High-risk human papillomavirus type 16 (HPV16) is a risk factor for cervical cancer. Previous studies suggest that polymorphisms in the E6 gene or the long control region(LCR)of HPV16 may alter the oncogenic potential of the virus. The aims of this study were to investigate the genetic variations of HPV16 E6 gene and LCR in isolates from Chinese population and correlation of the E6 and LCR polymorphisms with disease status of infected patients.HPV16 positive endocervical specimens were collected from 304 women living in Northeast of China. Sequences of E6 gene and LCR were analyzed by PCR-sequencing.Two lineages were found in the populations, including EUR lineage and As lineage. Based on the HPV16 prototype, the most frequent variation in the E6 gene was T178A/G (48.7%), followed by mutations of G94A (12.2%) and T350G (9.9%). The rank orders of incidence of E6 variations in amino acid were as follows: D25E (46.3%), L83V (9.9%) and H78Y (4.3%). Nucleotide variations in LCR were found in all the 304 isolates from HPV16 positive cervical samples. The most commonly observed LCR variations were the transition replacement G7193T, 7434CIns, G7521A and 7863ADel (100%). The As lineage was associated with HPV persistent infections and with disease status of ≥CIN2,3. The EUR lineage variants showed a negative trend of association with the severity of ≥CIN2,3. Among 41 variations found in LCR, 25 (61.0%) were located at the binding sites for transcription factors. Occurrence of ≥CIN2,3 was significantly associated with the mutations of R10G/L83V in E6 and the C7294T co-variation in LCR, after adjusting for ages of infected patients.Associations between As lineage and HPV persistent infections, and with disease status of ≥CIN2,3, and an association between the EUR lineage and negative trend of association with the severity of ≥CIN2,3 were found in this study. An association between a co-variation of R10G/L83V in E6 and C7294T in LCR and an increased risk for developing CIN-2,3 was found in a HPV16 infected population of Chinese women. These findings indicate that HPV16 polymorphism influences development of CIN-2,3. Persistent infections of high-risk human papillomavirus (HPV) are the necessary causes of cervical cancer. However, only a minority of women infected with high-risk HPV will develop persistent infection . DevelopHPV type 16 (HPV16) is the most prevalent high-risk (HR) type worldwide and is found in the majority of cervical cancer cases . The HPVBased principally on LCR sequences, several studies from Europe and American have suggested that HPV16 variants can influence viral persistence and development of cervical cancer -11. The Three hundred and four HPV16 positive cervical cytobrush samples detected with flow-through hybridization  were collected from women admitted to the Affiliated Shengjing hospital of China medical university. According to the cytological and histological evaluations of fresh specimens, cervical disease statuses of the women were categorized as normal, cervicitis, atypical squamous cells of undetermined significance (ASCUS), cervical intraepithelial neoplasia grade 1, 2 and 3  and cervical cancer (CC). Histopathological diagnosis was completed by a pathologist who was unaware of the HPV detection results. Informed consent was obtained from participation in the study and this study was approved by the ethics committee of the Affiliated Shengjing hospital.To obtain HPV16 E6 and LCR sequences, a half-nested PCR was performed as that previously described . AccordiThe size of the PCR products containing HPV16 E6–LCR sequence was confirmed by gel electrophoresis. The amplicons were purified using the QIAquick gel extraction kit . Direct double-stranded PCR sequencing was performed for the E6-LCR amplicons using the same primers as in PCR by fluorescent cycle-sequencing method . All amplicons were sequenced at least twice to exclude Taq-induced errors. Sequences of the amplicons were aligned by CLUSTAL W (version 1.8). The obtained sequences were compared to the reference sequence (GenBank KO 2718) using NCBI BLAST. The HPV16 sequences were classified into respective variant classes as: prototype (GenBank KO 2718), European (E), Asian (As), African 1 (Af-1), African 2 (Af-2) and Asian American (AA) -15. To aThe association of HPV16 polymorphisms with persistent infection and developing risks of CIN2,3 or cervical cancer of women was investigated. Persistent infection was conceptualized at least two times positive for HPV detection in a series of samples collected at more than three visits. The proportion of women infected with HPV16 prototype was compared between participants with transient and persistent infection by Fisher’s exact test. In this study, CIN1 or low-grade squamous intraepithelial lesion (LSIL) is not considered as a precursor lesion to cervical cancer but a morphologic consequence of HPV infection. So, the controls including women without CIN or with CIN1 and LSIL are designated as <CIN2,3. After classifying the isolates into variant lineages, the association of HPV16 polymorphisms in women with CIN2,3 or cancer  was analyzed. The rate of HPV16 variants in 167 women with ≥CIN2,3 were compared to that in 137 women with <CIN2,3. The magnitude of the associations of HPV16 variants with ≥CIN2,3 was assessed by calculating odds ratios (OR) and respective 95% confidence intervals (CI). The association of HPV16 polymorphism in isolates from women with ≥CIN2,3 was assessed individually for HPV16 each variations. Fisher’s exact test was used to assess the significance of the differences between dichotomous variables, and a Mann–Whitney test was used to assess the significance of the differences of age between groups. Unconditional multiple logistic regression analysis was performed to obtain maximum likelihood estimates of the OR while controlling for the confounding effects of study site, age and smoking. Statistical analyses were performed with STATISTICA version 6 Software .Information about the studied population is shown in Table Tables Nucleotide variations of HPV16 E6 were found in 92.1% (280 of 304) of the HPV16 positive cervical samples, whereas amino acid variations were found in 65.5% (199 of 304). Nucleotide variations of E6 included 4 silent mutations of G94A, G176A, C215T and T241G and 5 missense mutations of A131C, T178G/A, C335T, T350G and A442C, which led to amino acid variations of R10G, D25E, H78Y, L83V and E113D, respectively. The most frequent nucleotide variation was T178G/A , in which T178G was found in 113 strains and T178A in 38 strains  of the 41 variations were located in the binding sites for transcription factors. The variation of G7193T resulted in loss of a putative binding site for the cellular transcription factor SRY. Furthermore, an insertion of 7434C resulted in a new binding site for the cellular transcription factor Oct-1, while a deletion of 7863A resulted in loss of a binding site for the cellular transcription factor E2. In the co-variation of 10 sites, the variation of A7515C resulted in loss of a binding site for the human activator AP-1, while the variation of A7602G resulted in an addition of binding site for the cellular transcription factor Oct-1.Two lineages were found in the populations, including EUR lineage and As lineage. The association of HPV16 polymorphisms with persistent HPV infections was investigated by comparing the proportion of certain variant in women with persistent (n =168) and transient (n=106) HPV infections  and 167 women with ≥CIN2,3 . Controlling for study, older women  and those with a history of smoking  were not at increased risk for developing ≥CIN2,3. The number of HPV genotypes per sample was similar between participants with <CIN2,3 (median of two genotypes) and those with ≥CIN-2,3 (median of three genotypes) .The As lineage was more frequent in pre-malignant and cervical cancer, and showed a statistically significant association for disease progression . While, the EUR lineage showed a statistically significant negative trend of association with the severity of >CIN2,3 . Individual polymorphism that varies within lineages may make sense to look at differences between different disease groups. However, the 350T/G, the 94G/A polymorphism in the EUR lineage and the 131A/C polymorphism in the As lineage did not showed any particular association with comparing relative risk.p=0.04) was significantly associated with risk for developing ≥CIN2,3. The C7294T co-variation in LCR, which include 10 variations, occurred in 4 strains from the 137 women with <CIN2,3 and 18 strains from the 167 women with ≥CIN2,3, and was significantly associated with development of ≥CIN2,3  (Table Two nonsynonymous variations of A131G (R10G) and T350G (L83V) were observed in HPV16 E6 gene. The variations of R10G/L83V occurred in 1 isolate from the 137 women with < CIN2,3 and 8 isolates from the 167 women with ≥CIN2,3. Controlling for study site, age and smoking, detection of the variant R10G/L83V (OR 4.7, 95% CI 1.0-23.5; In the present study, it was examined whether intratype variations of E6 and LCR may affect persistent infection and have higher risk for developing cervical disease. Sequence variations in E6 gene and LCR were found in 304 HPV16 isolates from women with various grades of cervical lesions. Phylogenetic analysis of HPV16 variants with respect to the E6 gene and LCR was conducted to study the local distribution of different HPV16 lineages, according to the classification of Yamada et al. . In our Different biological behaviors have been previously proposed for HPV16 infection. In Europe, it was reported that the frequency of Non-E variants, of the AA variant, in particular, increases strikingly with severity of lesion in infected women, suggesting that they could have a more relevant role in oncogenesis ,18. AlthHPV16 E6 polymorphisms have been shown, although inconsistently, to be associated with CIN or cervical cancer ,24. As rHPV16 variants have been refined by the LCR, a variable non-coding segment of HPV genome . Non-codIn summary, the finding of this study is that the R10G/L83V variations in E6 and C7294T co-variation in LCR was significantly associated with risk for developing ≥CIN2,3. Whether these associations are explained by a direct effect of E6 or LCR variations on HPV infection need to be complemented by functional studies further.The authors declare that they have no competing interests.Zhengrong Sun carried out the molecular genetic studies, participated in the sequence alignment and drafted the manuscript. Zhitao Lu carried out the molecular genetic studies. Jianhua Liu participated in the sequence alignment. Guili Wang participated in performed the statistical analysis. Weiqiang Zhou, Lianxia Yang, Chao Liu, Bo Wang performed the clinical detection. Qiang Ruan conceived the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/13/459/prepub


A 53 year-old male gynecologist presented with human papillomavirus (HPV) 16 positive tonsillar squamous cell carcinoma. He had no identifiable risk factors with the exception of long term occupational exposure to laser plumes, having performed laser ablations and loop electrosurgical excision procedures (LEEP) on greater than 3000 dysplastic cervical and vulvar lesions over 20 years of practice. The second patient is a 62 year old male gynecologist with a 30 year history of laser ablation and LEEP who subsequently developed HPV 16 positive base of tongue cancer. He also had very few other risk factors for oropharyngeal cancer or HPV infection. HPV is a probable causative agent for oropharyngeal squamous cell carcinoma and has been reported as being transmittable through laser plume. This paper suggests that HPV transmitted through laser plume can result in subsequent squamous cell carcinoma. Furthermore, there are two case reports describing health care professionals contracting oropharyngeal HPV following long-term occupational exposure to laser plumes. Certain HPV subtypes are known to be oncogenic, therefore, laser plume exposure may put health care professionals at increased risk of oropharyngeal carcinoma development. This case report describes, to our knowledge, the first cases of HPV-16 positive oropharyngeal squamous cell carcinomas in two surgeons following long-term occupational laser plume exposure.Carbon dioxide (CO2) lasers are commonly used to excise lesions on the larynx, cervix, lower genital tract, and perianal regions. Although this is an effective treatment modality, there are concerns regarding potential adverse events associated with its use. Tissue destruction from the laser's energy produces a gaseous plume containing cell contents and other aerosols. Many potential risks have been associated with laser plume exposure including the risk of human (HPV) transmission; Patient A is a 53 year-old male gynaecologist who consulted the Department of Otolaryngology having noticed a lesion on his right tonsil and a lump in the right side of his neck. His symptoms had started a few months prior to his visit and included increased fatigue. The physical exam revealed right tonsil hypertrophy and a small node at the posterior aspect of the right mandible. A CT scan of the region demonstrated a 2.2 cm soft tissue lesion in the right tonsil extending to the right soft palate; it also confirmed the presence of a level 2 lymph node. A biopsy of the right tonsil was performed confirming invasive squamous cell carcinoma of moderate to poor differentiation. The cancer was staged at T2N1M0. Testing at the National Microbiology Laboratory in Winnipeg, Manitoba revealed that the lesion was positive for HPV type 16 by hybrid capture assay (Luminex Hybridization).Patient A had no identifiable risk factors for oropharyngeal cancer or HPV with the exception of occupational exposure to HPV-positive laser plumes, having performed laser ablation and later loop electrosurgical excision procedures (LEEP) of more than 3000 dysplastic cervical and vulvar lesions over 20 years. Most of these procedures were performed in an environment without proper ventilation or mask. Being in a long-term monogamous relationship with his wife, he denies any other sexual contacts. She was tested following patient A’s diagnosis and was found to be HPV negative. Unfortunately, she had no HPV testing prior to patient A’s cancer diagnosis. Additionally, the patient is a non-smoker and consumes alcohol only occasionally. He has never been vaccinated against HPV. The association of his diagnosis with workplace exposure warranted his receipt of worker's compensation.He received Intensity Modulated Radiotherapy over 35 fractions with adjuvant cisplatin for 3 courses. Treatment side-effects caused significant reduction in quality of life due to marked dysphagia and xerostomia, which resulted in 40 lbs weight loss despite percutaneous gastrostomy feeding, radiation-induced Lhermitte's sign, and 40% high frequency hearing loss. He returned to work 11 months following initiation of therapy. In the four years since treatment, he has had no recurrence of squamous cell carcinoma; he was subsequently vaccinated against HPV types 6, 11, 16 and 18. He continues to practice gynecology, but has not returned to laser surgery.Patient B is a 62 year old gynecologist who recently consulted his local otolaryngologist after having a foreign body sensation in his throat for many weeks. A biopsy of the base of tongue revealed a squamous cell carcinoma; the lesion was positive for HPV 16. The base of tongue lesion was excised by laser and he also had a bilateral modified neck dissection. He had been practicing for 30 years, of which he spent 15 doing weekly laser ablations with a CO2 laser. He reported poor ventilation in this clinic space and subsequently moved to a different area where he performed loop electrosurgical excision procedures for the next 15 years. He is a non-smoker, only drinks occasionally, and was married twice. Once again, this patient may have contracted HPV through occupational laser plume exposure.Squamous cell carcinomas are accountable for 90% of head and neck cancers . TobaccoThe potential for HPV transmission through laser plume has been explored by various authors. Many studies have reported the presence of intact HPV DNA in laser plume of HPV-positive lesions; however, other studies have not been able to recreate these findings . Two casThe relationship between HPV and oropharyngeal squamous cell carcinomas has been studied extensively. A study of the American SEER database from 1984 to 2004 reveals that HPV-positive head and neck cancers have increased 225% during the study period, while HPV-negative head and neck cancers have decreased by 50% . RetrospPatient A's lesion was a poor to moderately differentiated cancer with a stage of T2N1M0. His cancer responded well to treatment and he has not had any relapse. HPV-positive and HPV-negative oropharyngeal squamous cell carcinomas seem to be two distinct types of cancer as they have different histological appearances  and geneThis article reports on a case of HPV-16 positive oropharyngeal squamous cell carcinoma in two laser surgeons following occupational exposures to laser plumes. There is now a strong body of evidence supporting a causal relationship between oncogenic HPV types and head and neck squamous cell carcinomas. It is also recognized that HPV may be transmitted through laser plume. Therefore, long term occupational exposure to laser plumes may lead to HPV infection and oropharyngeal squamous cell carcinomas. There is significant morbidity associated with these lesions and their treatment. It would thus seem prudent to reduce laser plume exposure amongst healthcare professionals. Existing protective methods such as standard surgical mask and laser mask have been described as ineffective against viral pathogens . The NatWritten informed consent was obtained from the patients for publication of the Case report. Copies of written consents are available for review by the Editor-in-chief of this journal.The authors declare that they have no competing interests.MR has reviewed the patient's chart as well as the literature and drafted the manuscript. AG has conceived this case report, participated in its coordination and has helped draft the manuscript. DW has helped review the manuscript and provided substantial intellectual content to the original manuscript. ER has helped draft the manuscript. All authors have read and approved the final manuscript.


No association between the Human T-cell lymphotropic virus (HTLV), an oncogenic virus that alters host immunity, and the Human Papillomavirus (HPV) has previously been reported. Examining the association between these two viruses may permit the identification of a population at increased risk for developing cervical cancer.Between July 2010 and February 2011, we conducted a cross-sectional study among indigenous Amazonian Peruvian women from the Shipibo-Konibo ethnic group, a group with endemic HTLV infection. We recruited women between 15 and 39 years of age who were living in the cities of Lima and Ucayali. Our objectives were to determine the association between HTLV and: (i) HPV infection of any type, and (ii) high-risk HPV type infection. Sexually active Shipibo-Konibo women were screened for HTLV-1 and HTLV-2 infections. All HTLV-1 or -2 positive women, along with two community-matched HTLV negative sexually active Shipibo-Konibo controls were later tested for the presence of HPV DNA, conventional cytology, and HIV. We screened 1,253 Shipibo-Konibo women, observing a prevalence of 5.9% (n = 74) for HTLV-1 and 3.8% (n = 47) for HTLV-2 infections. We enrolled 62 (60.8%) HTLV-1 positive women, 40 (39.2%) HTLV-2 positive women, and 205 community-matched HTLV negative controls. HTLV-1 infection was strongly associated with HPV infection of any type : 2.10, 95% CI: 1.53–2.87), and with high-risk HPV infection . HTLV-2 was not significantly associated with either of these HPV infections.HTLV-1 infection was associated with HPV infection of any type and with high-risk HPV infection. Future longitudinal studies are needed to evaluate the incidence of high-risk HPV infection as well as the incidence of cervical neoplasia among HTLV-1 positive women. Human viruses are associated with approximately 10–15% of human malignancies worldwide or approximately 1.3 million new cancer cases each year Certain HPV genotypes are oncogenic. These high-risk types are associated with the development of cervical cancer and with cervical intraepithelial neoplasia (CIN) stage 3, a precursor to invasive cervical cancer HIV and Human T-lymphotropic virus (HTLV) are both oncogenic retroviruses that target CD4+ T cells, which play a role in controlling HPV infection HTLV infects 10 to 20 million people worldwide and is endemic in southern Japan, the Caribbean, the Melanesian islands, Papua New Guinea, the Middle East, Central and South Africa and South America To our knowledge, there have been no reports regarding the association between HPV and HTLV infections. Examining the association between these two viruses may permit the identification of a population at increased risk for developing cervical cancer. The objectives of this study were to determine the association between HTLV and: (i) any HPV infection, and (ii) high-risk HPV infection.Our proposal and this study were approved by the Institutional Review Board of the University of Washington in Seattle and the Universidad Peruana Cayetano Heredia and U.S. Naval Medical Research Unit No.6 (NAMRU-6) in Lima, Peru. All enrollees provided a written informed consent prior to their participation in our study.Between July 2010 and February 2011, we conducted a cross-sectional study among women from Shipibo-Konibo communities between 15 and 39 years of age who lived in two communities in Lima, the capital of Peru, or in 22 Shipibo-Konibo indigenous Amazon Jungle communities located within four hours by car or boat from the city of Pucallpa. Sexually active Shipibo-Konibo women with HTLV-1 or HTLV-2 infection, along with two community-matched HTLV negative sexually active Shipibo-Konibo controls were later tested for the presence of HPV DNA, conventional cervical cytology and HIV infection. We defined women as belonging to the Shipibo-Konibo ethnic group if they had at least one of the following characteristics: 1) they self-identified as Shipibo-Konibo, 2) they spoke Shipibo-Konibo language, or 3) at least one of their parents belonged to the Shipibo-Konibo ethnic group.In our study, we first established community advisory boards (CABs) in Lima and in Ucayali. The objective of these CABs was to provide advice regarding approaches to engage the communities in the project and to receive feedback regarding study procedures. These CABs included leaders of the communities involved in the study, key personnel from the indigenous health program of the Interethnic Association for the Development of the Peruvian Rainforest (AIDESEP), and personnel from the Ministry of Health and Non Governmental Organizations working with indigenous peoples.After receiving feedback from the CABs, we initiated visits to the communities. The study visits were divided into two phases. In the first phase, we visited the chief of each community selected for the project. If the chief agreed to permit the community to be involved in the study, he signed the informed consent; we then conducted a census of the community to identify women who met our inclusion criteria. Later, we individually approached all eligible women at their home to assess their interest in participation. If participants were at least 18 years old, or between 15 and 17 years of age and married/cohabitating for two or more years, and they agreed to participate, they were invited to participate and asked to sign the informed consent. Other consenting eligible women signed an assent and their parent or legal guardian signed an informed consent. Afterwards, we administered a questionnaire and took a venous blood sample to test for HTLV infection.Approximately two months after the first visit, we carried out a second visit to the communities to deliver HTLV test results. During the second phase, we selected all women who tested positive for HTLV and for each HTLV-positive women, two randomly selected HTLV-negative women from the same community. After these participants signed a second informed consent, a midwife performed a speculum examination of the cervix, collected a cervical sample with a polyester swab, a sample with an Ayre spatula, and a cytobrush specimen for cytology. The polyester swabs were placed in a 4.5 ml cryotube containing 1 ml of Specimen Transport Medium  and transported in a cooler to the laboratory where they were stored at −20° until shipment to the University of Washington. The Pap smears were fixed with 96% ethanol for at least 15 minutes and then placed in special boxes prior to their shipment to Lima. The stored blood samples from all participants selected for the second phase of the study were tested for HIV infection.All women with abnormal cytologic results were referred for colposcopy and offered free treatment, when indicated. Women testing positive for HIV infection were referred to the Ministry of Health, where free highly active antiretroviral therapy is provided.During the first and second visits, we performed a survey to obtain demographic characteristics, sexual and other risk behaviors for Sexually Transmitted Infections (STIs), STI history, smoking status, drug and alcohol use, parity, abortion, contraceptive use, history of previous Pap smears, history of cervical abnormalities, knowledge about HPV and willingness to get vaccinated against this virus, risk factors for HTLV acquisition and history of HTLV related symptoms. The survey was administered in Spanish since in previous studies researchers identified that most women under 40 years living in that area spoke Spanish. We piloted the survey for understanding of language and accurate interpretation of questions prior to initiating the surveys.Survey information was collected using EpiSurveyor Mobile HTLV testing was performed at NAMRU-6 in Peru. HTLV-1 and -2 antibody testing was conducted using ELISA  with confirmatory Western blot assay . A woman was considered HTLV-1 seropositive if the ELISA assay was positive and confirmatory Western blot assay revealed bands representing gag , gp46, and two env proteins (GD21 and rgp 46-I). An individual was considered HTLV-2 seropositive if the ELISA assay was positive and confirmatory Western blot assay revealed bands representing p24, GD21 and rgp46-II. If gag and env proteins were absent, but other HTLV specific bands, such as p53 or p19 were present, the individual was considered indeterminate. Subjects with indeterminate results were excluded from the analysis.For HPV testing we used linear array assay (Roche Molecular Systems). For HPV DNA preparation, samples were digested with 20 μg/mL proteinase K for 1–2 h at 37°C. For each sample, 2400 μL was used to isolate DNA following the manufacturer's protocol . Then, each DNA sample was amplified using HPV L1 consensus primers MY09, MY11, and HMB01 and β-globin primers PC04 and GH20. Five µl of PCR products were then dotted onto nylon filters and probed with biotin-labeled β-globin and HPV generic probes. Samples negative for β-globin were considered insufficient for HPV DNA testing.Samples testing positive by the generic HPV probe were typed using the line-blot assay developed by Roche Diagnostics. This assay recognizes 37 individual HPV types: high-risk types, 16, 18, 26, 31, 33, 35, 39, 45, 51–53, 56, 58, 59, 66–68, 73, 82, and IS39; low-risk types, 6, 11, 40, 42, 54, 55, 61, 62, 64, 69–72, 81, 83, 84, and CP6108. For the analysis of high-risk HPV infection, we included types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 as defined at the 2005 meeting of the International Agency for Research on Cancer Cervical slides were sent to a cytopathologist in Lima for conventional cytology. Presence of endocervical or metaplastic cells were rated according to the Bethesda 2001 criteria We used a fourth-generation enzyme immunoassay (EIA) to detect HIV-1/2 IgG and IgM antibodies or HIV-1 p24 antigen . Positive samples were confirmed by Western blot.A generalized linear model, matching by community of residence, was used to assess the association between HTLV and (i) any HPV infection, (ii) infection with high-risk HPV type, (iii) cytologic abnormalities in the cervix, and (iv) presence of a low-grade squamous intraepithelial lesion (LSIL) or higher lesion. Prevalence ratios (PR) estimates were adjusted for age, education, age of sexual partner, number of sexual partners within the last 12 months, and condom use at last sexual intercourse. Analyses were performed using Stata .Between August 2010 and February 2011, we identified 1,460 women, and 1,402 agreed to be screened for HTLV infection. Of these women, 149 were not Shipibo-Konibo women  and were excluded from the analysis. Of the 1,253 eligible Shipibo-Konibo women, 74 (5.9%) tested positive for HTLV-1, 47 (3.8%) for HTLV-2 infection, and 4 (0.3%) had indeterminate results. Of the 121 HTLV-1 or -2 Shipibo-Konibo positive women, four were non-sexually active women; 11 migrated to other communities between the HTLV screening visit and the HPV testing visit; two did not agree to undergo the gynecologic examination, and two did not have controls from their community and were excluded from HPV testing. This analysis includes 62 (60.8%) HTLV-1 positive, 40 (39.2%) HTLV-2 positive, and 205 HTLV negative women who underwent HPV, Pap and HIV testing. Three of these women tested positive for HIV infection (two HIV positive women were negative for HTLV and one was positive for HTLV-2). These women were included in the analysis.HTLV-infected women were more likely to be older and less educated than HTLV-negative women . HTLV-inAfter adjustment for potential confounding variables, there was an association between HTLV-1 or -2 infection and the presence of any HPV type infection. There is the possibility of residual confounding for lifetime number of sexual partners, as we only adjusted for number of sexual partners within the last 12 months. We found a non-significant association between HTLV-1 or -2 infections and the presence of high-risk HPV infection, cervical cytologic abnormalities or presence of LSIL or higher lesion in the cervix .Women with HTLV-1 were two times more likely to have HPV infection of any type and high-risk HPV infection than HTLV negative women, after adjusting for confounding variables. However, HTLV-1 positive women had a similar prevalence of epithelial cell abnormalities in the cervix or presence of LSIL or higher grade lesion than women without HTLV .Women with HTLV-2 infection had a similar prevalence of HPV infection compared to HTLV seronegative women . They also had a similar prevalence of high-risk HPV type infection , abnormal Pap smear , or Pap smear with a LSIL lesion or higher lesion than controls .Among women with HTLV-1 or -2 infection, the most frequent high-risk HPV type was HPV 16 , followed by HPV 31 , HPV 18 , and HPV 52 , followed by HPV 18 , HPV 31 , HPV 52 , HPV 53 , and HPV 73 . The prevalence of HPV 6 was 4.8% (n = 3) and the prevalence of HPV 11 was 0%. The combined prevalence of HPV 16 or 18 infections was 19.4% (n = 12) and the combined prevalence of HPV 6, 11, 16 or 18 infection was 21.0% (n = 13).To our knowledge, this is the first description of a potential association between HPV and HTLV infections. Women with HTLV-1 infection had increased prevalences of HPV infection of any type and with high-risk HPV infection. HPV prevalence in women with HTLV-2 infection was similar to that found in HTLV seronegative controls. Among HTLV-1 infected women, the prevalence of HPV infection with any type was 43.6% and the prevalence of high-risk HPV infection was 32.3%. These prevalences are higher than those reported in Peruvian women from the general population (7.5% for any HPV type and 12.6% for a high-risk HPV type) The higher prevalence of HPV infection among HTLV-1 infected women could be due to a higher incidence of HPV infection among HTLV-1 seropositive women or to a longer duration of infection in these women. A higher incidence could also be the result of both viruses sharing sexual intercourse as a route of transmission; although HTLV-2 infection is also transmitted via sexual intercourse and was not similarly associated with higher HPV prevalence We did not detect an association between HTLV infection and cervical cytologic abnormalities. It is important to mention that our study was not powered to detect this association. A case-control study conducted in Mexico, comparing women with and without cervical cancer also did not detect an association between HTLV infection and cervical cancer If HTLV increases the risk for cervical cancer it is most likely through HPV infection. Two hospital-based case-control studies in Jamaica that assessed if HTLV infection increased the risk of cervical cancer above and beyond HPV infection did not find an association HTLV has also been associated with extensive genital warts. Radja et al. reported two cases of severe condylomatosis associated with HTLV-1 infection in a Caribbean and Ghanaian women who lived in the UK Our study has some potential limitations: 1) the cross sectional nature of this study precludes the determination of the temporal relationship between HTLV and HPV infection, and 2) the inclusion of prevalent cases of HPV infection means that any association with HTLV may be due to higher incidence or longer duration of infection.In conclusion, HTLV-1 infection was associated with higher prevalence of any HPV type infection and with high-risk HPV type infection. Future studies to evaluate the persistence of high-risk HPV type infection as well as the incidence of cervical neoplasia among HTLV-1 positive women could examine the temporal relationship between HTLV and HPV infection and could have implications regarding whether HPV vaccination should be emphasized for HTLV-1 infected women.


The site of action of HPV in the cervix is the squamous junction cylinder and the transformation zone. Abnormal epidermoid metaplasia and basal hyperplasia is the starting point for epithelial atypia.We assessed the dysplastic lesions of the cervix which underwent extended biopsy in the past 3 years - 579 cases and herpes virus type 2 infection was present in 2 cases.HPV-HR (high-risk) was positive in 377 cases, HPV-LR (low-risk) was positive in 165 cases and in 35 cases no HPV typing was performed. The herpes virus type 2 infection was present in 2 cases.Cytology showed LSIL  in 329 cases and HSIL  in 189 cases.Histological examination revealed cervical intraepithelial neoplasia (CIN) I in 69 cases and CIN II, CIN III in 183 cases and carcinoma in situ in 6 cases.HSIL cases all presented infection with HR  strains.Concomitance of cervical dysplasia with HSIL cytology is a major risk factor in cervical neoplasia.


Information on HPV knowledge in patients with genital warts is scarse as is the information on factors related to the impact on self-esteem and sex life among them.2 test.We conducted a cross-sectional study in adult patients with a clinical diagnosis of genital warts (GW) attending a major private out-patient clinic in Bogotá, Colombia. Patients underwent biopsy for pathological diagnosis, HPV-DNA testing and completed a questionnaire assessing HPV knowledge, and the consequences of GW on self-esteem and sexual life. Differences in proportions were assessed with a chi106 men and 155 women had pathologic confirmation of GW. 51% of subjects had heard of HPV before consultation coming mainly from the media (82%). Less than half of the participants knew that HPV could be transmitted through non-penetrant sexual intercourse and only two thirds acknowledged HPV vaccine as a preventive measure against HPV infection. Impact on self-esteem was higher among women than men . In men, factors related to a higher impact on sexual life were HPV awareness and age; in women they were higher education and anatomic location; external GW had a higher impact on sexual life in women .We found a low awareness of HPV and low knowledge on the vaccine as a preventive measure for associated diseases even in patients suffering from genital warts, highlighting the need for communication and education on HPV. Greater impact on self-esteem in women might reflect higher health consciousness among Latin American women. Genital warts (GW) are caused by infection with certain types of human papillomavirus virus (HPV) and are one of the most prevalent sexually transmitted infections in the world ,2. The dAlthough information on the prevalence of genital warts is scarce, it is estimated to be approximately 1% among the sexually active populations in some high-income countries. Data also suggest that the cumulative lifetime risk of GW can reach around 10% ,4. In adThe recent development of prophylactic HPV vaccines that provide protection from HPV types 6 and 11   is a potIn the Latin American or Hispanic population, despite the high burden of cervical cancer in the region , little Importantly, the proportion of persons that have heard about HPV varies between studies 13% to 95%), but most report that, in general, the knowledge of the virus remains lacking  although% to 95%,Data indicate that patients with GW suffer anxiety, experience shame and carry the stigma of having a venereal disease. All of these disease sequelae affect sexual and love lives as well as health-related quality of life -18, thouThe aim of this study was to examine awareness and knowledge of both the HPV virus and the HPV vaccine, as well as explore how genital warts affects self esteem and sexual life in patients with genital warts attending an outpatient clinic in Bogotá, Colombia.a in Bogotá, Colombia.The present report was part of a broader study entitled “Prevalence of HPV genotypes in genital warts of a Colombian population” that was approved by the Ethical Review Board of Profamilia Between December 2009 and August 2010 we invited male and female patients, aged 18 to 45 years, who lived in Bogotá at the beginning of the study and who attended the clinic with lesions suspected of GW. Patients with clinical diagnosis of GW were invited by the gynecologist or urologist in the first consultation to participate in the study and consented their participation; all patients underwent a biopsy for the establishment of the associated HPV genotypes and afterwards a questionnaire was applied by a trained interviewer. Patients not willing to participate, having received the HPV vaccine or with a known diagnosis of an immune suppressive disease were excluded from the study. The questionnaire included sociodemographical aspects, awareness and knowledge of HPV and HPV vaccine, sources of HPV-related information, initiation of sex life and previous medical history of STDs. Clinical features of the lesions diagnosed were also registered.Affiliation to the Colombian Health System was defined according to the law in three broad regimes: ‘contributive’, corresponding to workers or their families, where a proportion of the salary goes to the payment of the insurance; ‘subsidized’ corresponding to persons and their families of lower economic conditions where the government subsidizes their basic health care and ‘non-affiliated’ for those who at the moment of the clinical diagnosis were not covered by the health system.Awareness of HPV and HPV vaccine was assessed through the question whether patients had heard about HPV (or HPV vaccine) before coming to the clinic. Knowledge was assessed through a series of “false or negative” statements on HPV transmission, on HPV related diseases  and on preventive measures . To evaluate the effect of GW on self-esteem we included the following question used in a qualitative study . “Have GPhysicians involved in the study were given a short training on general conceptual and operative aspects of the study. A pilot study with 20 patients was carried out for adjustment of questionnaires and general procedures.2 test. A multinomial lineal model was used to evaluate what variables were associated with self-esteem, calculating prevalence ratios and 95% confidence intervals. A multivariate model was used to check whether there was confusion between variables related to awareness of HPV and HPV vaccine. All statistical procedures were performed using STATA® software.Frequencies were presented as absolute values and percentages. Differences in proportions, were assessed with a chiThe study included 342 patients (139 men and 204 women) with genital lesions suspected to be GW, with a participation rate of 99% (Only three patients refused to participate). From these, 106 (77%) men and 155 (76%) women had pathologic confirmation of GW and all responded the survey and were included into the analysis45% of the study population was between 25 and 34 years old and 40% was 18 to 24 years old. The majority  pertained to the contributive health regime. Half of the men and women had heard about HPV before consultation  . When exploring the variables that might be associated with this outcome by sex, none of them showed a statistically significant association Table .p value 0.05) . Factors associated with this outcome were not the same by sex. In men, both age and knowledge of HPV were related to a severe impact on sex life. Absence of impact decreased to 25% in men aged 35–44 years from 63% in men aged 18–24 years. Likewise, absence of impact on sexual life decreased with HPV awareness from 43.4% to 26.4% . To our knowledge it is the first study in Latin America evaluating awareness, knowledge and attitudes towards HPV in male and female patients affected by GW. The majority of the studies in our region have focused on cervical cancer and pre neoplasic lesions. The main limitation of the present study is that we didn’t use a pre-established validated questionnaire nor any cognitive testing or focus groups to assess the impact of GW in individuals. As such, ratings of the impact on sexual life remain subjective and without explanation on the issues lying behind. However, we explored 2 of the domains included in such instruments  that havWe found evidence of low HPV awareness in patients with GW. No differences in HPV knowledge were seen among different gender or age strata. Despite HPV awareness, GW affect both, self-esteem and sexual life in women and in older men, who identify GW as a sexually transmitted disease. This information is valuable to highlight the burden of HPV beyond cervical cancer in Latin American populations and encourages further research in this field. It also clarifies the benefits and needs of HPV prevention through education and vaccination.aProfamilia (Asociación Pro-bienestar de la Familia Colombiana) is a private organization, specialized in sexual and reproductive health that offers medical services throughout the country.Marion Piñeros has been member of an Advisory Board of MSD for HPV vaccine and has received travel grants from MSD for attending scientific meetings; Gustavo Hernández has received travel grants from MSD for attending scientific meetings; Gonzalo Pérez is employee at Merck&Co, INC.; the other authors declared no conflicts of interest. IJME formats have been uploaded for each author.MP participated in the design of the study, supervision, analysis and writing of the present manuscript. GHS participated in the design of the study, conduction, supervision, data processing, analysis, critical appraisal of the manuscript and writing of some parts of the article. LO participated in the pilot study, data collection, interviewing of patients, quality review and critical appraisal of the manuscript. JCV participated in the pilot study, data collection and critical appraisal of the manuscript. GP participated in the conception of the study, design of the study, data review and critical appraisal of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/13/272/prepub


Papillomaviruses have been implicated in a variety of human diseases ranging from common warts to invasive carcinoma of the anogenital mucosa. Existing assays for genotyping human papillomavirus are restricted to a small number of types. Here, we present a comprehensive, accurate microarray strategy for detection and genotyping of 102 human papillomavirus types and validate its use in a panel of 91 anal swabs. This array has equal performance to traditional dot blot analysis with the benefits of added genotype coverage and the ability to calibrate readout over a range of sensitivity or specificity values. Papillomaviruses are a group of nonenveloped, epitheliotropic DNA viruses that infect the skin and mucous membranes of humans and animals. There are over 100 different human papillomaviruses (HPV), which are associated with disease of the skin, mucous membranes, and aerodigestive tract. HPV infection leads to lesions ranging from common and genital warts to laryngeal papillomatosis, to epithelial cancers, particularly cervical carcinoma and a subset of head and neck squamous cell carcinoma (HNSCC). Detection of papillomavirus is usually based on molecular assays for viral nucleic acid [papillomaviridae. The HPVs fall into five genera: alphapapillomavirus (α-PV), betapapillomavirus (β-PV), gammapapillomavirus (γ-PV), mupapillomavirus (μ-PV), and nupapillomavirus (ν-PV). The mucosal α-PV are the best described, due to their association with genital malignancy. There are thirteen types of high-risk mucosal α-PV associated with cervical and anal intraepithelial neoplasia: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68. It has recently been recommended that HPV 66 be added to this high-risk group [There is considerable sequence diversity within the family Most commercial assays for alphapapillomaviruses are limited to the high-risk types, which are relevant for use in the clinical setting but provide limited information in the research setting. The current FDA-approved tests for HPV are the Digene Hybrid Capture 2 (HC2) test and the Cervista HPV16/18 and HR test. HC2 combines antibody capture and chemiluminescent signal detection to detect 13 high-risk HPV types , while the Cervista HR uses invader chemistry to detect 14 high risk types  . While bThere are currently no genotyping assays that cover multiple HPV genera. The majority of laboratory tests use PCR amplification with degenerate or multiplex primer sets followed by genotype readout with sequencing or blot, or bead-based Multiplexing –13. A nuThe obstacle to a comprehensive detection strategy is the sequence diversity of HPVs. Standard PCR assays are sensitive, but it is difficult to design primers to detect a broad range of papillomavirus types. Here, we present a microarray strategy for the detection and genotyping of 102 HPV types representing all genera and validate its use in a large panel of anal swabs from an HPV cohort study.Ninety-one samples were assayed, drawn from a previously reported cohort study of HPV infection in the anal canal of homosexual and bisexual men . The stuhttp://derisilab.ucsf.edu/index.php?page=software). Ten probes were selected for each genome with a target GC content of 30% and a maximum binding energy cutoff of −45 kcal/mol. Sequences containing AT regions longer than 20 bp and less than 10% of non-AT nucleotides were excluded from probe selection. A second set of genotype-specific probes were generated using a tiling strategy, with an offset of 20 nt across the whole genome and an offset of 5 nt over the L1 region, the region traditionally used for genotyping. These probes were filtered for a predicted binding energy of <−45 kcal/mol with their source HPV species and a predicted binding energy of >−10 kcal/mol against all other HPV species. All probes were filtered against nonspecific binding to the human genome. 60 nt oligonucleotide probes were designed based on the full sequences of the 102 full-length HPV genomes listed in GenBank on January 6, 2009 . Genotype-specific probes were designed using ArrayOligoSelector , a desigAdditional probes were designed to cover conserved regions in each genus using a variation on a previously described strategy . Each fuOverall, 14,161 60-mer oligonucleotide probes and their corresponding reverse complements were designed. The array was synthesized on the Agilent 8x60K SurePrint G3 custom array platform . Each of the 28,322 probes was printed in duplicate (Supplementary File 2).DNA was randomly amplified and labeled with Cy3 using an adaptation of a previously described protocol without the reverse transcription step . In paraValues from replicate spots on the array were averaged for analysis, and data were analyzed using the E-Predict algorithm . An E-maA set of uninformative oligonucleotides were defined as probes with significant autofluorescence when hybridized to water and probes with cross-hybridization to human DNA in a testing set of HPV-negative specimens. These were excluded from E-Predict analysis.α-HPV PCR was performed using the nested primer sets MY09-MY011 and GP5-GP6, as previously described in [E. coli (Invitrogen), according to manufacturer's specifications. For each DNA sample, 12 transformant colonies were selected for PCR insert amplification using M13 forward-reverse primers followed by sequencing on the ABI 3130xl Genetic Analyzer .ribed in . The PCRA receiver-operating characteristic (ROC) curve was generated to assess the similarity score for each of the 102 possible HPV genotype outcomes on array with the actual genotype(s) detected by sequencing of the 91 samples, for a total of 9282 tests. Sensitivity and specificity for the array were calculated with similarity score cutoff values of 0.035 and 0.1. Sensitivity and specificity were calculated for the blot based on the 29 types covered, for a total of 2639 tests. Agreement and Cohen's kappa coefficients were calculated for the blot versus the array for the 29 types covered on the blot. All statistical analysis was performed with Stata 11 .28,322 60-mer oligonucleotide probes were designed and printed on the array. An E-matrix was generated to reflect the theoretical hybridization energies of each genome in the design set to each oligonucleotide on the array. This matrix was used for E-Predict analysis of biological samples hybridized to the array. Performance of the array for one representative HPV type, HPV18, is demonstrated in To characterize the performance of the array across all genomes, DNA from 91 anal swab samples was randomly amplified and hybridized to the array. For each specimen run on the array, a similarity score was generated for each of the 102 HPV genotypes in the E-matrix, generating 9,282 observations. These results were compared to a gold standard of PCR and sequencing. A ROC curve was generated with an area under the curve of 0.8287 .Based on the ROC curve, two similarity score cut-points were generated for sensitivity and specificity calculations . A scoreEach of the samples had previously been genotyped for HPV with a PCR and blot assay that covers 29 individual types and a probe mixture that detects ten additional types but does not discriminate between them. Compared to PCR and sequencing, the blot had a sensitivity of 0.41 and a specificity of 0.98 for the 29 specific types covered, similar to the array cut-point of 0.1. For the 29 types covered on the blot, the two assays had an agreement of 96.6%, kappa 0.6, representing good agreement . Among the 91 samples, 157 instances of 34 HPV types were detected by PCR and sequencing. The types detected were HPV 6, 11, 12, 16, 18, 26, 31, 33, 34, 35, 40, 44, 45, 52, 53, 54, 55, 56, 58, 59, 61, cand62, 66, 67, 70, 71, 72, 81, 82, 83, 84, cand85, 97, and 102. Among those, there were 22 instances of 11 types which were not specifically covered by the blot strategy. Eighteen of these 22 infections (82%) were detected on the microarray .We demonstrate the performance of a comprehensive microarray for detection and genotyping of HPV and validate this system on a large panel of anal swab samples. Previous microarrays for HPV genotyping were designed to read out multiplex or degenerate PCR amplification and targeted a small number of HPV types. The main benefit of this system is its accuracy for broad range of HPV genotypes, as demonstrated by an area under the ROC curve of 0.8287. Because the nucleic acid amplification is not reliant on degenerate or multiplex PCR, any HPV type present in the sample may be detected.s value cutoff of 0.1 yielded a sensitivity of 0.41 and specificity of 0.99. This cutoff favors specificity over sensitivity, a feature common to many HPV assays in clinical use. This is based on the prevalent assumption that extremely low levels of HPV in a sample are likely not clinically relevant. One example of this in practice is the blot assay used to characterize this sample set in a previously reported study [In this sample set, an ed study . Our arrAs well as covering more HPV types, our assay is more flexible than the standard array readout system. Because the similarity score is a continuous variable, the cutoff can be tailored to the needs of the assay system. The similarity score cutoff that optimized both sensitivity and specificity was at a score of 0.035 . This yiα-PV detection with the Digene Hybrid Capture II Assay, the Roche Linear Array Assay, and the Kurabo GeneSquare Assay and reported a sample proportion of agreement ranging from 0.86 to 0.89 between the three assays. In two studies assessing multiplex PCR and array primer extension (APEX), Gheit et al. compared their arrays to reverse line blot or to one-step PCR and reverse hybridization with overall kappas of 0.55 and 0.5. In those studies, only a few samples were selected for sequencing to confirm array detection of types not seen on the other assays [We validated the performance of this microarray by a comparison to a gold standard of sequence genotyping. No previous study has reported microarray sensitivity and specificity with this type of validation. Some studies reported limits of detection and the absence of cross-hybridization but did not address true sensitivity and specificity , 20, 21.r assays , 15. In α-PV have been evaluated by calculating the sensitivity and specificity of the test for a cytologic diagnosis of neoplasia, which may be clinically relevant for a screening diagnostic but does not address the performance of the assay in genotyping HPV [A number of arrays targeting the high-risk ping HPV , 23, 31.The theoretical E-matrix of energy profiles used to generate similarity scores was created from the 102 HPV genomes used for oligonucleotide probe design. However, the E-matrix can be expanded to include additional papillomavirus genomes if desired, allowing for an updated genotyping strategy without the synthesis of additional probes. This flexibility will lend itself to future studies as the number of complete papillomavirus sequences continues to expand. As vaccination for HPV types 6, 11, 16, and 18 increases, we may see a shift in HPV prevalence and the rise of less common types. This assay is well suited to detect these trends.Overall, this microarray provides a comprehensive and accurate solution for the detection and genotyping of a broad spectrum of HPV types. It will lend itself to high-throughput applications such as screening of tumor cohorts and large epidemiologic studies.Supplementary File 1: HPV genomes used for probe design. This list includes the 102 full-length HPV genomes listed in GenBank on January 6, 2009.Supplementary File 2: Oligonucleotide probes included on the array.Click here for additional data file.


Human papillomavirus (HPV) variants differ in their biological and chemical properties, and therefore, may present differences in pathogenicity. Most authors classified variants based on the phylogenetic analysis of L1 region. Nevertheless, recombination in HPV samples is becoming a usual finding and thus, characterizing genetic variability in other regions should be essential.We aimed to characterize the genetic variability of HPV 18 in 5 genomic regions: E6, E7, E4, L1 and the Upstream Regulatory Region (URR), working with both single infection and multiple HPV infection samples. Furthermore, we aimed to assess the prevalence of HPV 18 variants in our region and look for possible existence of recombination as well as analyze the relationship between these variants and the type of lesion.From 2007 to 2010, Clinical Microbiology and Infection Control Department analyzed 44 samples which were positive for HPV 18. Genetic variability was determined in PCR products and variants were assigned to European, Asian-amerindian or African lineage. Recombination and association of variants with different types of lesion was studied.Genetic analysis of the regions revealed a total of 56 nucleotide variations. European, African and Asian-amerindian variants were found in 25/44 (56.8%), 10/44 (22.7%) and 5/44 (11.4%) samples, respectively. We detected the presence of recombinant variants in 2/44 (4.5%) cases. Samples taken from high-grade squamous intraepithelial lesions (H-SIL) only presented variants with specific-african substitutions.Multiple HPV infection, non-european HPV variants prevalence and existence of recombination are considered risk factors for HPV persistence and progression of intraepithelial abnormalities, and therefore, should be taken into consideration in order to help to design and optimize diagnostics protocols as well as improve epidemiologic studies.Our study is one of the few studies in Spain which analyses the genetic variability of HPV18 and we showed the importance of characterizing more than one genomic region in order to detect recombination and classify HPV variants properly. Based on the epidemiologic classification in terms of their risk to induce cervical cancer, human papillomaviruses (HPV) can be divided into 3 groups: “high-risk” genotypes associated with a greater risk of developing cancer, “low-risk” genotypes associated with low grade cell changes or benign epithelium proliferations in the genital area, but not with cancer, and “probable high-risk” genotypes from which there is not enough data about their relationship with cervical cancer to classify them .About 15 genotypes are classified as high-risk types, and two of them 16 and 18) cause over 70% of all cervical cancer cases ,3. Nucle and 18 cMost authors classified variants based on the phylogenetic analysis of one genomic region nucleotide variations . NeverthThere are very few epidemiological national studies in Spain and all of them refer to HPV 16 which is the most investigated HPV type worldwide. However, there is no previous national work related to HPV 18 nucleotide variability, which is the second most prevalent HPV genotype found in cervical cancer.The aim of the present study was: i) to characterize the genetic variability of HPV 18 in 5 genomic regions: E6, E7, E4, L1 and the Upstream Regulatory Region (URR), working with both single infection and multiple HPV infection samples, ii) assess the prevalence of HPV 18 variants in our region and look for existence of recombination, and iii) analyze the relationship between variants and types of lesion.Since 2007 to 2010, a total of 1085 positive samples for HPV were received and analyzed. HPV 18 was detected in 65 samples (6%). Forty-four patients consented to have their samples analyzed and studied, so this study was based on their samples: 10 single HPV infections (22.7%) and 34 multiple HPV infections (77.3%).We were able to amplify HPV DNA in 43/44 samples for E6 region, 41/44 for E7, 35/44 for E4, 43/44 for L1 region and 44/44 for URR region. All PCR products were sequenced and sequences from each region were submitted to GenBank.Variant distribution was determined through E6, E7, E4, L1 and URR sequences. Genetic analysis of the regions revealed a total of 56 nucleotide variations , T251C (9/10), A548G, G266A and G374A  and C342T (5/10 African variants) which lead to a non-synonymous amino acid alteration His/Tyr. A non-synonymous substitution T318C (Tyr/His) was found to be specific to the European lineage (2/25 European isolates), while the synonymous substitution C549A was detected among the three different branches (35/43 sequenced E6 amplimers). In our study, C287G was observed in all HPV 18 isolates.In the E7 gene genetic variability analysis revealed five nucleotides substitutions. Three nucleotide variations were specific for the African lineage: C665T (3/10 African variants), C593T (His/Tyr), C640C and T864G (Asn/Ser). All of them but C665T and T864G were present in all African variants. One synonymous substitution (C751T) was detected in both European and Asian-amerindian isolates (26/34 non-African variants).E4 gene analysis presented most nucleotide variations (17 substitutions) and almost half of them (8/17) lead to amino acid changes. All African variants showed 4 non-synonymous substitutions , one synonymous variation (T3534C) and a deletion of 6 amino acids (3627–3632). European variants showed 4 specific non synonymous substitutions G3482A , T3563A , C3617T  and C3630G . Two non-synonymous substitutions were also detected in two European isolates: T3492A and C3615T.L1 gene and URR sequence analysis demonstrated the presence of substitutions C6842G and T7592C in all our isolates.Most nucleotide variations found in our study have been already described in literature except for T318C, C665T, C3615T, C3617T, G6897A, G6993A, A7000T/C, T7001C, T7007G and T7765G. Only substitutions in positions 318 and 3617 lead to amino acid changes . T318C substitution was present in 2 European isolates whereas C3617T nucleotide change was not specific to any lineage and was detected in 5 samples.In our study, the predominant variant found was the European (25/44 samples) followed by the African (10/44) and the Asian-amerindian variants (5/44).Phylogenetic analysis of the all regions studied showed that European and Asian-amerindian lineages formed closely related nodes as well as a maximal nucleotide diversity between African and non-African variants Figure  Additio.Isolates which showed nucleotide diversity from the three branches  - LSM3, LSCM, LSCE, LSMK1, LSCR, LSMH1, LSC5, LSM7 and LSCB – were analyzed for possible recombination (Table E6 (Asian-amerindian) due to the lack of one nucleotide substitution (C549A). This sample was also classified as European.Phylogenetic trees were constructed for each sequenced region from these isolates and we found that samples LSCR, LSMH1, LSC5 and LSM7 belonged to the European branch in all regions and therefore, were classified as Europeans. Isolate LSCB belonged to the European lineage in all regions but in Samples LSM3, LSCM, LSCE, LSMK1 belonged to the African branch in some regions but were classified as European in others , 9 samples were diagnosed as L-SIL while presence of H-SIL was detected in 3 specimens.High grade lesions only presented African variants  and variants that presented both African and European substitutions  whereas most European and Asian-amerindian variants were detected in negative cytologies . Nevertheless, there was not a statistically significant association between type of infections (single vs multiple) and presence of lesions (p = 0.18078).There are almost no epidemiologic studies about HPV 18 variants carried out in Spain and even though national HPV prevalence is low, it cannot be forgotten that this genotype together with genotype 16 cause 70% of cervical cancer cases.Many authors confirm that distribution of HPV variants is related to geographic or race distribution ,17 and tHPV 18 has been associated with both recurrent lesions with very bad clinical prognosis . This faMost nucleotide changes reported in our study have been previously described and some of them are of particular importance. In URR, the mutation A41G is located in the Sp-1 binding site and isolates with this nucleotide variation have shown to have an increased transcriptional activity . VariatiSubstitutions C287G, C6842G and T7592C were found in all our isolates. Variation C6842G has been previously reported as error in the original sequence , and H. Furthermore, ten “new” nucleotide variations have been detected and two of them were non-synonymous and lead to amino acid changes .Knowledge on HPV variants and their nucleotide variability is essential for three main reasons: i) nucleotide variations may interfere with the viral oncogenic potential, ii) host cellular immune response can be different when there are substitutions in the amino acids on the viral capsid which may be relevant for the vaccination, iii) HPV infections with a variant may not give immunological protection against a subsequent infection with other variant of the same genotype.Nowadays, HPV variants recombination has already been described and it is more often found since coinfection with more than one HPV type prevalence is not a unusual finding .In our study we detected 2 recombinant variants (4.5%) which might have been missed or wrong classified if only amplifying one genomic region. Furthermore, non-recombinant samples as LSCM or LSM3 showed specific-african substitutions in some regions (for example E6) whereas they would be classified as european variants when only analyzing nucleotide variation in URR. Therefore, characterizing more than one genomic region may be essential in order to detect recombination and classify HPV variants properly.E6, E7 and L1genes from at least 93% of samples. However, when characterizing E4 region, we were only able to amplify 35 samples. E4 gene is generally disrupted during DNA integration into the host genome and this disruption may explain the inability to amplify E4 gene in some of our samples.We amplified URR and In conclusion, data and knowledge on geographic HPV intratypic variants distribution might help to establish a data base about the diversity and pathogenicity of different HPV variants, which may help to design and optimize diagnostics protocols in order to reduce the disease.Clinical Microbiology and Infection Control Department at Basurto University Hospital  analyzed samples which were remitted from different Hospital Services, especially the Consultation of Sexually Transmitted Diseases and the Department of Obstetrics and Gynecology, from 2007 to 2010.All samples were collected from patients with clinical manifestations of HPV related infections. Lesions were classified by pathologists into three categories: negative (no lesion was found), low-grade squamous intraepithelial lesion (L-SIL) or high-grade squamous intraepithelial lesion (H-SIL).Molecular genotyping was carried out using “Linear Array HPV Genotyping Test” kit (Roche Molecular Diagnostics). In our study, we analyzed positive samples for HPV genotype 18 (both single infections and multiple HPV infections) from patients who had given written, informed consent.DNA extraction was performed by QIAamp DNA mini Kit , according to the manufacturer´s instructions. Extracted DNA was eluted with 200 μl AE buffer and stored at −20°C until amplification.E6, E7, E4 genes and the URR was performed using type-specific primers designed according to HPV 18 genome prototype sequence (GenBank accession number NC001357). The URR was amplified using 2 primer sets. In order to amplify L1 region, consensus HPV primers were used .PCR was performed in 30 μl of reaction mixture containing 10 × PCR buffer, 25 mmol/L MgClThe thermal program started with a pre-heat of 95°C for 15 min, followed by 35–40 cycles of suitable annealing temperature which depended on the primers and finished with a final extension at 72°C for 10 min (Table PCR products were confirmed based on specific bands of amplified DNA presence in agarose gel (2%). Afterwards, amplimers were automatically sequenced using the “Big Dye Terminator Cycle Sequencing kit” (Applied Biosystems) according to the manufacturer´s instructions.For E6, E7, E4 and URR amplicons the same forward specific primers as those used in amplification were chosen as sequencing primers. In the L1 region, a specific primer was used in order to sequence HPV 18 and not other HPV types present in cases of multiple infection Table .http://www.genome.jp/tools/clustalw/).HPV sequences were aligned and compared to the HPV 18 prototype sequence which belongs to the Asian-amerindian lineage (accession number NC001357), using BioEdit Sequence Alignment Editor v7.0.4.1 and Clustal W (Amplification and sequencing of the samples were repeated to confirm nucleotide variations which were present in less than three isolates.Sequences were assigned to a lineage on the basis of their similarity to HPV 18 known variant sequences  which beAssociation of lesions and variants or infection type (single HPV vs multiple HPV infection) was analyzed. Fisher exact test was used for statistically significant association.The following are the GenBank accession numbers for all the sequences used in this analysis. X indicates absence of nucleotide sequence data Table All procedures followed were approved by the appropriate Ethics Commitee related to our institutions  and complied with the guidelines and ethical standards for experimental investigation with human subjects of Helsinki Declaration of 1975, as revised in 2000. All study participants provided written, informed consent.HPV: Human papillomavirus; H-SIL: High-grade squamous intraepithelial lesion; L-SIL: Low-grade squamous intraepithelial lesion; SIL: Squamous intraepithelial lesion; URR: Upstream regulatory region.All authors declare no potential conflicts of interest.LSA carried out the molecular studies, participated in the sequence alignment and drafted the manuscript. MB and EA participated in the design of the study and its coordination and have been involved in drafting the manuscript and revising it critically for important intellectual content. SH, DA and VE have made substantial contributions to acquisition of samples and data and have revised the manuscript critically. KGE and BMJ performed phylogenetic and statistical analysis. RC conceived the study, participated in its design and coordination and has given final approval of the version to be published. All authors read and approved the final manuscript.E4, E6, E7, L1, LCR_bay. Phylogenetic trees were constructed for each region individually.Click here for file


Background. Liquid-based Pap (L-Pap) media are used for Pap and human papillomavirus (HPV) testing. Objectives. To compare RealTime High Risk (HR) HPV testing of a new collection kit (Cervi-Collect) and PreservCyt L-Pap specimens. To determine ease of use and safety of Cervi-Collect. Methods. L-Pap samples (n = 203) were tested with HC2 and RealTime HR HPV and Cervi-Collect with RealTime HR HPV. Discordant samples were genotyped. Results. L-Pap and Cervi-Collect specimens tested by RealTime HR HPV showed 93.1% agreement (Kappa 0.86). RealTime HR HPV and HC2 on L-Pap had 90.3% agreement (Kappa 0.80). RealTime HR HPV on Cervi-Collect and HC2 on L-Pap showed 88.2% agreement (Kappa 0.76). Sixteen of 21 samples which were HC2 negative and RealTime HR HPV positive on L-Pap or Cervi-Collect contained HR HPV genotypes. Eleven healthcare collectors were in strong agreement on a usability and safety questionnaire. Conclusion. Cervi-Collect samples were easy to collect and showed strong agreement with L-Pap samples tested with RealTime HR HPV or HC2. In cases where Pap testing is performed using non-L-Pap samples or HPV testing is performed as the primary screening method, a cervical specimen is collected for HPV testing. A collection brush and transportation medium kit (Cervi-Collect) was designed by Abbott Molecular for testing with the Abbott RealTime HR HPV assay. The principles and analytical performance of this assay have been described [High-risk human papillomaviruses (HR HPV) are a major cause of cervical cancer . HR HPV escribed , and theescribed –6 and toescribed –9. me HR HPV assay by testing Cervi-Collect and PreservCyt L-Pap specimens, (b) to compare the RealTime HR HPV and HC2 assays on L-Pap specimens, (c) to test discordant samples in a linear array (LA) assay, and (d) to analyze the strength of agreement of healthcare workers on ease of use and safety of the collection device and its package insert using a questionnaire.The aims were as follows: (a) to compare the performance of the RealTiA total of 203 women attending a women's health clinic undergoing a routine gynecological exam or a follow-up exam due to an abnormal Pap or positive HR HPV test signed consent to have 2 cervical specimens collected: the first was collected with a Cervex-Brush  and placed into an L-Pap PreservCyt collection medium tube  and the second was collected with the Cervi-Collect brush and placed into a Cervi-Collect transportation tube. Specimen collection was performed according to the respective manufacturers' instructions. The PreservCyt sample was processed for cytology in the Pathology Laboratory at the Juravinski Hospital, Hamilton, ON, Canada, and the remainder of the sample was sent to the Infection Research Laboratory (IRL) at the St. Joseph's Healthcare Hamilton, Hamilton, ON, Canada. Both samples were received within 24 hours in the IRL. The L-Pap sample was tested for HR HPV with the HC2 test  at the IRL according to the package insert. Previous positive and negative clinical samples were included with each run as controls. Samples were scored negative if relative light units/cutoff (RLU/CO) ratios were <1.0, indeterminate when ≥1.0 and <2.5, and positive when ≥2.5. Indeterminate samples were repeated in duplicates: a sample with an RLU/CO ratio ≥1.0 in either replicate was considered positive.me HR HPV assay on the Abbott m2000 instrument. The automated test procedure consisted of sample preparation, reaction assembly, real-time PCR, and result reporting [m2000sp, 0.4 mL of sample was processed using the Abbott mSample Preparation SystemDNA where it was lysed with chaotropic reagents, allowing the DNA to be captured on magnetic microparticles. The bound purified DNA was washed and then eluted. An amplification master mix was created with AmpliTaq Gold enzyme , magnesium chloride, and an oligonucleotide reagent containing primers, probes, and dNTPs. The PCR reaction was then assembled in a 96-well optical reaction plate by combining aliquots of the master mix and the extracted DNA eluate. Thermocycling and fluorescence detection of the amplified products were carried out in the Abbott m2000 real-time PCR instrument, and results were automatically reported. The assay detects 14 HR HPV genotypes  with type specific detection for types 16 and 18 and detection of the other 12 non-HPV 16/18 types as a group. A separate detection category of β-globin is included as an internal control to validate sample adequacy, DNA recovery, and PCR efficiency. Results for each sample were reported based on all three HPV signals, corresponding to HPV16, HPV 18, and non-HPV 16/18 HR types, as well as the internal control signal. The Cervi-Collect sample and one milliliter of the L-Pap sample were packaged and shipped to Abbott Diagnostics in Wiesbaden, Germany, where they were tested in a blinded fashion with the RealTieporting . During me HR HPV assays were tested using the LA HPV Genotyping Test  following the manufacturer's protocol. PCR was performed in a final reaction volume of 100 μL containing 50 μL of kit master mix. The genotyping strips were visually interpreted using the HPV reference guide provided in the kit package insert. The same high-risk genotypes represented in the Abbott assay were considered high risk.Samples which showed discordant results after testing by HC2 and RealTiSample collectors (physicians and nurses) were asked to complete a questionnaire rating whether the product labeling information was adequate and easy to understand in the following areas: the intended use statement, the instructions for safe use, collection, storage, and transport, and limitation of use statement in the package insert. They also evaluated the usability aspects  as well as the safety aspects for the collection kit and instructions. In total, eleven questions were answered by each of the eleven collectors. Each question was answered on a scale of 1 to 5, with 5 indicating strong agreement with a statement and 1 if there was strong disagreement. The overall rating across all collectors for each question was calculated as the combined score as a percentage of a maximal score of 55 .κ).Agreement between tests was assessed by kappa statistic (me HR HPV (me HR HPV). me HR HPV and HC2 performed on 195 L-Pap specimens. The assays agreed on 73 positives and 103 negatives. There were 15 samples which were positive by the RealTime HR HPV test and negative by HC2, and 4 other samples which were positive by HC2 but negative by the RealTime HR HPV test. The positive agreement was 79.3% (73/92), negative agreement was 84.4% (103/122), and the overall agreement was 90.3% (176/195) (Kappa 0.80). When the RealTime HR HPV test was performed on Cervi-Collect specimens and HC2 was performed on L-Pap . Samples from 16 of 21 patients with a negative HC2 result and a positive RealTime HR PCR result obtained either from L-Pap or Cervi-Collect samples contained HR HPV genotypes. Samples from 4 patients  which were positive by HC2 and negative by the RealTime HR HPV assay in the L-Pap and Cervi-Collect samples contained low-risk HPV genotypes. Three patients , which were HC2 and RealTime positive in L-Pap but were negative in the Cervi-Collect sample, contained HR genotypes. me HR HPV assay  genotypes by LA  as the primary input tube. This study demonstrated excellent performance of the Cervi-Collect samples for the detection of HR HPV when tested with RealTime HR HPV compared with the PreservCyt L-Pap samples tested with RealTime HR HPV or HC2. The Cervi-Collect samples were not evaluated as a source for cytological examination. Healthcare collectors showed strong agreement with the usability and safety design features of Cervi-Collect and its package insert. Further studies need to be conducted to determine the versatility of this new collection kit for other anatomical sites such as the vagina [Because cervical samples may be collected specifically for HPV testing, a system suitable for the collection, transportation, and storage of specimens for the detection of HR HPV DNA by the Abbott RealTie vagina , anus [1e vagina , and oroe vagina  as well e vagina , 16.


HPV infection is considered the most frequent sexually transmitted infection. The clinical manifestations of infection are various and variable, including the ones in ENT, such as recurrent laryngeal papillomatosis and tonsillar cancer.We present the possibilities of diagnosis of HPV infection in ENT, and the therapeutic, prophylactic and curative modalities, as well as the assumptions and prevention goals through vaccination within this specialty. We communicate the management of three clinical cases of laryngeal papillomatosis in adults.The first case, a 75-year-old woman, 4 times under surgical intervention with “cold surgery”, vaccinated with Silgard. The second case, a 55-year-old woman, also 4 times under surgical intervention receives Gardasil after the last intervention. The third case, a 35-year-old man, 2 times surgically operated after diagnosed with papiloma scuamosis and vaccined with Silgard.All the above mentioned cases don’t present any regression of the disease during the last three years.The involvement of HPV in ENT cancer pathology is undeniable. Laryngeal papillomatosis is a precancerous condition caused by HPV strains 6 and 11. HPV vaccination can be useful both to prevent HPV-induced pathology in otolaryngology and after surgery, to prevent recurrence.


Squamous cell carcinoma (SCC) of the oral cavity and pharynx represents the sixth most common form of malignancy worldwide. A significant proportion of these cases are related to human papillomavirus (HPV) infection. In general, HPV-associated SCC is more commonly nonkeratinizing and poorly differentiated, whereas non-HPV-associated SCC is typically keratinizing and moderately differentiated. Nevertheless, significant overlap in morphology is seen between these two forms of SCC. The purpose of this paper is to highlight the utility of ancillary studies in the establishment of HPV status of oropharyngeal SCC, including p16 immunohistochemistry, high-risk HPV in situ hybridization, polymerase chain reaction, and newer HPV detection modalities. Malignancy of the oral cavity and pharynx constitutes the sixth most common form of malignancy worldwide . In the More recently, human papilloma virus (HPV) infection has been implicated as a major etiologic agent for SCC development –12. HPV Evidence suggests that there is a causal association between HPV infection and SCC of the oropharynx, with molecular characteristics that distinguish it from non-HPV-related SCC, including alterations of p16 and c-myc expression –15. The Distinction between HPV- and non-HPV-related SCC is important in relation to clinical outcome. A study by Ang et al. found three-year survival rate of 82.4% for HPV positive tumors versus 57.1% for HPV negative tumors . A numbeDespite the importance of establishing the HPV status of SCC, no consensus has been reached on the optimal way to identify HPV-associated SCC . The focIHC staining for p16 is frequently used as a surrogate marker of HPV infection. It has the advantage of being easy to perform on formalin-fixed, paraffin-embedded (FFPE) tissue, and monoclonal antibodies against p16 are commercially available. HPV protein E7 binds to Rb, a negative regulator of p16 expression. Thus, HPV infection leads to increased nuclear p16 expression. As a result, IHC staining for p16 has a sensitivity approaching 100% for detecting HPV-associated SCC Figures  13, 15], 1513, 1However, p16 is overexpressed in a subset of tumors apparently lacking evidence for the presence of HPV DNA , 22, 23.It is possible, then, that there is a subset of non-HPV-associated tumors with histologic phenotype, molecular characteristics, and prognosis similar to HPV-associated SCC. The percentage of these p16 positive, HPV negative tumors varies significantly between the Lewis and Thavaraj studies. Whether this represents differences in sensitivities of the HPV tests used or true differences in HPV prevalence in different populations is not definitely clear.In any event, p16 positivity is a sensitive marker for nonkeratinizing, poorly differentiated yet prognostically favorable SCCs. While p16 may not be a specific marker of HPV infection, it can provide important prognostic information, and future therapies aimed at targeting this pathway of HPV tumorigenesis may well be effective in treating p16 positive, HPV negative SCC.ISH testing for HPV has the benefit of being the only molecular method allowing for direct identification of HPV in topographical relation to the pathologic lesion in tissue Figures  26]. Un. Un26]. Numerous technically validated HPV ISH assays are commercially available, most containing a cocktail of probes targeting multiple types of HPV. Though probes for individual types can be used if subtyping is clinically relevant, HPV subtype 16 is by far the most commonly found in oropharyngeal SCC , 10, 26.The most common technical difficulties experienced with ISH are background and an absence of signal . BackgroThe sensitivity of the assay is increased by signal enhancement techniques. One such technique is tyramide signal amplification, which has been shown to have a 10 to 100-fold increase on sensitivity . In thisNevertheless, the sensitivity of ISH appears to be less than that seen in PCR analysis, as a metaanalysis by Termine et al. found HPV in 39.9% of cases by PCR compared to 29.8% by ISH . HoweverPCR is a process in which a signal sequence of DNA or mRNA is amplified several orders of magnitude through several rounds of denaturing at high temperature (~95°C), annealing of complimentary oligonucleotide primers at a lower temperature (~55°C), and DNA replication at an intermediate temperature (~72°C) by a heat-resistant DNA polymerase. In theory, it can be used to detect as few as one copy of a DNA sequence, making it a highly sensitive detection assay .Material for PCR can be obtained from FFPE tissue by scraping tissue from a tissue block, digesting, centrifuging, and using the resultant supernatant for PCR studies . In addiPCR has the advantages of being highly sensitive for HPV detection, widely available, and cost effective. However, standard PCR techniques have a number of drawbacks. PCR has lower specificity than ISH and is technically cumbersome to perform . In contSeveral PCR amplification techniques are commercially available. These PCR screening assays commonly have primers designed to amplify a region of DNA that is present in multiple HPV types (most commonly within the highly conserved L1 gene) , 33. SinThe use of real-time PCR has also been assessed in HPV detection. Real-time PCR allows for quantification of target DNA via colorimetric markers that accumulate during PCR amplification, allowing a mechanism of identification of HPV DNA as well as an estimation of viral load , 23. ThiRecent studies have looked into the ability of PCR to distinguish between episomal and integrated HPV DNA. The HPV gene for E2 protein is a common break site prior to viral integration into the host genome . E2 protFinally, there are a number of commercially available assays for the detection of HPV by PCR by reverse transcriptase PCR. These kits target mRNA of the oncogenic E6 and E7 proteins. Thus, they have the advantage of detecting transcriptionally active HPV , 26. It Overall, PCR is a reliable, sensitive marker of HPV DNA. Nonetheless, ISH still has a number of advantages over PCR, including higher specificity, the ability to reliably distinguish episomal from integrated HPV DNA, and the ability to localize HPV to the area of neoplasia.Over the past two decades, a technique has been developed for combining PCR and ISH, referred to as PCR in situ hybridization (PISH) , 39, 40.Another hybridization technique, coined hybrid capture II (HC-II) has been developed and utilized in the detection of HPV. This is an FDA-approved method for HPV detection in cervical pap smears, and studies have demonstrated its utility in demonstrating the presence of HPV in lesions of the cervix and oropharynx , 43, 44.While IHC staining against p16 is frequently used as a surrogate marker of HPV, to date, IHC staining against specific HPV proteins has generally not been performed. Nevertheless, the development of IHC stains against the oncogenic E6 and E7 proteins would have a number of potential advantages over other HPV detection methods. It would have the ability to prove that HPV DNA is being expressed and directly demonstrate that important HPV oncogene proteins are present . DevelopA number of tests for the detection of HPV in oropharyngeal SCC are available, each possessing its own strengths and weaknesses. At the present time, IHC staining for p16 and PCR for HPV appear to be the most sensitive markers of HPV, while ISH confers the greatest specificity. For most clinical laboratories, the combination of a sensitive test  and a specific test  allows for the best potential to accurately establish the presence or absence of HPV in a given case of SCC. The study by Thavaraj et al. utilizes an algorithm originally developed by Weinberger et al. , 45 in wGiven the relative frequency of discordant p16 and HPV results  when using p16 in conjunction with a single HPV test, Thavaraj et al. suggest a three-tiered algorithm. Tumors are still categorized as p16 positive or negative by IHC. Those that are negative are then assessed for HPV by ISH and ultimately categorized accordingly as Class I (p16−/HPV−) or Class II (p16−/HPV+). Likewise, tumors that are p16 positive are initially assessed for HPV by ISH, and if ISH is positive, the tumor is categorized as Class III (p16+/HPV+). However, if the tumor is negative by ISH, a confirmatory PCR test is performed. If the tumor is still negative by PCR, it is classified as Class IV (p16+/HPV−). On the other hand, if the tumor is positive for HPV by PCR, the tumor is considered Class III (p16+/HPV+) despite the negative ISH result.In their study, this three-tiered approach resulted in just a small minority of cases falling under one of the discordant categories , with concordance of p16 and HPV results in 97% . This study suggests that with the three most commonly utilized tests, HPV status can be confidently determined in the vast majority of cases of oropharyngeal SCC.In the future, more recently applied molecular technologies, such as PISH and HC-II, may offer even more accurate diagnosis of HPV in the clinical laboratory, and development of IHC against important viral proteins may ultimately provide the optimal test for active HPV genomic transcription and translation in SCC. Nonetheless, using current ancillary tests in combination with clinical clues and morphology, such as the presence of nonkeratinizing, poorly differentiated lesions, HPV status can be accurately assessed in the vast majority of cases of oropharyngeal SCC.


Ureaplasma spp. and M. hominis was performed using Mycoplasma IST2 , Identificaton of U. parvum and U. urealyticum was performed by Kong. Primers described by Jensen were used for M. genitalium. For detection of high-risk HPV types Amplicor HPV  was used. Prevalence of urogenital mycoplasmas in the hemodialysed women (53.1%) was significantly higher (P = 0.0059), compared with controls (25%). In both groups, U. parvum was the most frequently isolated. Cooccurrence of urogenital mycoplasmas was shown in 75% of the HPV-positive hemodialysed women and in 30.4% of HPV-positive controls (P = 0.0461). Cooccurrence of urogenital mycoplasmas with HPV was significantly higher in hemodialysed women. The need to take into account these microorganisms in routine diagnostic, especially for hemodialysed patients, was demonstrated. Further studies to demonstrate the role of this cooccurrence in etiopathogenesis of infection in hemodialysed patients are required.Bacterial infections, especially endogenous, are the frequent complications among hemodialyzed and renal transplant patients. In this study we assumed the prevalence of urogenital mycoplasmas and HPV among hemodialysed women. We examined 32 hemodialysed women aged 20–48 (mean 35.6 ± 8.23) and 100 healthy controls of the same ages. Two swabs were collected for detection of mycoplasmas and HPV. Culture of  M. genitalium, U. parvum and U. urealyticum. Thanks to this, studies on the epidemiology and etiopathogenesis of urogenital mycoplasmas in human diseases were intensified [Bacterial infections are the frequent complications observed in hemodialyzed and renal transplant patients, a significant risk factor for transplant rejection and an essential mainspring of mortality in this population. Increased susceptibility to disease and severe infections are due to impairment of the immune system caused by primary diseases and immunosuppressive therapy. Common problems are endogenous infections caused by own microflora. Urogenital mycoplasmas occur in 20–50% of sexually active women. Molecular biology techniques allowed detection of ensified . RecentlIn the current study, we assumed the prevalence of urogenital mycoplasmas and HPV among hemodialysed and healthy asymptomatic women.Examination included 132 sexually active women. The study group consisted of 32 hemodialysed women aged 20–48 (mean age 35.6 ± 8.23 yr) under care of Clinic of Obstetrics and Gynecology, Medical University of Warsaw. The control group included 100 women without diseases and subjectively experienced symptoms from the urogenital tract. The age of the control group was in the range of 20 to 48 years (mean age 33.5 ± 7.49 yr). This study was approved by Bioethical Committee of Medical University of Silesia (KNW/0022/KB1/88/09) and Medical University of Warsaw (KB/117/2007). Exclusion criteria of patients were based on lack of consent, antibiotic therapy and/or chemotherapy and antifungals (at least 4 weeks before examination), pregnancy, diagnosed STI, and vaginal discharge.First swab from posterior vaginal fornix was taken in order to make a Gram-stained direct smear and evaluated vaginal biocenosis according to the 10-point Nugent score .Ureaplasma spp. and M. hominis was performed using Mycoplasma IST2 test , according to manufacturer's instructions.Second endocervical swab immediately after collection was cultured on Columbia agar with 5% defibrinated sheep blood (for aerobic and anaerobic incubation), Chapman, MacConkey, Sabouraud, de Man, Rogosa, and Sharpe agars. Identification of isolated strains was performed using microbiological analyzer Vitek Compact 2 . Next two endocervical swabs were collected aseptically for determination of urogenital mycoplasmas and HPV, respectively. Culture of U. parvum and U. urealyticum was conducted in accordance with Kong et al. [M. genitalium primers for adhesin genes MgPa-1, MgPa-3 and for 16S rRNA gene MG16-45F, MG16-447R, MG16-1204F, and MG16-1301R by Jensen et al. were used [Taq PCR Core Kit (Qiagen Inc.) in thermocycler Mastecycler (Eppendorf AG). Negative samples were checked for presence of amplification inhibitors. The reference strains Ureaplasma urealyticum ATCC 27618, Ureaplasma parvum ATCC 27815 and genomic DNA of Mycoplasma genitalium ATCC 33530 were used as positive controls.Isolation of mycoplasmal DNA was performed from pellet of cells obtained from centrifuged  mycoplasmal culture using DNeasy Tissue Kit (Qiagen Inc.). Identification of Detection of high-risk human papilloma virus (HPV) types 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, 82 and 83 was conducted using Amplicor HPV Test , according to manufacture's instruction.Obtained results were subjected to statistical analysis in the STATISTICA programme, 9.1.210.0 version .P = 0.0059), compared with the control group, 53.1% and 25%, respectively . Presence of HPV was detected in 8 women of the study group (25%) and in 23 of the control group (23%) .Significant cooccurrence of HPV and mycoplasmas was demonstrated in the control group, in contrast to individual presence of HPV , Gram-negative rods, and Candida spp. was similar in both groups  was also shown by other authors [Majority of positive outcomes in our study concerned  authors –19.Ureaplasmas into two new species U. parvum and U. urealyticum and demonstration of differences in the frequency of individual species detection, studies were undertaken to determine pathogenicity of these microorganisms. U. urealyticum is the commonest etiologic agent of infections, including severe infections in renal transplant patients. U. urealyticum detection in patients without infections and subjectively experienced symptoms from the urogenital tract is low (1.2%) [ U. urealyticum. M. genitalium DNA detection in our study was 3% in control group and 9.4% in hemodialysed women. Although it is a high percentage, our study was limited by small number of patients in study group. Many studies confirm low percentage of M. genitalium in healthy women without symptoms: 4.5% of positive cases were described by English authors [M. genitalium DNA was demonstrated in 2.3% women and in 1.1% men [M. genitalium is clearly defined as an etiologic agent of STI. Clinical indications for next studies of this species are urethritis, epididymitis, and prostatitis in men and cervicitis, urethritis, and PID in women [M. genitalium cases among studied hemodialysed women suggest importance of this microorganism as an etiological agent of possible complications.After the division of w (1.2%) , 21 and w (1.2%) –22 and ww (1.2%) . Besides authors . In Denm1.1% men . M. geniin women . More thFairley et al. demonstrated 20% HPV positive cases in hemodialysed women versus 4.5% cases in control group . This suWe summarize that because of demonstrated significant differences in occurrence of urogenital mycoplasmas in hemodialysed women and controls these microorganisms can cause future complications especially in patients preparing for renal transplantation and receiving immunosuppressive therapy.Significantly higher incidence of urogenital mycoplasmas and cooccurrence with HPV was demonstrated in hemodialysed women. The need to take into account these microorganisms in routine diagnostics, especially for hemodialysed patient, was demonstrated. Further studies to demonstrate the role of this cooccurrence in etiopathogenesis of infection in hemodialysed patients are required.