We assessed the knowledge and awareness of cervical cancer, HPV and HPV vaccine, and willingness and acceptability to vaccinate in sub-Saharan African (SSA) countries. We further identified countries that fulfill the two GAVI Alliance eligibility criteria to support nationwide HPV vaccination.We conducted a systematic review of peer-reviewed studies on the knowledge and awareness of cervical cancer, HPV and HPV vaccine, and willingness and acceptability to vaccinate. Trends in Diphtheria-tetanus-pertussis (DTP3) vaccine coverage in SSA countries from 1990–2011 were extracted from the World Health Organization database.The review revealed high levels of willingness and acceptability of HPV vaccine but low levels of knowledge and awareness of cervical cancer, HPV or HPV vaccine. We identified only six countries to have met the two GAVI Alliance requirements for supporting introduction of HPV vaccine: 1) the ability to deliver multi-dose vaccines for no less than 50% of the target vaccination cohort in an average size district, and 2) achieving over 70% coverage of DTP3 vaccine nationally. From 2008 through 2011 all SSA countries, with the exception of Mauritania and Nigeria, have reached or maintained DTP3 coverage at 70% or above.There is an urgent need for more education to inform the public about HPV, HPV vaccine, and cervical cancer, particularly to key demographics, , to leverage high levels of willingness and acceptability of HPV vaccine towards successful implementation of HPV vaccination programs. There is unpreparedness in most SSA countries to roll out national HPV vaccination as per the GAVI Alliance eligibility criteria for supporting introduction of the vaccine. In countries that have met 70% DTP3 coverage, pilot programs need to be rolled out to identify the best practice and strategies for delivering HPV vaccines to adolescents and also to qualify for GAVI Alliance support. The introduction of vaccines has been one of the most effective public health interventions for combating infectious diseases Worldwide, 15% of all cancer cases and nearly 26% of cancer cases in developing countries are attributable to infectious agents, particularly viruses Several recent developments have emphasized HPV vaccine as an important prevention strategy. The 2009 WHO position paper on HPV vaccines recommended they be included in routine national immunization programs as a public health priority http://www.gavialliance.org/support/nvs/human-papillomavirus-vaccine-support). GAVI Alliance also announced that in 2014 it will support the first nationwide introduction of HPV vaccine in Rwanda for girls of all eligible ages, as well as other HPV demonstration projects in Mozambique, Zimbabwe, Ghana, Madagascar, Malawi, Niger, Sierra Leone, and Tanzania .Currently, the GAVI Alliance uses two criteria to determine eligibility for vaccination support, including HPV vaccine: 1) a DTP3 threshold of 70% national coverage (WHO/UNICEF estimates) and 2) a pilot demonstration of the ability to deliver a complete multi-dose series of vaccines to at least 50% of the target vaccination cohort in an average sized district in a country These developments, coupled with SSA's recent success in reaching 70% coverage for other routine vaccines, namely measles, hepatitis B, Influenza, tuberculosis and polio since 1990 Nevertheless, introducing HPV vaccine in SSA offers unique challenges, especially due to limited awareness of cervical cancer, its relationship to HPV, concerns about safety and future fertility, and political factors In this study, we assessed the knowledge and awareness of cervical cancer, HPV and HPV vaccine, willingness and acceptability to vaccinate through a systematic review of peer-reviewed literature. We further identified the fulfillment of GAVI Alliance eligibility criteria among countries in SSA. To the best of our knowledge this is the first systematic review of the potential readiness for introduction of HPV vaccine in the sub-continent, which reveals further insight into some of the unique challenges that need to be addressed.Studies examining awareness, knowledge of cervical cancer, HPV and acceptability of HPV vaccines in SSA were identified by searching PubMed/MEDLINE (NCBI), Embase (Elsevier), African Index Medicus (AIM), and POPLINE  from their earliest dates through July 11, 2013. Bibliographies of relevant reviews and eligible studies were examined for additional sources. The search was performed by a librarian (PAB) using terms for papillomavirus vaccines and vaccination, acceptability or awareness, and the names of all SSA countries . The seaThe review was conducted using methodology reported in the National Health Service Centre for Reviews and Dissemination report, supplemented by Harden's recommendations for systematic reviews of qualitative studies Studies included in the review needed to meet the following criteria: 1) it occurred in a SSA country; 2) it was published in 2006 or later, after HPV vaccination was introduced; 3) it focused on one or more of the three key demographics ; and 4) it examined at least one or more of the following key themes: a) level of awareness of HPV and/or cervical cancer; b) level of knowledge of cervical cancer and/or awareness; c) willingness to vaccinate; d) and acceptability of HPV vaccine. Studies that conducted a pilot HPV vaccination program were automatically included in the review. Animal studies were excluded from the review.A systematic review of the identified studies was then performed summarizing key results. The authors' findings were treated as primary data and studies were synthesized using a framework approach. Data were extracted and organized by key information such as demographic, method and sample size, and key findings of the study about levels of knowledge, awareness, willingness and acceptability. Any strategies for vaccination or increasing awareness (whether recommended or implemented strategies) were also extracted along with factors influencing acceptability of HPV vaccine.Levels of awareness, knowledge, acceptability and willingness were broken down into these categories: 1) awareness of cervical cancer, HPV and/or HPV vaccine; 2) knowledge of cervical cancer, HPV and/or HPV vaccine; 3) acceptability of HPV vaccine ; and 4) willingness to vaccinate or get vaccinated. Willingness was broken down further into categories based on how each individual article defined it as seen in The literature search returned 142 unique records as summarized in the flow chart . Review Two qualitative studies were considered to be of good standard while one was considered moderate and one was considered poor. Nearly all of the surveys were well conducted and 17 of these had a sample size greater than 200 respondents. Sixteen of those had a response rate greater than 70%. The remaining studies varied in quality from moderately good to poor Fifteen studies examined awareness of cervical cancer, HPV and/or HPV vaccine among specific demographic groups. Levels of awareness were mixed with 11 studies demonstrating high awareness, nine studies demonstrating low awareness and two studies demonstrating moderate awareness. Levels of knowledge of cervical cancer and HPV were consistently low. Of the 16 studies examining knowledge of cervical cancer, HPV and HPV vaccine, all noted low levels of knowledge, three reported no knowledge, and two reported moderate knowledge. Only one study specified a moderately high level of knowledge of HPV vaccine.Categories of willingness varied across studies: willingness to recommend HPV vaccine (five studies); willingness to get vaccinated (four); willingness to get daughter vaccinated (four); willingness to participate in vaccine trials (one); “interest” in the vaccine for daughters (one); and “interest in learning more about the vaccine” (one). All studies reported high rates of willingness in their respective categories. Twelve studies examined acceptability levels of HPV vaccine and one study examined acceptability of cervical cancer screening . All 12 Of the 27 studies, 26 discussed strategies for vaccination and increasing awareness. Studies in the six countries in the review where HPV vaccination pilot programs were conducted discussed the implementation strategies used for vaccination while the remaining countries in this review discussed recommended strategies for implementation. Rwanda, South Africa Tanzania and Uganda used school-based strategies achieving 93.2%, 97.8%, 71.6% and 88.9% vaccine uptake, respectively Education for increasing awareness was a strong theme throughout the majority of studies. Recommended strategies to implement sensitization programs included community health education programs, continuing medical education for nurses, midwives, doctors and other healthcare workers, and health promotion and policy programs including awareness through social and mass media .Factors influencing acceptability also varied. Twelve studies addressed reasons for acceptability of HPV vaccine. Two studies in Nigeria Of the 13 countries included in this review, 12 have achieved 70% coverage or higher of the DTP3 vaccine since 2003, one of GAVI Alliance's criteria for vaccine support; the exception is Nigeria . Six of This review identified low levels of knowledge and mixed levels of awareness on cervical cancer, HPV and HPV vaccine, and high levels of acceptability of HPV vaccine among all key demographics. In particular, high acceptability of HPV vaccine despite the lack of knowledge about cervical cancer and HPV represents an opportunity for increased education and awareness strategies about cervical cancer, HPV and HPV vaccine. This is important to help key demographics understand the transmission of HPV, its characteristics and associated risks Engaging all key demographics through improved and increased education will elevate public trust, which is a critical component of successful implementation of widespread vaccine coverage. Incidences such as those seen in Japan, India and Rwanda Anti-vaccination groups frequently post inaccurate information about vaccine side effects on the web, which is publicized by both local and national media Tailored community-based interventions and sensitization programs are a viable means to achieve this for multiple reasons. They have the potential to curb concerns about safety and effectiveness of the vaccine while dispelling negative myths or controversies In addition to understanding levels of knowledge, awareness and acceptability, this review uncovered encouraging trends concerning SSA countries' eligibility for GAVI Alliance support. Only six countries have currently met the two criteria required by the GAVI Alliance to support introduction of the HPV vaccine at a lower cost of US$4.50 per dose Further, our analysis of national vaccine coverage for DTP3 in SSA shows rapid expansion from just one country reaching 80% in 1980 to 35 countries in 2010 Such consistently high DTP3 coverage levels among the majority of SSA countries indicates structural health system capabilities to deliver simple vaccines to the population As this review shows, engaging all stakeholders at multiple levels in countries that meet these requirements is necessary for successful HPV vaccine implementation. Governments should leverage the high levels of acceptability and willingness to vaccinate by increasing education for healthcare workers, women/girls and parents/guardians. Governments should also determine appropriate strategies for disseminating information and vaccine delivery by building upon current infrastructures, such as existing EPIs and school-based programs. This has been shown to be an effective approach because it relies on the involvement of multiple critical stakeholders from the government down to the community level, as seen in Rwanda, South Africa, Uganda and Tanzania The objective of this study was to provide a systematic review of knowledge and awareness of HPV vaccine, willingness to vaccinate, and acceptability of the vaccine, as well as fulfillment of GAVI Alliance's eligibility criteria for vaccine assistance in SSA. To the best of our knowledge, this is the first systematic review of the potential readiness for introduction of HPV vaccine in the sub-continent. Examining the region collectively offers insight into its readiness and ability to implement HPV vaccination on a broader scale while shedding light on the successes, challenges, and lessons of implementation.From this review, three important themes have emerged. (1) There are high levels of acceptability and willingness to vaccinate. These should be harnessed by national governments to establish and implement HPV vaccination strategies that build upon existing infrastructures. (2) Overall, six SSA countries qualify for GAVI Alliance assistance to introduce HPV vaccine at a national level. (3) Lastly, there is a need for increased education and awareness among all three key demographics about HPV, HPV vaccine and the burden of cervical cancer as a disease.The combination of required DTP3 coverage, high acceptability of HPV vaccine and high willingness to vaccinate, indicates the readiness and potential for SSA countries to introduce HPV vaccine to the population. Successfully doing so will depend on implementing tailored delivery strategies that fit each country's needs and engaging the government at all levels. Building upon the lessons learned from GAVI Alliance eligible SSA countries will pave the way for those SSA countries still working to meet the criteria. These countries should also take advantage of and benefit from the “decade of vaccines” and the GIVS when there is a re-dedication by donors to meet these goals There were some limitations in this review. The studies reviewed lacked consistency in regards to psychometric characteristics used. Not all of the studies used a theoretical framework, consistent labeling of themes examined, or rigorous testing and validation of the measures as previously outlined Figure S1Flow Chart Diagram.(TIFF)Click here for additional data file.Table S1Quality appraisal of qualitative studies.(DOCX)Click here for additional data file.Table S2Quality assessment of surveys.(DOCX)Click here for additional data file.Checklist S1PRISMA Checklist(DOC)Click here for additional data file.Appendix S1Search strategies for bibliographic databases.(DOCX)Click here for additional data file.


Human Papillomavirus (HPV) infection in men may produce cancer and other major disorders. Men play an important role in the transmission of the virus and act as a reservoir. The aim of this study was to determine the HPV-genotypes and their prevalence in a group of men attending a Sexually Transmitted Infection service.Between July 2002 and June 2011, 1392 balanopreputial, 435 urethral, 123 anal, and 67 condyloma lesions from 1551 men with a mean age of 35.8±11.3 years old (range: 17–87) were collected for HPV-DNA testing.A fragment of the L1-gene and a fragment of the E6/E7-genes were amplified by PCR. Positive samples were typed by hybridization.The HPV genome was detected in 36.9% (486/1318) balanopreputial and in 24.9% (101/405) urethral (p<0.0001) swabs from 38.1% (538) of 1469 men. Co-infections were present in 5.4% (80/1469) of cases. HPV was found in 43.9% (373/850) of men younger than 35 vs. 31.7% (187/589) of men aged >35. HPV was found in 59.4% (104) of 165 men with lesions (macroscopic or positive peniscopy), and in 22.8% (61/267) without clinical alterations. HPV was also detected in 71.4% (40/56) men with condylomata and in 58.7% (64/109) of men with positive peniscopy.HPV prevalence in men was high and decreased with age. HPV was found more frequently in balanopreputial than in urethral swabs. There was a low rate of co-infections. Low-risk HPV vaccine genotypes were the most recurrent especially in younger. Although HPV has been associated with clinical alterations, it was also found in men without any clinical presentation. Inclusion of men in the national HPV vaccination program may reduce their burden of HPV-related disease and reduce transmission of the virus to non-vaccinated women. Papillomaviruses are the most sexually transmitted viruses. Experimental and epidemiological data imply a causative role for HPVs and they appear to be the second most important risk factor for cancer development in humans, exceeded only by tobacco usage The most common cause of mortality related to human papillomavirus (HPV) infection is cervical cancer . HPV infection is also an important concern for men, due to disease burden and the risk of transmission. HPV is associated with anal cancer (approximately 90%) and a subset of penile (50%) and oral cancers (10–72%) The aims of this study were: 1) to assess the prevalence of Papillomavirus infection and the circulating genotypes in men who attended a Sexually Transmitted Disease service by examining urethral and paraurethral swabs; 2) to analyze the variables which may play a role in HPV acquisition and persistence; 3) and to determine which would be the best sample to perform this kind of diagnosis; 4) determine the percentages of HPV-infections in men that may be prevented by the bivalent and quadrivalent HPV vaccine.The HPV genome was detected in 36.9% (486/1318) balanopreputial swabs, and in 24.9% (101/405) urethral swabs (p<0.0001) from 38.1% (560) of 1469 men. In 5.4% (80/1469) of males a mixed infection was found. The incidence of HPV infection found throughout the study period was studied . It becoIn addition, HPV was present in 50.9% (34/67) lesion swabs, and in 49.6% (61/123) anal swabs (p = no significant).If HPV infection is analyzed according to age, HPV was found in 48.7% (98/201) individuals younger than 25, in 42.4% (275/649) of individuals in the 25–35 range, in 31.7% (111/350) in the 36–45 range, and in 31.8% (76/239) individuals over 45 (p<0.0001). Mixed infections were detected in 9.2% (9/98) men younger than 25, in 16% (44/275) men in the 25–35 range, in 13.5% (15/111) men in the 35–45 range and in 15.8% (12/76) men over 45 years .On the other hand in Low-risk genotypes  were detected in 47.1% (317/674) of patients, as opposed to 41.2% (278/674) of individuals who exhibited high-risk genotypes. High-risk genotypes are more frequent in men younger than 25 years old. However the most frecuent genotype in men younger than 25 was HPV 6 (p = 0.0013) .The involved genotype distribution by age groups was analyzed , table 1Vaccinal genotypes, notably those with low oncogenic risk were more frequent than non-vaccinal types, except in men older than 45 years old.As for mixed infections, The mixed infection rate of positive samples was 14.3% (80/560) and was not related with age . To 2007On the other hand, 15.9% (89/560) of patients presented HPV-16 and/or HPV-18 without other genotypes. And 40% (224/560) presented HPV-6 and/or HPV-11 and/or HPV-16 and/or HPV-18 without other genotypes.The relationship between HPV infection and several factors associated to it or its progression is shown in The results of macroscopic penis examinations were known for 151 men; 56 featured alterations compatible with condylomata at the time of exploration, and HPV was detected in 71,4% (40/56); however, only 16,8% (16/95) out of males with negative exploration were HPV positive (p<0.0001). After performing a peniscopy, HPV was present in 58.7% (64) out of 109 patients with lesions compared to 26.6% (45) of 172 patients without lesions (p<0.0001).Of all the studied variables, only marital status was related to the infection: single and divorced men have a higher viral infection rate than married men (p = 0.05).While much is known about the natural history of cervical HPV infection and its consequences, including cervical intraepithelial neoplasia and cervical cancer, relatively little is known about the natural history of genital HPV infection and diseases in men. In part this reflects difficulties in penile sampling and visual assessment of penile lesions. Although HPV is transmitted sexually and infects the genitals of both sexes, the cervix remains biologically more vulnerable to malignant transformation than the penis or anus in men. However, as more precise and sensitive methods become available and wider studies are performed, the number of HPV-associated pathologies has also increased The HPV infection rate increased steadily throughout the years of the study in the male population in our area. In the first years of the study (2002–2005), it was 28.3% to 43% in 2007–2009. This rate remained stable in the latest years after solving some issues related to sample types and sample gathering, and improving the assessment of individuals with a potential pathology. The 36.3% incidence rate in men attending a STI clinic is close to the highest values described Data on infection incidence take into account the results of the urethral and paraurethral swabs. As other authors have already described, paraurethral swabs exhibited a greater diagnostic performance, which facilitates the performance of this type of studies, since these samples can be easily obtained In this study, swabs obtained from anal lesions or penile condylomata were also processed. The performance of these types of samples was lower than expected (50%), but it must be taken into account that no biopsies were carried out. Biopsies would be the preferred samples to perform an etiologic diagnosis of lesion samples et al.No evident influence of age on infection rate has been found in the published literature on HPV infection in men The more frequent viral genotypes in the male population were the vaccinal genotypes, notably HPV-6 and HPV-11, a fact already mentioned by other authors According to these results, male vaccination with the tetravalent vaccine would be more effective than with the bivalent one, since it could reduce the risk of anal-genital warts, which, although not a severe pathology, do cause significant psychological morbility.In the present study, to 2007 a 3.1% of mixed infections were detected. The prevalence of multiple-type HPV has been reported to be between 2.1% and 34.8% in men The individuals from this study that would have benefit by the bivalent vaccine will be 15.9% and by the quadrivalent vaccine 40%.The virus was more frequently detected in those men showing a clinical presentation compatible with HPV infection than in those who did not. In these cases, however, the percentage of HPV found was not higher than 51%. Obtaining cellular samples by exfoliation of the keratinized epithelium was difficult in those instances, thus compromising the diagnosis The risk of contagion is related to sexual behaviour , sexual intercourse with risk groups (prostitution), polygamy, having suffered other STDs and parenteral drug use. In our study, none of these factors seemed to be involved in the increase of the HPV infection rate, with the exception of marital status: single or divorced individuals were associated to a higher HPV infection rate. These males also exhibit a sexual behaviour different to that of married men.Although many males get infected with HPV, most of such infections do not develop into cancer. It is thus probable that other co-factors, such as tobacco usage, alcohol intake and coinfection with other sexually transmitted diseases play a role in the pathologic process. No relationship between these co-factors and the presence of oncogenic genotypes was established in this study.On this study, urethral and paraurethral swabs are not collection simultaneously, and clinical and pathological data were reviewed only from 151 patients due to the medical history confidentiality. These limitations could have any influence in the data.To sum up, although HPV infection do have worse consequences for women, men play an active role in it, as the high incidence rates found demonstrate. Men are involved in its reservoir and transmission, and the virus may end up causing major disorders and severe consequences. The high prevalence in this collective suggests the need of more exhaustive assessments, even though most individuals do not feature high-risk oncogenic viruses. Most countries have only included girls in their national vaccinations programs Between June 2002 to June 2011, HPV genome detection was performed prospectively in 1551 males  which were attended in the Sexually-transmitted Infections Service of the Hospital Monte Naranco . From them 1318 paraurethral swabs, 405 urethral swabs, 123 anal and 67 condyloma lesions were recovered.Clinical data together with HPV infection-related risk factors  were gathered from 515 patients. From them, in 151 macroscopic penis data, and in 286 peniscopy data were also recovered.5′-ACACAACTTGTGTGTTCACTAGC-3′and reverse 5′-CAAACTTCATCCACGTTCACC-3′). If there were problems with PCR, an automated extraction with a TNAI kit  from stored samples was performed. From 2009, in all the samples automated extraction was used.Half the volume of the samples was homogenized and concentrated by centrifugation at 6000 rpm (1800 g) for 5 min and supernatant removed. Then, 200 µl of lysis solution  was added, mixed and incubated for 45 min at 56°C, then for 10 min at 96°C to inactivate proteinase K, and finally stored at −20°C until it was analyzed. The quality of extracted DNA was checked by PCR amplification of the β-globin gene (the primer forward Standard protocol of PCR with MY11/MY09 primers (site-directed L1 fragment of HPV) 5′-TGTCAAAAACCGTTGTGTCC-3′, and onco2- antisense-5′-GAGCTGTCGCTTAATTGCTC-3′). PCR was done in 25 µl of reaction mixture containing 1× PCR buffer, 2 mmol/L MgCl2, 50 µmol/L of each deoxynucleoside, 0.5 µmol/L of sense and antisense primer, 10 µl of DNA template and 1 U Taq DNA polymerase  by thermal profile at 40 cycles: 95°C for 30 sec, 55°C for 30 sec, and 72°C for 30 sec. This was followed by a final extension at 72°C for 5 min. An initial denaturation at 95°C during 5 min was also carried out. Products about 250 bp in sizes were expected.Another PCR was performed with a pair of primers against the E6/E7 fragment , HPV-11 (MY13: 5′-TCTGTGTCTAAATCTGCTACA-3′), HPV-16 (MY14: 5′-CATACACCTCCAGCACCTAA-3′), HPV-18 (WD74: 5′-GGATGCTGCACCGGCTGA-3′), HPV-31 (WDB128: 5′-TTGCAAACAGTGATACTACATT-3′), HPV-33 (MY16: 5′-CACACAAGTAACTAGTGACAG-3′), HPV-45 (MYB69: 5′-ATACTACACCTCCAGAAAAGC-3′) as previously described by Ting and Manos22, and an “in-house” design probe against HPV-58 (SANTI58: 5′-TGAAGTAACTAAGGAAGGTACA-3′).All positive specimens for L1 fragment were tested by hybridization assays using type-specific probes for HPV types 6 (MY12: 5′-CTGCTGTGTCTTCTAGTGA-3′), HPV- 39 (5′-ATAGAGTCTTCCATACCTTC-3′), HPV- 51 (5′-TGCTGCGGTTTCCCCAA-3′), HPV- 52 (5′-GAATACCTTCGTCATGGC-3′), HPV- 56 (5′-TGTCTACATATAATTCAAAGC-3′), HPV- 66(5′-AGCTAAAAGCACATTAACTAA-3′), HPV- 68 (5′-CTGAATCAGCTGTACCAAT-3′).From 2007 were added probes against HPV types 35  were performed using GraphPad InStat version 3.00 for Windows 95  following the manufacturer's instructions.All experiments were performed in compliance with relevant laws and institutional guidelines and in accordance with the ethical standards of the Declaration of Helsinki. Information concerning the research project was provided to all participants, and all signed a free and informed consent from approved by Ethical committee of Clinical Investigation of the HUCA.Table S1Distribution of patient detected genotypes in mixed infections according to age.(DOC)Click here for additional data file.Table S2Distribution of patient detected genotypes in both periods of study.(DOC)Click here for additional data file.


Cervical cancer is caused by high-risk human papillomaviruses (HPVs), and a unique characteristic of these is a PDZ ( PDZ domain-containing proteins are ubiquitous protein interaction modules and possess multiple functions that are essential to maintain cellular homeostasis. PDZ domain recognition motifs are often encoded by pathogenic viruses, including high-risk HPVs, and through this motif they target various cellular proteins via protein-protein interactions; these HPV-PDZ interactions are in part associated with virally induced cancer progression. However, as this two-way interplay exhibits diverse characteristics and the activities are often combinatorial, depending upon various cellular contexts, extensive understanding of their roles in diverse aspects of cancer has a long way to go. In this paper we focus on the multiple functions that HPV E6-PDZ interactions potentially have in relation to HPV-induced malignancy, depending upon their localizations, stages along the time-axis of malignant progression, and various posttranslational modifications such as phosphorylation by various kinases and we also consider their potential for both predictive and therapeutic applications in HPV-induced malignancy.When exposed to infectious viruses, humans may eliminate infection by their innate immune system, or the infection may progress in stages from mild symptoms to serious, long-term impairment. Some human viruses have been found to cause cancer, accounting for 10–15% of human cancers worldwide [Cervical cancer is the third most common female cancer, with 530,000 new cases and 275,000 deaths in a year . In 10% in vitro, that the removal of either E6 or E7 from cells derived from cervical tumors results in growth arrest and apoptosis, highlighting their potential for novel diagnostic and therapeutic targets in cervical cancer [The key feature of HPV-induced malignancy is the sustained high-level expression of E6 and E7, which are encoded by early genes of HPVs. Previous studies have provided abundant evidence of a direct role for E6 and E7 oncoproteins in both the development and maintenance of the malignant state [Mechanisms underlying the abilities of both proteins to induce malignancy are diverse and complex. Certainly, the ability of E7 to override cell cycle regulation (stimulate S-phase progression) by interaction with the pocket protein family, including the retinoblastoma tumor suppressor (pRb) is critical , togetheDrosophila have shown that some PDZ domain-containing substrates regulate cell growth and polarity, and loss of these substrates results in excessive overproliferation and neoplastic transformation of epithelial cells, thereby defining this type of PDZ domain-containing proteins as potential tumor suppressors [PDZ domains are ubiquitous protein interaction modules conserved throughout evolution  and are pressors . HoweverInterestingly, the E6 proteins of high-risk or oncogenic HPVs uniquely contain a class I -PBM (sequence x-S/T-x-V/I/L-COOH) located at the C-terminus, and this is absent in those HPV E6 proteins that are only associated with benign disease, a fact suggestive of the PBM being a distinctive signature for malignant potential among HPVs , 18 Fig. ThroughDrosophila discs large 1) [Drosophila Scribble) [Many potential PDZ-containing proteins targeted by E6 have been identified and intensively studied, including hDlg1 (a human homologue of large 1) , 18, hSccribble) , MAGI-1 cribble)  and manycribble) . WhetherIn addition to its contribution to the E6-related acquisition of malignant transforming characteristics, the PDZ-PBM interaction has a crucial role in the HPV life cycle. Studies in human foreskin keratinocyte (HFK) cell lines transfected with HPV-31 genomes have demonstrated that mutant viruses defective in PDZ binding lead to reduced growth rates, loss of viral episomes, and increased numbers of unstable viral genomes that are either lost or become integrated into the host chromosomes . ConsideIn the discussion below, we focus on what effect these varieties of HPV E6-PDZ interactions potentially have in relation to HPV-induced malignancy, depending upon “where”  and “when”  they take place and upon various posttranslational modifications to which they are subject.The existing model for the progression from HPV infection towards malignancy is shown in Studies on NIKS cells, a spontaneously immortalized human keratinocyte cell line that can support the entire life cycle of HPV-16 , have shThis is consistent with the previous study on HFK cells transfected with HPV-31, where mutation of PBM resulted in significant retardation in their growth rates and reduction in their viral copy number, together with the unusual morphological differentiation of cells . It is somewhat unexpected, but appears to be a curious paradox, that the same series of proteins which E6 targets for degradation during malignant progression fulfill an important role for the stabilization of E6 and the episomal maintenance in earlier stages of the HPV life cycle, suggesting the possibility of the multiple functions for PDZ-PBM interactions, depending upon different cellular contexts.As definitively discussed in previous studies, both hDlg and hScrib are intimately linked to homeostatic maintenance of several epithelial tissues, including regulation of cell polarity and proliferation. Deregulation of these proteins has been shown in a number of human epithelial cancers: mislocalized and deregulated hScrib expression is seen in colorectal , breast Interestingly, changes in the levels of expression and localization of Dlg and Scrib occur dependently upon different stages of malignant progression to cervical cancer. For Dlg, analysis by immunohistochemistry showed that the expression of Dlg in cervical intraepithelial precursor lesions displays a distribution pattern different from what is seen in normal epithelium, that is, an unusual increase in the cytoplasm and loss at the sites of cell-cell contact . It was only in the invasive form of cervical cancers that a severe decrease in the amount was observed , suggestIn addition, the analysis of Scrib showed redistribution during cervical carcinogenesis, from cell contact sites in the squamous cells towards the cytoplasm in dysplastic cells, with a gradual reduction in the Scrib expression levels along with the tumor progression .in vitro and in vivo, has revealed that specific forms of Dlg, which remain bound by E6 but not degraded, may actually have oncogenic potential [A recent study, showing that SGEF (Src homology 3 domain-containing guanine nucleotide exchange factor) is a strong binding partner of Dlg and that the interaction of the two induces activation of RhoG both otential , which iThus, PDZ domain-containing proteins are implicated in diverse aspects of tumor growth, development, and metastasis.In addition to the two distinct aspects of HPV E6-PDZ interactions at different stages along the time axis of HPV-induced malignant progression, as mentioned above, recent studies have shown that these PDZ domain interactions can be modulated by a multitude of posttranslational modifications, the most important of which is phosphorylation. Indeed, specific phosphorylation states have been shown to greatly affect the localization and the susceptibility of each PDZ-containing protein to proteasome-mediated degradation by E6 .For instance, hyperphosphorylation of hDlg by Jun N-terminal kinase (JNK), which occurs in response to osmotic shock, results in the rapid accumulation of Dlg at the sites of cell contact and renders it more susceptible to degradation induced by HPV-18 E6 . AnotherThe ERK/MAPK cascade, another notable cell signaling pathway, has been reported to be activated in cervical cancers , and hScin vivo. Thus, the E6-PBM phosphorylation by PKA greatly inhibits E6-Dlg recognition and subsequent degradation, thereby maintaining high Dlg protein levels under conditions of high PKA activity [ζ and that this E6/14-3-3ζ interaction is essential to maintain E6 levels [ζ in high-risk HPV positive cervical tissues could be considered as a potential biomarker for predicting the development of cervical cancer.Interestingly, not only are the PDZ-containing proteins regulated through phosphorylation, but so also is HPV E6. Embedded within the HPV E6 PBM there is a consensus recognition site for protein kinase A (PKA) phosphorylation on the Thr/Ser residue at the −3 position . Previouactivity . A strikactivity . However6 levels . Thus, tAt present, no information is available about at which stage, either during malignancy or during the course of viral life cycle, and to what degree, this E6 phosphorylation by PKA or AKT takes place, but this feature assumes great importance for the prospective designs for both progression-risk prediction and treatment for cervical cancer, considering the correspondent fact shown by many previous studies that mutations that disrupt normal PKA activity are frequently found in many forms of malignancy.Thus, modifications by kinases and changes in cellular signaling pathways can profoundly influence, and sometimes dramatically alter, the efficiency of E6-PDZ interplay.There are some fascinating recent studies that indicate PDZ targeting by E6 might be related to viral evasion of the host innate immune response. The important virulence factor of the Influenza A virus, NS 1 , has no oncogenic potential but does have a PBM at the carboxyl terminus and binds to PDZ-containing proteins as HPV E6 does . The impκB), is well known as a driving force for generating chronic inflammation in the pathogenesis of many cancers, in addition to being implicated in a variety of other processes including proliferation, migration, angiogenesis, and prevention of apoptosis. Toll-like receptors (TLR) are possible signal initiators for NFκB activation, due to inflammation-inducing carcinogenesis, and are also known to be the forefront receptors in response to microbial infection. Therefore, activation of NFκB through stimulated TLR in local chronic inflammation may serve as an initiator of carcinogenesis [κB in HPV-related cancers, and again the PBM of E6 appears to be required for this process [The relationship between cancer progression and chronic inflammation has long been reported, and a transcription factor, nuclear factor-kappa B , another crucial hallmark of cancer progression, especially in the progression of metastasis . TherefoWhilst previous studies have revealed much of the requirement for the oncoproteins of E6 and E7 in the development and maintenance of cervical cancer, much still merits further investigation to precisely understand the functions of these two viral proteins. In this review, we have attempted to feature one particular aspect of E6 function that may help to provide some clues to the molecular mechanisms that underlie HPV-induced development of cancer. Now the subject we must focus on is the prospective application of PBM-PDZ interactions for both the prevention and treatment of cervical cancer.Focusing on diagnostic tools, there is an urgent demand for the development of new biomarkers that can discriminate lesions with a high risk of progression towards cancer from those that will spontaneously regress. As noted above, most infections even with the high-risk HPVs occur without any clinical symptoms, are eliminated by the host immune system within the short period, and do not progress to cancer. High-grade cervical intraepithelial neoplasia (CIN) is normally the subject of therapeutic intervention, involving either removal or destruction of the lesions, to prevent malignant progression, and while this intervention certainly reduces the cancer development rates, the number of patients subject to overmedication increases at the same time. It is fairly reasonable to suggest that a more direct CIN predictor for malignant progression would be desirable. Among the PDZ-containing proteins, MAGI-1, hDlg, and hScrib are degraded to varying degrees by high-risk HPV E6 oncoproteins, both in cells derived from cervical tumors and in a variety of experimental settings. However, although Dlg is targeted by E6 for degradation and is lost during the later stages of cervical cancer progression, only certain pools of the Dlg are degraded; therefore substantial amounts of Dlg protein, though often with unusual distribution pattern or phosphorylated forms, are found in HPV-positive tumor cells . As mentMoreover, one of the hallmarks of cervical cancer progression is the frequent integration of the viral DNA into the host genome, and although there is considerable debate as to the role of this in disease development, the presence of integrated DNA sequences is likely to be of concern due to the concomitant high levels of E6 and E7 gene expression . Therefoin silico, but the problem is that a large proportion of candidates identified by in silico screening prove to be false positive results [in silico approach to drug design could be a valuable tool in identifying novel inhibitors of the E6-PDZ interactions, potentially leading to significant advances in the treatment of cervical cancer.Whether any of these PDZ targets of E6 might have therapeutic potential is also an intriguing possibility. In general, protein-protein interactions provide attractive possibilities for therapeutic intervention, and disruption of specific protein interactions with high affinity is a key concept for producing a molecularly targeted drug. However, targeting protein interactions are clearly more difficult than those targets that naturally bind molecules , 56. Tha results . Additio results , 58. ThuAlthough studies are still in their infancy, the HPV-PDZ interactions could have vast potential as both predictive and therapeutic targets in HPV-induced malignancies. And as these proteins appear to be multifunctional, sometimes exerting completely opposite influences on cancer progression depending on contexts, a comprehensive analysis of the PDZ protein expression at different stages along the time axis of malignant progression, and in various posttranslationally modified states, is essential to precisely elucidate the functions of HPV-PDZ interactions, before novel molecular biomarkers or drugs targeting these interactions come into practical use.


Human Papilloma Virus (HPV) is a major leading cause of Human Cancer. Through the HPV Prevention series we would like to highlight the quality and the breadth of the research being carried out on the Control and Prevention of HPV and HPV related disease. This series aims to bring together a diverse range of HPV related specialties featuring research that has as ultimate goal insights into HPV related disease reduction. Articles within a wide range of topics such as natural history studies, impact of screening interventions or impact of HPV vaccines will be most welcome. Human Papillomavirus (HPV) is responsible for over five per cent of all cancers worldwide and of 15% of cancer in women in developing countries, and is Research on HPV and cancer has drawn a scenario in which a major reduction of HPV related disease is feasible through population based prevention strategies. These include routine HPV vaccination of pre-adolescent and young women, improvement of cervical cancer screening strategies through HPV based technologies, adaptation of preventive strategies using cost-benefit models and inclusion in the health priority lists the wide range of HPV related disease.While our knowledge on the natural history and prevention mechanisms of cervical cancer has increased enormously during the last 30 years, there remain major unanswered issues. A wide HPV related disease is not an entity that navigates on its own. It represents a complex mixture of genetics, micro-environment, behaviour and social influences. With the HPV Prevention series we hope to wider our minds, to increase our capacity for action and ultimately, to increase the health of our population.


Surgical excision of the primary tumor with safe margins remains the mainstay of treatment for oral cavity squamous cell carcinoma (OSCC). The standard of care for assessment of intraoperative margins is frozen section histopathology. Unfortunately the facility is not available at most centers in limited resource countries. Toluidine blue, a metachromatic dye, has been well described in clinical identification of malignant and premalignant lesion in the oral cavity. Considering this we decided to explore intraoperative use of toluidine blue staining, in comparison with frozen sections, for the assessment of tumor-free margins.After obtaining clearance from the in-house ethical review committee, a prospective study was conducted at Aga Khan University Hospital, Karachi, from August 15, 2009 to March 14, 2010. A sample of 56 consenting patients with biopsy-proven OSCC were included in the study, giving us 280 tumor margins. Margins were analyzed using toluidine blue staining and frozen section histopathology. A receiver operator curve (ROC) was then applied to compare assessment of margin status by toluidine blue and frozen section.Of the 280 examined margins 11 stained positive with toluidine blue, three were positive on frozen section biopsy, and three were positive on final histopathology. Toluidine blue staining had sensitivity and specificity of 100% and 97%, respectively. The diagnostic accuracy of toluidine blue was found to be 97.1% with a positive predictive value (PPV) of 27.2% and a negative predictive value (NPV) of 100%.Toluidine blue can be used as an effective screening modality for the assessment of intraoperative margins in resource limited environments and reducing the number of frozen section biopsies performed. Further by providing real-time clinical information within minutes it can reduce indirect costs such as operating room time. It may also be used as an ad hoc for frozen section biopsies where frozen section facilities are available. Contrib disease . In the  disease . Given t disease ,3. The aThe mainstay of treatment of OSCC involves surgical excision of the primary tumor with safe margins . This maToluidine blue is an easily available, economical, metachromatic dye known to bind DNA of dividing cells. It has previously been described to stain malignant and premalignant cells but not normal mucosa -7. MultiAfter ethical clearance from the institutional ethics review committee, a prospective study at The Aga Khan University Hospital, Karachi, Pakistan was conducted from August 15, 2009 to March 14, 2010 on 56 consenting patients with biopsy-proven OCSCC, undergoing primary excision. Patients with prior history of head and neck malignancy, tumors of the oro-pharynx or having previously undergone treatment (surgery and/or radio-/chemotherapy) for the current oral squamous cell carcinoma were excluded from the study as increased inflammation and scar tissue could possibly lead to errors while interpreting the staining results. The patients were recruited regardless of the age, gender, ethnicity, and tumor stage.All procedures were performed by experienced head and neck surgeons with assistance from senior residents. The operative protocol was essentially the same with minor differences due to the individual tumor status and attending surgeon's preference. After excision of the primary tumor a trained senior resident stained the tumor margins as per the defined protocol. Once the senior was done painting the margins and had recorded his findings, the primary surgeon took frozen biopsies from clinically suspicious areas regardless of staining pattern as per institutional protocol. In addition if any margin stained positive with toluidine blue the primary surgeon was requested to take a frozen section biopsy from the area. Each frozen section biopsy was sent in a different container with labeling of the site from which it was obtained. A log was maintained of the staining results. The senior resident and the histopathologist were blinded to each other's findings . However the staining may have persisted after processing and may have had an influence on the histopathologist's decision. The results of the frozen section biopsy were directly conveyed to the attending physician via the operating room (OR) phone. The senior resident performing the staining was not present in the OR at the time these results were conveyed. The results where then compared and contrasted with the findings of frozen section biopsy and final histopathology by another author. No intraoperative action, other than frozen section biopsy, was taken on the basis of positive staining margins on toluidine blue, due to the inherent surgical protocol of our institution. However if a margin was declared positive on frozen section histopathology it was further excised and sent for frozen section again till the time it came out negative.et al. was kept in the OR. Once the primary tumor was excised a designated senior resident irrigated the tumor margins with normal saline, followed by 1% acetic acid. The margins were then gently dried with gauze and painted with the 1% toluidine blue solution with a cotton swab. The stain was left in place for 30 s after which the tissue was once again irrigated with normal saline followed by 1% acetic acid. The staining patterns were observed and noted by the senior resident independently. Margins which stained dark blue were considered to be positive, whereas those which stained light blue or did not take the stain at all were considered negative. In order to establish staining patterns we used simultaneous frozen sections in two pilot patients and revealed the results to the interpreter during the surgery, the results of which were not included in the study.One per cent toluidine blue solution, as described by Meshberg The data were entered and analyzed using Statistical Package for Social Sciences (SPSS) version 17 (IBM Corporation). Means with standard deviations were calculated for all continuous variables while proportions and percentages were calculated for categorical variables. A 2 × 2 table was constructed from the acquired data, and the measures of yield (sensitivity and specificity) and reliability  were calculated using Microsoft Excel 2010 (Microsoft Corporation). Separate 2 × 2 tables were constructed using SPSS for each category of T stage and tumor site to calculate above measures. A receiver operator curve (ROC) was then applied to compare the margin status between toluidine blue and frozen section.n = 23) of the lesions. This was followed by the tongue 23.2% (n = 13), lower alveolus 12.5% (n = 7), retro-molar trigone 8.95% (n = 5) and the floor of the mouth 5.3% (n = 3). When considering the extent of the lesion 19.6% (n = 11) patients were staged as T1, while 33.9% (n = 19) were staged as T2, 19.6% (n = 11) as T3 and 26.7% (n = 15) as T4.Out of 56 patients the majority (75%) were men. About 280 margins were sampled from this pool of patients. Mean age of the patients was 50.07 ± 15.73 years. Buccal mucosa tumors accounted for 41%   and final histopathology (gold standard). We found the findings of frozen section biopsies in our sample to be 100% concurrent with the final histopathology. So comparison of toluidine blue with either was equivalent. In our study sample staining with toluidine blue had an intraoperative sensitivity of 100% and a specificity of 97% with a NPV of 100% and a PPV of 27.2%. These figures are in agreement with previous studies done in clinical settings; with slightly higher sensitivity. This could probably be explained by the fact that all the studies quoted above present staining in a clinical scenario whereas our study toluidine blue was used intraoperatively, which allowed a uniform staining protocol to be followed with adequate time for observationP value of 0.004. False-positive results have also been shown to be associated with inflammation secondary to increased mucosal trauma during excision [et al. pointed out that staining with toluidine blue identified all positive tumor margins with few false-positives and no false-negatives [et al. also reported that toluidine blue staining could not only be used to effectively localize tumor borders but also detect satellite lesions within 1 cm of the primary lesions [In our study, 11 margins stained positive with toluidine blue, of which three were positive on frozen section biopsy and final histopathology. This low PPV was significant. ROC analysis revealed a excision , indicategatives . Furtheregatives . Missman lesions . We cannIn resource-limited environments where facilities for frozen section biopsies are not readily available surgeons often rely on either their clinical judgment or on the final postoperative histopathology for assessing microscopic extension of the disease. This leads to a major disadvantage for patients and may account for high recurrence rates of OCSCC in developing countries.Coupled with the huge burden of OCSCC in Pakistan ,22, the The authors declare that they have no competing interests.MJ came up with the idea, wrote the protocol and obtained the ethical committee approval, MC, ZA, MJ did most of the literature review and were mainly involved in the preparing of the manuscript, MK and AS were the primary surgeons and supervised the manuscript writing, SQ and NA were responsible for prepration of dye, obtaining informed consent, application of the dye intra-operatively, observing the results and maintaining the log, GM provided with the necessary statistical input and evaluation of data and results. All authors read and approved the final manuscript.


COMP). Only a small number of studies have documented the clinical phenotype and genetic basis in Chinese PSACH patients.Pseudoachondroplasia (PSACH) is caused exclusively by mutations in the gene for cartilage oligomeric matrix protein . A heterozygous TGTCCCTGG insertion in exon 13, between nucleotide 1352T and 1353G, were identified in the patients except the unaffected individuals, which resulted in a three-amino-acid insertion (451V_452P ins VPG) in the sixth calmodulin-like repeat of the COMP protein.We investigated a four-generation PSACH pedigree of Chinese Han origin. Two patients and two unaffected individuals were recruited for clinical evaluation and molecular genetic analysis. The genomic DNA was extracted from peripheral blood leukocytes. Polymerase chain reaction (PCR) was adopted to amplify the 8-19 exons of This c. 1352_1353ins TGTCCCTGG is a novel mutation responsible for severe familial PSACH. Pseudoachondroplasia  is a rare autosomal dominant osteochondrodysplasia characterized by typical disproportionate short stature, brachydactyly, lower limbs anomalies, joint laxity, scoliosis, early onset osteoarthritis, epiphyseal and metaphyseal abnormalities. It is estimated to affect at least 1 in 20,000 persons -5.COMP) gene were subsequently found to cause PSACH and another skeletal dysplasia, multiple epiphyseal dysplasia [COMP gene have been found to date, most of them occur in the CLR regions (~85%), while others in the C-terminal globular domain (~15%) . Although the normal function is not fully known, the pathological link between gene mutation, protein alteration, abnormal cell growth and PSACH has been established [COMP mutations have been identified to be with PSACH [COMP protein [COMP gene for mutations in a Chinese family affected by severe PSACH.The PSACH gene was initially localized to chromosome 19 in 1993 ,7. Mutatysplasia ,9. COMP ablished -17. Moreth PSACH ,18, but th PSACH . Severe  protein . Elliott protein . In the This family was initially contacted in the Sichuan province through a member hospital in the Chinese Birth Defects Monitoring Network. PSACH disorder was transmitted through at least four generations in this family. Figure  After inCOMP exons 8-19 (accession number NM000095) and PCR is outlined previously [Using a commercially available kit , genomic DNA was isolated from blood samples utilizing the standard protocol provided by the manufacturer of the kit. The procedure used for amplifying eviously . The resPedigree analysis revealed six individuals affected by severe short stature within a span of four generations and the disorder exhibited dominant autosomal transmission. All affected individuals demonstrated the typical characteristics of PSACH: disproportionate short-limb dwarfing, short upper limbs, lower limb abnormities, waddling gait, but normal facial features and intelligence. The abnormalities of the affected members in this family appeared at about one year of age, usually presenting as difficulties in learning to walk and delayed growth compared with normal children. The proband  was identified between nucleotide 1352T and 1353G in exon13, which is located at the end of the sixth repeat of the type-3 calcium-like repeat region of the COMP gene. . A recent analysis indicated that the residues 451V and 452P are in a calcium-binding pocket domain of COMP protein [After sequencing the  protein . The resCOMP is the only gene known to be associated with pseudoachondroplasia. COMP mutations and pathological consequences have been extensively studied in Caucasians, but a few in the Chinese population. Besides the c.815C→T in exon 8 [COMP gene reported lately in Chinese PSACH patients, we identified the c. 1352_1353insTGTCCCTGG within exon 13 of COMP that contributes to severe PSACH phenotype in a Han Chinese pedigree. This novel trinucleotide expansion was located in the region coding the sixth CLR (p. 451V_452PinsVPG) and expands the knowledge of COMP mutations leading to PSACH.Up to now, n exon 8  and the n exon 8  in exon COMP gene encoding the type III CLRs of COMP protein [COMP mutations involve the substitution of one amino acid for another in the COMP protein. Trinucleotide expansion at the region encoding type III CLRs of the protein has now been reported in this study and one other previous study [COMP gene.PSACH mutations are located in exons 8-14 of  protein ,18,28. T protein ,8,29,30.us study . Genotypus study . The earCOMP protein [COMP protein, where a calcium ion is embedded. Calcium supports the binding of COMP to fibronectin which reportedly influences the conformation of COMP [COMP and other proteins in the rER of affected chondrocytes [COMP protein, and missense mutations or in-frame deletions in the calmodulin-like repeats region reduces this ability [COMP protein, the proliferation and apoptosis of chondrocytes. This study suggests that the 9 bp insertion can be responsible for the severe phenotype in the Chinese family in despite no direct functional evidence. In conclusion, the c.1352_1352insTGTCCCTGG within exon 13 of COMP expands the spectrum of mutations that causes PSACH. It could be of value to the molecular diagnosis, genotype-phenotype relationship understanding and pathogenesis of this disorder.COMP is a multifunctional structural protein that affects cellular attachment, proliferation and chondrogenesis. It also participates in the ECM assembly through interactions with numerous proteins including types I, II, IV, XI, XII collagen, decorin, fibronectin and matrilin-3 ,33. Acco protein , the p.4 of COMP . Specifi of COMP ,32,34-37drocytes ,38-40. Idrocytes . A mousedrocytes . Previou ability . The lonPSACH: pseudoachondroplasia; COMP: cartilage oligomeric matrix protein; CLR: calmodulin-like repeat domain; ER: endoplasmic reticulum; ECM: extracellular matrix.The authors declare that they have no competing interests.LD studied the family, designed research plan and prepared manuscript. LX and NL performed the molecular genetic studies, participated in sequence alignment. YW and MM participated in clinical evaluation. JZ participated in collecting specimens and supervised study. CK and YZ reviewed and revised the manuscript. All authors had read the final manuscript and approved the publication of the clinical images.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2350/12/72/prepub


To examine the relationship between human papillomavirus (HPV) and giant cell arteritis (GCA) of the temporal artery.The study group consisted of 22 cases of histologically positive/biopsy confirmed GCA. The control groups consisted of 21 histologically negative temporal artery biopsies and fifteen cases of vascular margins of nephrectomies. For detection of the presence of HPV, two methods were used: 1) polymerase chain reaction (PCR) with INNO-LiPA HPV Genotyping Extra, 2) Cervista™ HPV HR. All cases were from the files of the Barnes-Jewish Hospital and Washington University in St. Louis.TM) confirmed the aforesaid results with 100% concordance with the exception of three cases which had low genomic DNA for which it was not possible to perform the test. The differences in HPV positivity between the histologically positive and negative temporal artery biopsies and between the histologically positive temporal artery biopsies and controls were both statistically significant .HPV DNA was detected by PCR and genotyping in 16 of 22 (73%) histologically positive cases of GCA and in only five of 21 (24%) histologically negative temporal artery biopsies. Among the vascular margin controls, only three of 15 (20%) were positive for HPV DNA. The second, independent method . Further studies are necessary to elucidate the pathophysiology underlying this association. Giant cell arteritis is one of the most common causes of vasculitis involving the temporal artery. Other potential causes are Wegener granulomatosis, polyarteritis nodosa (PAN), and Buerger disease . GCA genHistologically, GCA shows transmural inflammation with mixed inflammatory cell infiltrate mostly consisting of lymphocytes, histiocytes, plasma cells, occasional neutrophils and rarely eosinophils Figure . This caInfectious agents have long been considered as a possible etiology of GCA. The concept is that GCA represents a chronic inflammatory response, triggered by an infectious agent, with subsequent inappropriate tissue response to injury. However, studies looking for organisms have had conflicting results. Some have demonstrated organism DNA, such as Herpes Simplex Virus, while the vast majority have failed to demonstrate an association. It is speculated that the inflammatory response may be triggered by an infectious agent. If a connection between GCA and HPV infection were to be established, the potential clinical implications are great, as GCA, with its possible vision loss -5, polymHuman papillomavirus (HPV) is an increasingly common human pathogen in recent decades. It is a mucosotropic virus which is not thought of as spreading systemically. However, HPV genotype 16 has been found integrated into the genome of bacteria isolated from cervical cancer biopsies, and there is also published data showing HPV viral particles within peripheral nerves and small vascular endothelial cells adjacent to oral and cervical cancers as demonstrated by transmission electron microscopy ,10. In tWith approval by the Washington University Human Research Protection Office (HRPO), we searched the Copath database of Barnes Jewish Hospital for temporal artery biopsy specimens from 1995 to 2008 and retrieved all specimens for which material was available. We identified approximately 60 cases, of which 43,  had material available for review. There were five males and 17 females in the histologically positive GCA group with a mean age of 78.9 years. In the histologically negative group, there were four males and 17 females with a mean age of 67 years, and the control kidney vascular margin group had six males and nine females with a mean age of 38.2 years. There were no statistically significant difference in gender among the groups (p = 0.176). The clinical diagnosis of GCA was based on the criteria of the American College of Rheumatology . HoweverTM HPV HR  assay.Two methods were utilized to evaluate for HPV DNA. The first, INNO-LiPA HPV Genotyping Extra, was used to perform the testing in our research laboratory of the division of anatomic and molecular Pathology, Department of Pathology and Immunology, Washington University School of Medicine, St. Louis. All blocks were then submitted to an outside laboratory  for independent preparation of genomic DNA and for testing employing the CervistaIn both laboratories to avoid potential contamination, a maximum physical separation between the pre- and post-amplification steps was used. Separate pipettes and other lab materials were used as a part of good laboratory practice. The FFPE blocks were cut and processed under strict conditions to prevent DNA from being carried over from one case to the next during microtomy. Also a new blade was used for each case and the area was cleaned. Ice cubes used to cool blocks were discarded between cases.With regards to the technical limitations of this study, it is well known that formalin fixation randomly fragments DNA in a duration-dependent manner, resulting in a partial degradation. The degree of fragmentation depends on the type and age of the sample and the conditions used for fixation. Due to this degradation, FFPE tissue is not suitable for amplification of large DNA segments. Nevertheless, PCR amplification of segments ranging up to 1300 base pair has been reported. On the other hand, incubation at an elevated temperature after proteinase K digestion partially removes formalin cross-linking of the DNA, improving yield as well as DNA performance in assays. Furthermore, in one of the techniques used in this study (INNO-LiPA HPV Genotyping), short-PCR-fragment (SPF 10) primers are employed, which amplify a 65 base pair segment of target DNA and this testing is considered to be one of the most sensitive PCR assays for the detection of HPV DNA.http://www.Gentra.com) as per the manufacturer's instructions from 10 μm sections cut from the paraffin blocks. The concentration of the prepped DNA was measured spectrophotometrically using Nanodrop. Detailed procedure information is available at their web site, but briefly, we placed five 10 micron sections of tissue into a 1.5 mL microcentrifuge tube and added 1.0 mL of xylene, vortexed, and incubated for five minutes with constant gentle mixing. Then we centrifuged it for five minutes at 13,000-16,000 x g. In the fume hood, discarded by pipetting the xylene supernatant and left behind the visible pellet (tissue). We repeated this xylene wash twice. We then added 1.0 mL 100% ethanol, vortexed, and incubated five minutes with constant gentle mixing at room temperature, centrifuged at 13,000-16,000 x g for five minutes to pellet the tissue, and discarded the ethanol. We repeated these ethanol washes twice. Subsequently we added 1.0 mL 70% ethanol, gently mixed, and centrifuged at 13,000-16,000 x g for five minutes at 4°C, removed all residual ethanol and allowed tissue pellet to dry by centrifugation under vacuum for five minutes. For cell lysis, we added 300 μl Cell Lysis Solution (Gentra Puregene™ kit) and gently vortexed for 30 seconds. Then we added three μl Puregene Proteinase K (20 mg/ml), and mixed by inverting 25 times and incubated the lysate at 55°C for three hours to overnight. We inverted the tube periodically during the incubation. Then was added three μl RNase A Solution to the cell lysate, and mixed by inverting the tube 25 times and incubated at 37°C for 15 min to one hour. For Protein Precipitation we cooled quickly the sample to room temperature by placing on ice and added 100 μl Protein Precipitation Solution (Gentra Puregene™ kit) to the cell lysate. The volume should be 1/3 of the Cell Lysis Solution in the tube. Subsequently, it is vortexed vigorously at high speed for 20 seconds to mix the Protein Precipitation Solution uniformly with the cell lysate. Then, we centrifuged it at 13,000-16,000 x g for five minutes. The precipitated proteins formed a tight pellet. If the protein pellet was not tight, we vortexed vigorously for 20 seconds at high speed, and then incubated on ice for 5 min. We then centrifuged at 13,000–16,000 x g for three minutes. Using a pipette, we removed the supernatant containing the DNA (leaving behind the precipitated protein pellet) into a clean 1.5 mL microcentrifuge tube. We added two μl (for 400 μl supernatant) of a DNA carrier  to aid recovery of small DNA quantities and then vortexed them. We then added 400 μl 100% of isopropanol. Subsequently, it was mixed by inverting gently ~50 times until the white threads of DNA formed a visible clump, and then it was centrifuged at 13,000-16,000 x g for 10 minutes and the supernatant was poured off. We added 500 μl of 70% ethanol and inverted the tube to wash the DNA pellet. We centrifuged at 13,000-16,000 x g for 5 minutes, and carefully poured off the ethanol and inverted and blotted the liquid from the tube on clean absorbent paper and allowed to air dry for 10-15 minutes. Finally, we added 50 μl DNA Hydration Solution and incubated at 65°C for one hour to dissolve the DNA. We incubated at room temperature overnight with gentle shaking. Samples could then be centrifuged briefly and transferred to a storage tube. The concentration of the DNA used for INNO-LiPA HPV Genotyping Extra testing in each case was 50 ng.DNA was extracted using PureGene Kit  genome is amplified using short-PCR-fragment assay (SPF10 primers), and the resulting biotinylated amplicons are then denatured and hybridized with specific oligonucleotide probes.The INNO-LiPA HPV Genotyping An additional primer pair for the amplification of the human HLA-DPB1 gene is added to monitor sample quality and extraction. The length of the HLA-DPB1 fragment is 280 base pairs. All probes are immobilized as parallel lines on membrane strips. After hybridization and stringent washing, streptavidin-conjugated alkaline phosphatase is added, which binds to any biotinylated hybrid previously formed.Extra Amp) is used for standardized preparation of biotinylated amplified material. This amplification kit is based on the polymerase chain reaction (PCR) using SPF10 primers.Incubation with BCIP /NBT (Nitro-Blue Tetrazolium Chloride) chromogen yields a purple precipitate, and the results are visually interpreted using the reference guide provided. An amplification kit  as described by the manufacturer  was used for purification of genomic DNA from formalin-fixed, paraffin-embedded tissues according to the manufacturer’s instructions with exception of incubation time. Overnight incubation in proteinase K for digestion of proteins/contaminants is not recommended by Qiagen but is something that CPA Laboratory has found useful to increase the nucleic acid elution.Slides were cut from the original blocks, and The QIAamp DNA FFPE Tissue kit  bind to the target DNA recognition site. When those sequence specific oligonucleotides overlap by at least one base pair on the target sequence, an invasive structure forms that acts as a substrate for the Cleavase® enzyme. The enzyme cleaves the 5’ portion (flap) of the probe at the position of the overlap.CervistaM HPV HR  is a qua6– 107 fold signal amplification per hour. The reagents for this test are provided as three oligonucleotide master mixtures, which identify the 14 types of HPV arranged according to phylogenetic relatedness. Master mixture 1 identifies the positivity of genotypes 51, 56 and 66. Master mixture 2 (MM2) shows the positivity of genotypes HPV 18, 39, 45, 59 and 68 and master mixture 3 (MM3) reveals the positivity of HPV genotypes 16, 31, 33, 35, 52 and 58. By design, the released 5'-flaps bind only to their respective FRET oligonucleotides to generate target-specific signal. A positive result indicates that at least one of the 14 high-risk types is present in the DNA sample. For each case 150 to 200 ng of DNA prepped from the FFPE tissues at CPA Labs were utilized for the Cervista HPV testing. The sensitivity of this test is set at 5000 copies of HPV DNA. The Cervista assay has an internal control to verify if the assay worked. If the internal control does not pass, it suggests either the sample is degraded or extraction failed or the PCR did not work.The probes are present in large molar excess and cycle rapidly on and off the target sequence generating many cleaved 5’ flaps per target sequence. The cleaved flaps then bind to a universal hairpin fluorescence resonance energy transfer (FRET) oligonucleotide creating another invasive structure that the Cleavase® enzyme recognizes as a substrate. The enzyme cleaves the FRET oligonucleotides between the fluorophore and quencher molecule and produces a fluorescence signal as the cleaved flaps cycle on and off. For each copy of target, the combined primary and secondary reactions result in 10T-test and Fisher's exact tests to evaluate the correlation between the presence of HPV DNA and other variables. P values < 0.05 were considered significant. All statistical analyses were performed using the SAS v.9.1 system software .Statistical analyses were performed using the Chi-Square, Extra, 16 of the 22  biopsy-confirmed GCA cases were positive for HPV. Twelve out of the 16 (75%) HPV-positive GCA-positive patients were female. Among these 16 positive cases, 5 had a HPV 6 alone (31%), 3 cases had HPV 16 alone (19%), 7 cases had both types 6 and 16 (44%), and only one case was extensively "multi-genotype" showing types 6, 16, 31, 33, and 40 (6%) . Two cases which were HPV type 6 by INNO-LiPA (cases # 4 and 6) had low genomic DNA when prepped for the CervistaTM HPV HR testing, so could not be evaluated by this method. All positive cases of the CervistaTM HPV HR were from the Master Mix 3 which includes the HPV genotypes 16, 31, 33, 35, 52 and 58. As expected, all cases for which genotype 6 was present in the INNO-LiPA method were negative with the Cervista method since low risk types are not evaluated in that assay. All negative cases with INNO-LiPA HPV Genotyping Extra were negative also with CervistaTM HPV HR testing. As such, there was 100% agreement between the two testing methods on the histologically positive temporal arteritis cases.Using the INNO-LiPA HPV Genotyping Extra, of which 3 (60%) were genotype 16, one (20%) was genotype 6 and one (20%) was genotype 52. CervistaTM HPV HR testing revealed positivity of Master Mix 3 for all 4 cases which were positive by INNO-LiPA HPV Genotyping Extra for high risk HPV. Case number 2 which was negative with INNO-LiPA was of low genomic DNA with Cervista method. As such, there was 100% agreement between the two testing methods on the histologically negative temporal artery biopsy cases.Among the 21 cases of GCA histologically negative biopsies, 5 were positive for HPV by INNO-LiPA HPV Genotyping Extra. Two cases showed genotype 16 (1 case of Wilms' tumor and 1 of acute and chronic pyelonephritis), and one genotype 6 . CervistaTM HPV HR testing revealed positivity of Master Mix 3 for both cases which were positive for high risk HPV by INNO-LiPA HPV Genotyping Extra. As such, there was 100% agreement between the two testing methods on the 15 vascular margin control cases.Among the 15 nephrectomy vascular resection margins (controls), 3 were positive for HPV by INNO-LiPA HPV Genotyping The association between HPV positivity and histologically confirmed GCA was statistically significant (p = 0.0013). There was also a statistically significant association between HPV positivity and Caucasian race (p = 0.0339). No other associations were statistically significant.In multivariate analysis Table , subjectGiant cell  arteritis is a chronic vasculitis involving medium and large size arteries that typically affects individuals older than 50 years of age.,3,4 AlthHistologically, the disease progresses from minimal involvement of vessels, with only collections of lymphocytes confined to the internal or external elastic lamina or adventitia to a panarteritis with segmental areas of necrosis of the arterial wall and extensive destruction of the elastic laminae, a feature that can be clearly demonstrated with special stains for elastic fibers Figures  and 2.The etiology of the inflammatory reaction in the GCA has not been identified. However, it has been demonstrated that there is a restricted clonal expansion of tissue-infiltrating T cells in these lesions, which suggests that they are reacting to a specific antigen located within the affected arterial wall which is thus eliciting the disease . The mosChlamydia pneumoniae, Mycoplasma pneumoniae , and Parvovirus B19 [C. pneumonia and herpes simplex virus (HSV). However, further studies were unable to detect the presence of these infectious agents. Cooper et al. [C. pneumonia, HSV, varicella zoster virus (VZV), Epstein–Barr virus (EBV), or human herpesvirus 7 (HHV7). In addition, Rodriguez-Pla et al. [C. pneumoniae in temporal artery GCA. Due to this conflicting and contradictory data, no conclusive link has, to date, been demonstrated between a GCA and a specific infectious agent.Infectious agents have been considered in the past as the etiology of GCA. Elling et al., by indirect serological evidence, found high incidence of GCA within a population associated with irus B19 . Publishirus B19  and Poweirus B19  reportedr et al.  and Cankr et al. , could na et al.  and othea et al. -23 couldThe role of human papillomavirus (HPV) in the development of cervical, head and neck, anal and skin cancers is well known. Molecular and epidemiological studies have shown that persistent HPV infection is the most important risk factor for cervical cancer ,25. HighEnterococcus , Staphylococcus , Bacillus and Corynebacterium) and demonstrated the HPV viral particles by transmission electron microscopy in those bacteria. In addition, HPV type 16 has been detected in peripheral nerves, and vascular endothelial cells [Interestingly, Ma et al.  reportedal cells . Anotheral cells . The rolal cells -32.In this study, we found that HPV DNA, of both high and low risk types, is present in formalin-fixed, paraffin-embedded temporal artery biopsy specimens, and in statistically significantly higher numbers in the arteritis specimens compared to histologically-negative temporal artery biopsies and nephrectomy vascular margin controls. The validity of these results was confirmed with exactly matching results by an outside laboratory that independently prepped DNA from the paraffin blocks and utilized a completely different detection method. Our results are admittedly surprising and bring many questions about what HPV's role, if any, would be in temporal arteritis. HPV has been associated with tumorigenesis, and many studies have investigated HPV-related modification of the immune system to establish infection. However, we are not aware of any literature citing HPV as an inciting agent for inflammatory diseases. Our findings do support the notion that HPV can disseminate systemically, but do not, by themselves, tell us anything specific about the pathophysiological relationship between the virus and temporal arteritis, if there even is one. Further studies are necessary to address this.It is important to mention that a negative temporal artery biopsy does not rule out the diagnosis of temporal artery GCA, since the changes can be patchy with skip areas of uninvolved artery. Although serial sectioning of the temporal artery biopsy is recommended, not all cases will be diagnosed histologically . ActuallIt is also worth noting that of the 16 histologically-proven GCA cases that had HPV, 11 were high risk genotype 16. Only five cases were positive for low risk HPV genotype 6. Although, the importance of distinguishing low risk from high risk HPV genotypes has been established in the pathogenesis of neoplasms, what its significance might be in inflammatory disorders such as GCA is currently unknown.In summary, we have identified HPV DNA in the majority of histologically-proven giant cell  arteritis specimens and validated these results by a completely independent outside laboratory assay. The association raises questions regarding the biology of HPV infections, when dissemination occurs, and what this dissemination means clinically. Such studies are required to understand the significance of the association in our series.The authors declare that they have no competing interests.AM conceived the study, carried out the molecular testing, analyzed the data, and drafted the manuscript. JDP and JSL, designed the study, collected data, helped interpret findings and critically revised 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/1471-2474/13/132/prepub


Human papillomavirus (HPV) is known to be more prevalent in spontaneous abortions than in elective terminations of pregnancy. More recently, placental infection with HPV was shown to be associated with spontaneous preterm delivery. However, no study has evaluated the prevalence of HPV infection in pregnant Korean females and its association with adverse pregnancy outcomes.We conducted a cross-sectional study of 311 females who gave birth at Korea University Medical Center. Our sample included 45 preterm deliveries, 50 cases of premature rupture of the membranes (PROM), 21 preeclampsia cases, and 8 gestational diabetes mellitus (GDM) patients. We used the Hybrid Capture II system to detect high-risk (HR)-HPV infection at six weeks postpartum.The prevalence of HR-HPV infection was 14.1%. Women with HR-HPV infection had a higher incidence of PROM than those without HR-HPV. HR-HPV infection was associated with an increased risk of PROM . The prevalence of preterm delivery, preeclampsia, or GDM was not different between the two groups.We observed a high prevalence of HR-HPV infection in pregnant women. Moreover, HR-HPV infection was associated with a risk of PROM at term. Further studies are needed to evaluate mechanisms by which HR-HPV infection induces PROM. If cervical carcinoma in Korea .Human papillomavirus (HPV) is a small, double-stranded DNA virus. High-risk (HR)-HPV is considered to be the main cause of cervical cancer. Pregnancy is known to be an independent risk factor for HR-HPV infection . AlthougWe conducted a cross-sectional study of 311 women who gave a birth at Korea University Medical Center (KUMC) from February 2010 to January 2011 and came to KUMC for follow-up at 6 weeks postpartum. Our analysis included 45 preterm deliveries, 50 women with premature rupture of the membranes (PROM), 21 cases of preeclampsia, and 8 gestational diabetes mellitus (GDM) patients. All women provided written informed consent, prior to participation in the study, which was approved by the ethical committee  at our institution.We used the Hybrid Capture II system  for detection of HR-HPV infection  at six weeks postpartum. The samples were obtained in the cervix. We collected the basic characteristics, such as age, parity, number of abortions, body weight and height of pregnant women, gestational age at delivery, Apgar score and birth weight of each neonate, smoking status, history of PROM, history of preterm birth, and delivery mode by medical chart review. Body mass index (BMI) is defined as the individual's body weight (kilogram) divided by the square of their height (meter).Preterm delivery was defined as delivery before the 37th week of gestation. PROM is the rupture of membranes at any given time during pregnancy prior to the onset of labor. Patients were diagnosed with preeclampsia if they had a maternal blood pressure of ≥ 140/90 mmHg recorded on two occasions at least six hours apart after 20 weeks of gestation without a previous history of hypertension and also if they had concomitant proteinuria (≥ 300 mg/24 hours urine or ≥ 1+ in dipstick test). GDM was diagnosed according to the American Diabetes Association (ADA) criteria .p-Values were two-tailed. Statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS) software, version 12.0 .Data are reported as mean ± standard deviation for continuous variables and as a percentage for categorical variables. Clinical characteristics in participants both with and without HR-HPV infection were compared using Student’s t-test and the Chi-square test or Fisher’s exact tests, when the variables were continuous and categorical, respectively. We conducted a multivariate logistic regression analysis to investigate the relationship between adverse pregnancy outcomes and HR-HPV infection. All reported HR-HPV infection was present in 14.1% of the study participants. The characteristics of these participants are presented in Table Table Figure A multivariate logistic regression analysis was used to evaluate the relationship of PROM with HR-HPV infection Table . After cTo our knowledge, our study is the first to report on an association between HR-HPV infection and PROM. Women who experienced PROM were more likely to experience HR-HPV infection than those without PROM.Although it is widely accepted that PROM is multifactorial, infection has been cited as a major cause of membrane damage . One posIn this study, the prevalence of HR-HPV infection was 14.1%. Inconsistencies exist in the literature on the incidence of HR-HPV infection in pregnant women. The reported prevalence of HR-HPV infection in pregnancy varies between 5% and 40% ,16. ThisOur findings have several limitations. First, PROM was not classified according to the gestational age. PROM at term does not have the same clinical impact as PPROM. But, because PROM at term also has several complications, such as increased maternal infections and neonatal intensive care unit admissions, we think that our study has enough meaning . In addiIn this study, we observed a high prevalence of HR-HPV infection in pregnant women. HR-HPV infection was also associated with a higher risk of PROM at term. Further studies are needed to evaluate the mechanism by which HR-HPV infection induces PROM.The authors declare that they have no competing interests.GJC, KJM, HRH, and MJO conceived of the study, and participated in its design and coordination. JHH, JKL, SWK, and HJK performed the statistical analysis. GJC and KJM participated in the sequence alignment and drafted 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/1471-2393/13/173/prepub


Men who have sex with men (MSM) especially those who are HIV positive are at risk for HPV-associated anal cancer. We systematically reviewed studies with data on the prevalence of vaccine preventable anal HPV among men who have sex with men aged 25 or younger and identified 6 studies. None of these studies were specifically designed to determine the prevalence of HPV in this population. Available data, albeit limited, suggest many young MSM may not already be HPV infected. Further studies using representative sampling focused on teenage MSM are required to confirm this. Genital human papillomavirus (HPV) infection is widespread -3 and usThe prophylactic quadrivalent HPV vaccine is effective in preventing infection with HPV types 6, 11, 16 and 18 and in reducing the incidence of anogenital warts and anal intraepithelial neoplasia in males ,15. HoweWe systematically reviewed studies by searching MEDLINE and abstracts from the European Research Organisation on Genital Infection and Neoplasia (EUROGIN) Conferences and the International Papillomavirus (IPV) Conferences. Key words used in the search included: “men who have sex with men” or “MSM” or “homosexual” or “bisexual” or “gay” and “human papillomavirus” or “HPV”.The following data were extracted: author, year of publication, study setting, number of MSM, number of MSM aged 25 or younger, sample collection method, HPV DNA testing method, and HPV type tested. HPV 6/11/16/18 prevalence were extracted, and where possible, HPV 6/11/16/18 prevalence stratified by age group .The outcome of the review is summarized in Figure The 6 studies included provided data on anal HPV for a total of 698 MSM aged ≤ 25. Among men aged ≤ 20, the prevalence of anal HPV 6, 11, 16 and 18 ranged from 0 to 17%, 0 to 17%, 0 to 14% and 0 to 20%, respectively. Among men aged 21 to 25, the prevalence of anal HPV 6, 11, 16 and 18 ranged from 8 to 19%, 0 to 8%, 0 to 18% and 3 to 8%, respectively.Through this review, we found that there have been no published studies that have been designed to specifically determine the prevalence of HPV among young MSM using a representative sample of young MSM. Of the six studies identified, all had potential biases because of the way the studies were designed. These could have led to either an overestimate or an underestimate of the true HPV prevalence among young MSM. Several studies recruited men from clinics including STD clinics ,17,18 whDespite these important limitations, the available data suggest HPV infection may still be uncommon among teenage same sex attracted males, although the data are currently insufficient to draw this conclusion with confidence. A community-based, representative sample is needed to determine the prevalence of HPV infection, defined rigorously, among mainly teenage same sex attracted males.Given that HPV 16 and 18 are responsible for around 80% of anal cancers  and HPV HPV: Human papillomavirus; MSM: Men who have sex with men; EUROGIN: European Research Organisation on Genital Infection and Neoplasia; IPV: The International Papillomavirus Society; STD: Sexually transmitted diseases.CF has received honoraria from CSL Biotherapies and Merck and research funding from CSL Biotherapies. CF owns shares in CSL Biotherapies the manufacturer for Gardasil. JH has received an honorarium from CSL Biotherapies and is an investigator on an Australian Research Council funded project (LP0883831) that includes CSL Biotherapies as a research partner. AG has received honoraria and untied research funding from CSL biotherapies, and has received honoraria from Merck. SG has received advisory board fees and grant support from CSL and GlaxoSmithKline, and lecture fees from Merck, GSK and Sanofi Pasteur; in addition, she has received funding through her institution to conduct HPV vaccine studies for MSD and GSK. SG is a member of the Merck Global Advisory Board as well as the Merck Scientific Advisory Committee for HPV. None of this relates to this specific work. MC and HZ have no conflicts of interest.HZ and MC carried out the literature review and drafted the article. JH, SG, KF and AG participated in the coordination and editing of the article. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/12/341/prepub