thWe conclude from our present study data that hr-HPV infection may be more prevalent in tobacco chewers, alcohol drinkers and they act as risk factors for HPV infection in HNC cases of North-East India. Hybrid Capture 2 (HC2) assay due to its high negative predictive value, specificity and sensitivity can be applied for hr- HPV detection in HNC clinical specimens.


HPV 16 is the cause of cervical carcinoma, but only a small fraction of women with HPV infection progress to this pathology. Besides persistent infection and HPV integration, several studies have suggested that HPV intratype variants may contribute to the development of cancer. The purpose of this study was to investigate the nucleotide variability and phylogenetically classify HPV 16 E6 variants circulating over a period of 16 years in women from Southern Mexico, and to analyze its association with precursor lesions and cervical carcinoma.This study was conducted in 330 cervical DNA samples with HPV 16 from women who were residents of the State of Guerrero, located in Southern Mexico. According of cytological and/or histological diagnosis, samples were divided into the following four groups: no intraepithelial lesion (n = 97), low-grade squamous intraepithelial lesion (n = 123), high-grade squamous intraepithelial lesion (n = 19) and cervical carcinoma (n = 91). HPV 16 E6 gene was amplified, sequenced and aligned with reference sequence (HPV 16R) and a phylogenetic tree was constructed to identify and classify HPV 16 variants. Chi squared was used and data analysis and statistics were done with SPSS Statistics and STATA softwares.Twenty seven HPV 16 E6 variants were detected in women from Southern Mexico, 82.12% belonged to the EUR, 17.58% to AA1 and 0.3% to Afr2a sublineages. The most common was E-G350 (40%), followed by E-prototype (13.03%), E-C188/G350 (11.82%), AA-a (10.61%), AA-c (6.07%) and E-A176/G350 (5.15%). Eight new E6 variants were found and 2 of them lead to amino acid change: E-C183/G350 (I27T) and E-C306/G350 (K68T). The HPV 16 variant that showed the greatest risk of leading to the development of CC was AA-a , followed by E-A176/G350 , AA-c , E-G350  and E-C188/G350 .The variants more frequently found in women with cervical carcinoma are E-G350, AA-a, AA-c, E-C188/G350 and E-A176/G350. All of them are associated with the development of cervical carcinoma, however, AA-a showed the highest association. This study reinforces the proposal that HPV 16 AA-a is an oncogenic risk for cervical carcinoma progression in Mexico.The online version of this article (doi:10.1186/s12985-015-0242-3) contains supplementary material, which is available to authorized users. Infection by high risk human papillomavirus (HR-HPV) is necessary for the development of cervical carcinoma (CC)  and HPV Like other HR-HPV, HPV 16 has well preserved distinctive intratypic variants by geographical origin , their gSeveral reports have shown the presence of common polymorphisms that generate amino acid changes in the E6 oncoprotein, one of them is T350G, and is present in the four lineages. T350G causes a leucine to valine change (L83V), that leads to the split of the EUR sublineage into three classes, 350 T (prototype sequence), 350C and 350G. Other polymorphisms including A131G, G132C, C143G, G145T, G176A, T178G and C335T generate the amino acid changes R10G/I, Q14H/D, D25E/N, I27R and H78Y, respectively .Epidemiologic data shows that regions with high incidence of cervical carcinoma like Latin America, Africa and Asia, also have a high prevalence of sublineages AA and Af . StudiesSocial disparities like access to social security health care services, ethnic groups, residence and socioeconomic level are factors associated with cervical carcinoma development . The StaWe have previously shown that HPV 16 was the most commonly identified HPV genotype in cervical carcinoma and high grade squamous intraepithelial lesions in women from the State of Guerrero. We studied a sample of HPV 16 positive women and found E and AA variants . It has Knowing the regional variants of HPV 16 is of great value for evolutionary, phylogenetic, epidemiological and biological analysis . To furtThe most dominant HPV variants were detected in low and high-grade squamous intraepithelial lesion, cervical carcinoma and no intraepithelial lesion, and 8 novel HPV 16 variants were found. An association between E-G350, E-A176/G350, E-C188/G350, AA-a and AA-c variants and the risk of developing cervical carcinoma was shown in this study.http://www.ncbi.nlm.nih.gov/nuccore/NC_001526.2) as a reference sequence, a total of 27 variants were detected, 8 of them were new. Sequence analysis showed substitution in 29 nucleotides located between positions 104 and 559 in the E6 sequence with a predicted amino acid change  belonged to the EUR sublineage, followed by AA1 (17.58%) and Afr2a (0.3%) sublineages. From sublineage EUR, 15.45% was class E-T350 which includes E-Prototype (13.03%), and 66.67% was class E-G350 finding 19 subclasses. From sublineage AA1, AA-a class was the most frequent (10.91%) followed by AA-c class (6.67%). From the 27 variants found in this study, the most common E6 variant was E-G350 (40%), followed by E-prototype (13.03%), E-C188/G350 (11.82%), AA-a (10.61%), AA-c (6.07%) and E-A176/G350 (5.15%). The 8 novel variants are subclasses, 6 are subclasses of class G-350, one is subclass of class AA-a and the last one is subclass of class AA-c  as squamous cell carcinoma (SCC), 13 (14.3%) as adenocarcinoma (ADC) and 2 (2.2%) as other epithelial tumors. The majority of cases of cervical carcinomas were found in the FIGO stage IIB (34%). Additionally, 19 samples were HSIL, 123 LSIL and 97 with non-IL .HPV 16 AA variants were more common in ADC (46.15%) than in SCC 30.27%). AA-a variants increased their frequency according to the degree of evolution of cervical lesion: 38.46% in ADC, 15.79% in SCC, 15.79% in HSIL, 8.94% in LSIL and 3.09% in non-IL. HPV 16 E variants were the most common in SCC (69.75%), HSIL (78.94%), LSIL (87.79%) and non-IL (89.69%). HPV 16 E-G350 class was the most frequent in all groups, E-Prototype, on the other hand, was not detected in ADC and other epithelial tumors, and only 2.63% of SCC, 5.26% of HSIL, 18.7% of LSIL and 17.53% of non-IL . Data from a follow up cytological diagnosis of 5 women with HPV 16 E6 novel variants were collected. Follow up information shows that two with the variant E-C183/G350 evolved from non-IL to LSIL, whereas those with E-C188/G310/G350 and E-G189/T256/G350 maintain LSIL status  is presented in Table Of the 27 HPV 16 variants found in Southern Mexico in 16 years, 8 of them were new and may be considered to be variants specific to this Mexican region.Previous data suggests that HPV 16 variants with E6 sequence variation are biologically distinct and may confer different pathological risks for development of squamous intraepithelial lesions and invasive cervical carcinoma. E6 specific sequence variations may modify its linkage to cellular targets changing its ability for p53 degradation, inhibiting keratynocyte differentiation, modifying signal transduction -34.It was proposed that there might be a strong relation between AA variant and cervical carcinoma development ,25,35,36According to the HPV variants found in this study, the amino acid substitutions circulating in Southern Mexico are: Q3E, R10G, Q14H, D25N, I27L, I27R I27T, L28V, E29K, E29Q, E29G, I52L, K68T, F69L, H78Y, L83V (the most frequent) and E113D. The amino acid substitutions present in the HPV 16 E6 variants in descending order of association with CC were: Q14R/H78Y/L83V (AA-a variant), D25N/L83V (E-A176/G350 variant), Q14R/I27/H78Y/L83V (AA-c variant), L83V (E-G350 variant) and E29Q/L83V (E-C188/G350 variant). Q14R/H78Y/L83V amino acid changes gender AA variants with increased oncogenic potential and more efficient evasion of the host’s immune surveillance ,38. L83VIt was not possible to analyze the association of novel E6 variants for the risk of developing cervical carcinoma because of the low number of positive samples; however, by having nucleotide changes may also be associated with the development of cervical carcinoma.Current findings show that in 16 years, at least 27 HPV 16 E6 variants were present in Southern Mexico and 8 novel variants were found, which may be considered to be variants specific to this Mexican region. The variants more frequently found in women with cervical carcinoma are E-G350, AA-a, AA-c, E-C188/G350 and E-A176/G350. All of them are associated with the development of cervical carcinoma, however, AA-a showed the highest association. This study reinforces the proposal that HPV 16 AA-a is an oncogenic risk for cervical carcinoma progression in Mexico. This represents the largest study carried out in Mexico analyzing all classes of European and non-European variants in the whole spectrum of disease, from intraepithelial lesion-free cytology to cervical carcinoma including squamous cell carcinoma and adenocarcinoma. Further studies are needed to clarify the pathogenicity of HPV 16 E6 variants.The database and biobank, with 7,480 cervical DNA samples collected from 1997 to 2012, of the Molecular Biomedicine and Cytopathology Laboratories at the School of Chemistry and Biology of the Autonomous University of Guerrero in Chilpancingo Guerrero, Mexico, was searched for all cervical DNA samples with HPV 16 and 330 were found in appropriate conditions for analysis. The samples were studied to investigate circulating HPV 16 variants in Southern Mexico and to do a comparative analysis between these variants and the different grades of cervical lesion.Cervical samples came from women who were residents of State of Guerrero, seeking cytological screening or for other gynecological complaints, which attended public health centers of Acapulco, Chilpancingo, and Iguala, the three biggest cities in this state of Southern Mexico. Based on the diagnosis, samples were divided into: (1) no intraepithelial lesion (non-IL) (n = 97), (2) low-grade squamous intraepithelial lesion (LSIL) (n = 123), high-grade squamous intraepithelial lesion (HSIL) (n = 19) and (4) cervical carcinoma (CC) (n = 91). Non-IL and LSIL samples have cytological diagnosis; HSIL and CC samples have histological diagnosis. Cytological diagnosis was done according to the Bethesda System  and histThis study was approved by the Bioethical Committee the Autonomous University of Guerrero. Informed consent was obtained from women participants.HPV DNA was detected and identified by three methods depending on the year of in which the sample was taken and analyzed: (1) from 1997 to 2010, HPV detection was done by the MY09/11 system and typing by restriction fragment length polymorphism (RFLPs); (2) from 2005 to 2010, detection was done by general GP5+/6+ PCR system and typing by sequencing analysis  when sam2, and 1.25 U of Platinum Taq DNA Polymerase (Invitrogen). The DNA amplification was done in a DNA Eppendorf AG 22331 Hamburg [The HPV 16 E6 gene was amplified using type-specific primers E6-F048 (5′GAACCGAAACCGGTTAGTAT3′) and E6-R622 (5′CAGTTGTCTCTGGTTGCAAA3′) that amplify a 575-bp region . PCR amp Hamburg  as follohttp://www.ebi.ac.uk/Tools/psa/emboss_stretcher/nucleotide.html and http://embnet.vital-it.ch/software/LALIGN_form.html) and the Finch TV program respectively. Sequences were aligned with reference sequence (HPV 16R) [PCR products were purified with 75% isopropanol  and ZR DNA Sequencing Clean-up Kit™ (ZYMO RESEARCH). These were sequenced using Big Dye Terminator Chemistry v3.1 Ready Reaction Kit  in an automated sequencer DNA ABI Prism 310 Genetic Analyzer  using primers previously described . SequencHPV 16R) . Using tHPV 16R) . The subHPV 16R) . When neHPV 16 E6 sequences were compared by multiple sequence alignments using the CLUSTAL W method . A phylop values were less than 0.05. Age-adjusted odds ratios and 95% confidence intervals were used to estimate associations. Data analysis and statistics were done using IBM SPSS Statistics V.22.0 and STATA V.11 softwares.The Chi squared test was used to compare HPV 16 variant frequencies and cervical lesion grade. Differences were considered to be statistically significant when