Reverse Hybridization Line Probe Assay Research Articles

Evaluation of the Roche MagNA Pure 96 nucleic acid extraction platform for the SPF10 line probe assay system.

To evaluate the MagNA Pure 96 (MP96) nucleic acid extraction system as an alternative for cervical specimen processing for human papillomavirus (HPV) genotyping detection by reverse hybridization line probe assay (LiPA-25), compared to the well-established extraction system MagNA Pure LC 2.0 (MPLC). A total of 200 cervical samples preserved in ThinPrepCyt® solution were extracted by MP96 and MPLC, respectively, and then purified nucleic acids were amplified using the SPF10 PCR primer set. Amplification products were subjected to SPF10-DNA enzyme immunoassay (DEIA) and LiPA-25. The concordance between different extraction methods in this study was reflected in the comparison of the results of the DEIA and LiPA-25. Agreement of HPV-positive results (DEIA) was 97.5% (Kappa=0.932). Pair-wise analyses of either HPV grouping (any HPV genotypes, any high-risk HPV genotypes, any low-risk HPV genotypes, any nonavalent vaccine-targeted HPV, and any non-vaccine-targeted oncogenic HPV genotypes) identified >95% agreement (all Kappa>0.900). For the two extraction methods, there was no statistical difference (chi-square test: P=0.690) for single versus multiple genotypes, and concordant, compatible, and discordant genotypes were observed in 87.0%, 9.5%, and 3.5% of 200 samples, respectively. HPV genotyping results of the MPLC system and the MP96 system showed a high degree of concordance. Combined with the advantages of high-throughput and anti-contamination of MP96, the MP96 extraction system could be more suitable for the testing of samples in future studies.

Identification of nontuberculous mycobacteria isolated from household showerheads of patients with nontuberculous mycobacteria

This study aimed to examine whether nontuberculous mycobacteria (NTM) inside household showerheads are identical to those in patients with NTM-pulmonary disease (PD) since household water is one of the potential NTM sources. Samples were obtained from 32 household showerheads of patients with NTM-PD recruited through the Pulmonary Outpatient Department at the Severance Hospital between October 2018 and October 2019. All isolates from patients with NTM-PD were diagnosed using a reverse-hybridization line probe assay based on the ropB gene. To determine the mycobacterial compositions, the washing fluids were collected and investigated using multiplex polymerase chain reaction assay and NTM culture; suspected microbial isolates in these fluids and culture were identified using sequencing analysis of 16S rRNA gene. NTM species causing the PD in the patients were Mycobacterium avium, M. intracellulare, M. abscessus, M. massiliense, and M. fortuitum complex. The mycobacteria isolated from the showerhead were M. lentiflavum, M. gordonae, M. triplex, M. phocaicum, M. mucogenicum, M. florentinum, M. gilvum, M. llatzerense, and M. peregrinum. However, the species identified in the showerheads did not match those of the patients. Despite NTM species in the showerheads, clinical implications in the main pathogenesis associated with the disease in the patients studied were not elucidated.

Association of high-risk human papillomavirus with ocular surface squamous neoplasia: a case-control study in Mexico.

To investigate the association of high-risk hu-man papilloma virus (HR-HPV) and other risk factors with ocular surface squamous cell neoplasia (OSSN). We obtained DNA from 22 fresh frozen OSSN tissues and 22 pterygia as controls, we used a broad-spectrum HPV DNA amplification short PCR fragment to identify HPV infection in all specimens and then genotyped HPV by a reverse hybridization line probe assay. We also obtained demographic, sun exposure, and tobacco consump-tion information. HR-HPV frequency was 40.9% in the OSSN group and 4.5% in the pterygia group (p=0.009). After covariate adjustment, OSSN was associated with HR-HPV (OR=16.3, 95%CI=1.2,218.1, p=0.03) and sunburn (OR=10.8, 95%CI=1.8,86.0, p=0.02). Ocular surface squamous cell neoplasia is a multifactorial disease. The strong association between HR-HPV and OSSN, suggests that HR-HPV could play an etiological role in OSSN development.

Human papillomavirus genotyping in high-grade squamous intraepithelial lesion.

Human papillomavirus infection is one of the most common sexually transmitted infections. Long-term exposure to the HPV leads to high-grade squamous intraepithelial lesions affecting cervical cancer. Knowledge about the distribution of HPV genotypes is crucial to guide the introduction of prophylactic vaccines. We aimed the genotype distribution in patients reporting due to abnormal Pap - smear tests. We provide a prospective observational cohort study. We obtained material from 428 women registered to Provincial Hospital in Poznan and Specialist Medical Practice in 2018-2020. In the current study, we analyze results from the first 110 inclusions with the diagnosis of HSIL from a cervical biopsy. The probe for the molecular test was collected with a combi brush and passed to an independent, standardized laboratory. HPV detection was done using PCR followed by DNA enzyme immunoassay and genotyping with a reverse hybridization line probe assay. Sequence analysis was performed to characterize HPV-positive samples with unknown HPV genotypes. The molecular test detected DNA of 41 HPV genotypes. We performed statistical analyzes using the STATISTICA package 13.3. We found that 98.2% of patients received HPV-positive test results. The most frequent HPV genotype was 16, which assumed for 54.1%. In patients negative for HPV 16, the percentage decreased with increasing age. We detected that the following HPV types are next most common: HPV 31 (16.2 %), HPV 52 (11.7%), HPV 51 (9.9%), HPV 18 (9.0%), HPV 33 (9%). Moreover, thyroid diseases were the most common comorbidities and occurred in 15 patients CONCLUSIONS: To our knowledge, this study is the most extensive assessment of HPV genotypes in HSIL diagnoses in Poland.

Human papillomavirus genotyping in low-grade squamous intraepithelial lesions.

Human papillomavirus infection is one of the most common sexually transmitted infections. Histological LSIL in 70-80% of cases will regress spontaneously, while a subset is associated with residual risk for a future precancerous lesion. This study evaluates the performance of HPV genotypes for LSIL preceded by normal or mildly abnormal Pap smear. We provide a prospective observational cohort study. We obtained material from 428 women registered to Specialist Medical Practice and Provincial Hospital in Poznań in 2018-2021. In the current study, we analyze results from the first 112 inclusions with the diagnosis of LSIL from a cervical biopsy. The probe for the molecular test was collected with a combi brush and passed to the independent, standardized laboratory. HPV detection was done using PCR followed by DNA enzyme immunoassay and genotyping with a reverse hybridization line probe assay. Sequence analysis was performed to characterize HPV - positive samples with unknown HPV genotypes. The molecular test detected DNA of 42 HPV genotypes. We performed statistical analyzes using the STATISTICA package 13.3. We found that 77.7% of patients received HPV-positive test results. The most frequent HPV genotype was 16, which was assumed for 22.3%. We detected that following HPV types are next most common: HPV 56 (11.6%), HPV 52 (8.9%), HPV 31 (8.0%) and HPV 51 (8.0%). Among HPV 16-negative women, the vast majority are those living in the town (p = 0.048). Moreover, thyroid diseases were the most common comorbidities. To our knowledge, this study is the most extensive assessment of HPV genotypes in LSIL diagnoses in Poland.

Genotyping of human papillomavirus DNA in Wielkopolska region.

Human papillomavirus infection is one of the most common sexually transmitted diseases. Long-term exposure to the HPV leads to development of high-grade squamous intraepithelial lesions that can eventually transform into cervical cancer. The aim of the study was to assess the HPV genotype distribution in patients with abnormal pap smear and provide prospective study. We obtained material from 674 women who registered to Specialist Medical Practice in the years 2008-2020. The sample for the molecular test was collected using combi brush and forwarded to the independent, standardized laboratory. HPV detection was done using PCR followed by DNA enzyme immunoassay and reverse hybridization line probe assay for virus genotyping. Sequence analysis was performed to characterize virus genotypes in HPV - positive samples. We found that 53% of patients tested positive for HPV. The percentage decreased with age. The following HPV types were the most common: HPV - 16 (24.5%), HPV - 53 (13.1%), HPV - 31 (10.3%), HPV - 51 (9.7%), HPV - 56 (9.5%). To our knowledge, this study is the largest assessment of HPV genotypes in Poland. Our results suggest that type-specific, high-risk HPV DNA - based screening should focus on HPV types 16, 31, 51, 56.

Age distribution of human papillomavirus infection and neutralizing antibodies in healthy Chinese women aged 18–45 years enrolled in a clinical trial

ObjectivesData from clinical trials of human papillomavirus (HPV) vaccines showed that women naïve (negative for both type-specific antibodies and DNA) to vaccine types would derive benefit from vaccination; therefore, an understanding of the proportion of naïve women in different age groups is important for developing HPV vaccination strategies. MethodsFrom November 2012 to April 2013, a total of 7372 healthy women aged 18–45 years were recruited in five provinces in China. Cervical specimens and serum samples were collected for each woman at entry. Cervical specimens were first tested by the HPV DNA enzyme immunoassay method; if positive, the specimens were then tested by reverse hybridization line probe assay and HPV-16 and HPV-18 specific polymerase chain reactions. Neutralizing antibodies against HPV-16 or HPV-18 were tested with a pseudovirion-based neutralization assay. ResultsThe overall prevalence of high-risk HPV DNA was 14.8% (1088/7367, 95% CI 14.0–15.6), and the seroprevalence of neutralizing antibodies against HPV-16 and HPV-18 was 12.6% (925/7367) and 4.9% (364/7367), respectively. In younger women (18–26 years) and middle-aged women (27–45 years), 83.8% (3116/3719) and 81.4% (2968/3648) were naïve to both HPV-16 and HPV-18 (both neutralizing antibodies and DNA were negative), respectively. In addition, 98.5% (3664/3719) and 98.0% (3575/3648) of the younger or middle-aged women were naïve to at least one HPV type (HPV-16 or HPV-18). DiscussionThis study revealed that the majority of Chinese women aged 18–26 years and 27–45 years were naïve to both HPV-16 and HPV-18 and would thus derive full benefit from bivalent HPV vaccination.

HIGH PREVALENCE OF HUMAN PAPILLOMAVIRUS (HPV) INFECTIONS AND HIGH FREQUENCY OF MULTIPLE HPV GENOTYPES IN HUMAN IMMUNODEFICIENCY VIRUS-INFECTED WOMEN IN INDIA

Groups of 208 human immunodeficiency virus (HIV)-infected women in India were studied for the presence of human papillomavirus with the general SPF10 PCR primer set. Virtually all (98%) women were found positive for human papillomavirus (HPV) DNA. Genotyping by the reverse hybridization line probe assay (HPV-LiPA) revealed a high prevalence of multiple genotypes (78.9% of the cases), with an average of 3.1 genotypes per patient (range, 1 to 10 genotypes). HPV 6 was the most prevalent genotype and was observed in 80 (39.2%) patients, followed by types 51 (31.9%), 11 (26.0%), 18 (24.0%), and 16 (22.5%). Of the genotypes detected, 40.9% were low-risk genotypes. Twenty-two (10.5%) patients showed normal (Pap I) cytology, 149 (71.6%) patients had inflammation (Pap II), and 28 patients (13.4%) had a Pap III score. The prevalence of high-risk genotypes increased with the cytological classification. There were no significant associations between the number of HPV genotypes detected and the cytological classification, HIV viral load, and CD4 count in these patients. In conclusion, the highly sensitive SPF10 LiPA system shows that very high proportions of HIV-infected women in Brazil is infected with HPV and often carry multiple HPV genotypes.
 Key-words: HIV, Human Papillomavirus, Genotypes, Women

NS3 genomic sequencing and phylogenetic analysis as alternative to a commercially available assay to reliably determine hepatitis C virus subtypes 1a and 1b.

ObjectiveTo evaluate the use of hepatitis C virus (HCV) NS3 sequencing as alternative to the comercially available Versant HCV 2.0 reverse hybridization line-probe assay (LiPA 2.0) to determine HCV genotype 1 (HCV-1) subtypes.Patients and methodsA cohort of 104 patients infected by HCV-1 according to LiPA 2.0 was analyzed in a cross-sectional study conducted in patients seen from January 2012 to June 2016 at an outpatient clinic in Buenos Aires, Argentina.ResultsThe samples were included within well supported subtype clades: 64 with HCV-1b and 39 with HCV-1a infection. Twenty of the HCV-1a infected patientes were included in a supported sub-clade “1” and 19 individuals were among the basal sub-clade “2”. LiPA 2.0 failed to subtype HCV-1 in 20 (19.2%) individuals. Subtype classification determined by NS3 direct sequencing showed that 2/18 (11.1%) of the HCV-1a-infected patients as determined by LiPA 2.0 were in fact infected by HCV-1b. Of the HCV-1b-infected according to LiPA 2.0, 10/66 (15.2%) patients showed HCV-1a infection according to NS3 sequencing. Overall misclassification was 14.3% (κ-index for the concordance with NS3 sequencing = 0.635). One (1%) patient was erroneously genotyped as HCV-1 and was revealed as HCV genotype 4 infection.ConclusionsGenomic sequencing of the HCV NS3 region represents an adequate alternative since it provides reliable genetic information. It even distinguishes between HCV-1a clades related to resistance-associated substitutions to HCV protease inhibitors, it provides reliable genetic information for genotyping/subgenotyping and simultaneously allows to determine the presence of resistance-associated substitutions to currently recommended DAAs.

Association between HLA-DRB1*01 and HLA-Cw*08 and outcome following HTLV-I infection.

Human T cell lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is an inflammatory disease which occurs in less than 2% of HTLV-I -infected individuals. High proviral load, high HTLV-I-specific CD8+ cytotoxic T lymphocyte frequency (CTL) and host genetic factors such as HLA all appear to be associated with HTLV-I infection. Previous studies have shown that HLA-DRB1*01 increases the risk of HAM/TSP in Japanese HTLV-I infected individuals. To investigate the association between HLA class II DRB1 alleles and HLA class I alleles (HLA-Cw*08, B54, A*02 and A-30) in HTLV-I infected individuals in Mashhad. Here we determined the frequency of HLA class II DRB1, using INNO-LIPA reverse hybridization line probe assay, and HLA class I alleles (HLA-Cw*08,B54, A*02 and A-30) by PCR-SSCP method in healthy controls, HAM/TSP patients and HTLV-I infected individuals born and resident in Mashhad. The frequency of HLA-DRB1*01 alleles in this population was different from other areas of Iran. The frequency of HLA-DRB1*01 was significantly increased in HAM/TSP patients compared with carriers (p 0.028; OR=9.4). The frequency of HLA-Cw*08 was also significantly increased in HAM/TSP patients compared with controls (p=0.03; OR=13.5). Our results may suggest that possession of HLA-DRB1*01 increases the risk of HAM/TSP in HTLV-I-infected individuals and HLA-Cw*08 correlates with low CTL immune response in HAM/TSP patients.

Molecular Assay and Genotyping of Hepatitis C Virus among Infected Egyptian and Saudi Arabian Patients.

Hepatitis C virus (HCV) infection is a major health problem recognized globally. HCV is a common cause of liver fibrosis that may lead to liver cirrhosis or hepatocellular carcinoma. The aim of this study was to estimate the prevalence of HCV infection and genotyping among Egyptian and Saudi Arabian chronic patients using different molecular techniques. HCV RNA viral load was assessed by real-time polymerase chain reaction (RT-PCR) technology. For HCV genotyping, RT-PCR hybridization fluorescence-based method and reverse hybridization line probe assay (INNO-LiPA) were used. A total of 40 anti-HCV-positive patients with chronic hepatitis C were examined for HCV RNA, genotyping, and different laboratory investigations. In the present study, HCV genotypes 4, mixed 4.1b, and 1 were detected in patients of both countries, while genotype 2 was only detected in Saudi Arabian patients. Genotyping methods for HCV showed no difference in the classification at the genotype level. With regard to HCV subtypes, INNO-LiPA assay was a reliable test in HCV genotyping for the detection of major genotypes and subtypes, while RT-PCR-based assay was a good test at the genotype level only. HCV genotype 4 was found to be the predominant genotype among Egyptian and Saudi Arabian chronic patients. In conclusion, data analysis for detecting and genotyping HCV was an important factor for understanding the epidemiology and treatment strategies of HCV among Egyptian and Saudi Arabian chronic patients.

Susceptibility and progression of end stage renal disease are not associated with angiotensin II type 1 receptor gene polymorphism

Context: The role of the angiotensin II type 1 receptor (AT1R) gene polymorphism, A1166C, has been shown to be associated with end stage renal disease (ESRD) and its progression. There is also some evidence that HLA class II alleles are associated with ESRD independent of other factors. Objective: To examine the association between AT1R gene polymorphism in the susceptibility and progression to ESRD in patients with chronic renal failure and to investigate if the AT1R genotypes and HLA-DR alleles predict the time to ESRD. Materials and methods: Genotyping was performed in 50 ESRD patients and 44 control subjects for the AT1R A1166C gene polymorphism using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). ESRD patients were examined for HLA-DRB1 alleles according to a reverse hybridization line probe assay. Results: Allele and genotype frequencies of the AT1R polymorphism did not differ significantly between ESRD patients and controls. Furthermore, there was no association between the AT1R gene polymorphism or HLA-DRB1 alleles with the time to the occurrence of end stage failure. Discussion and Conclusion: We concluded that the AT1R genotype does not contribute to the genetic susceptibility of ESRD and is not associated with progression of chronic kidney failure to ESRD.

Type distribution of human papillomavirus among adult women diagnosed with invasive cervical cancer (stage 1b or higher) in New Zealand.

BackgroundHuman papillomavirus (HPV) is the necessary cause of cervical cancer. Published data on the epidemiology of HPV in women with invasive cervical cancer (ICC) in New Zealand (NZ) are limited. This cross-sectional study investigated the distribution of high-risk and low-risk HPV types in cervical specimens collected from women throughout NZ who had been diagnosed with ICC between 2004 and 2010.MethodsWomen aged ≥18 years, with ICC International Federation of Gynecology and Obstetrics stage Ib or greater were identified from the five tertiary public hospitals in NZ regularly treating women with ICC. Women were enrolled in the study only after obtaining informed consent. Stored, formalin fixed, paraffin-embedded cervical specimens were retrieved and histopathologically reviewed to confirm the diagnosis of ICC. Cervical specimens were tested for HPV using polymerase chain reaction-short fragment10; HPV DNA was detected using DNA enzyme immunoassay and typed by reverse hybridization line probe assay.Results242 women were enrolled and ICC was histologically confirmed in 227 samples. HPV infection was detected in 88.5% (n = 201; 95% CI: 83.7–92.4) of women with ICC; high-risk HPV types were detected in 87.2% of women. The most commonly detected HPV types were HPV-16 (51.1%) and HPV-18 (20.7%), followed by HPV-31 (4.0%), HPV-45 and HPV-52 (3.1% each). Overall, HPV distribution was highest (94.3%) in women aged 30–39 years at diagnosis and a higher distribution of HPV-16 (68.8%) was observed in women younger than 30 years. The overall distribution of HPV types between Maori and non-Maori women were similar. HPV-positive women with ICC stage II or greater were less likely to be infected with HPV-16/18 (P = 0.002) or HPV-18 (P = 0.029) compared with the other high-risk types. Single type infection and multiple infections were detected in 93.5% and 5.5% of women, respectively.ConclusionsHPV-16, HPV-18, HPV-31, HPV-45 and HPV-52 were the most commonly detected high-risk HPV types. Findings from the study fill an important data gap on HPV type distribution from NZ which will help facilitate better understanding of the epidemiology of HPV in NZ women.Clinical trial registrationNCT01328028.

Evaluation of p16INK4a Overexpression in a Large Series of Cervical Carcinomas

The aims of this study were to assess the overexpression of p16 in formalin-fixed paraffin-embedded cervical carcinoma tissue blocks and to determine its concordance with the human papillomavirus (HPV) status using the SPF10-LiPA25 polymerase chain reaction System and its correlation with the histologic type of invasive cervical cancer (ICC) and individual HPV genotypes. A total of 205 retrospectively collected ICC cases were analyzed by p16 immunohistochemistry. HPV detection was performed by polymerase chain reaction using SPF10 broad-spectrum primers, followed by deoxyribonucleic acid enzyme immunoassay and genotyping by reverse hybridization line probe assay (LiPA25). Of 205, 188 analyzed (91.7%) ICC cases showed p16 overexpression, whereas 181 (83.3%) cases were HPV positive using HPV LiPA testing. One hundred and seventy four (84.9%) cases were both p16 and HPV LiPA positive, indicating a positive concordance of 89.9% between both techniques (κ index agreement of 0.43; P<0.001), and no statistically significant difference (McNemar test, P>0.05). Squamous cell carcinomas were strongly positive compared with the adenocarcinomas (93.6% vs. 75% of the cases, respectively). When performed on formalin-fixed paraffin-embedded cervical tissue specimens, the higher positivity rate of p16 immunohistochemistry as compared with HPV DNA testing may allow identifying HPV-related ICC cases in which HPV testing was negative.

Hepatitis C virus genotype 6: Virology, epidemiology, genetic variation and clinical implication

Hepatitis C virus (HCV) is a serious public health problem affecting 170 million carriers worldwide. It is a leading cause of chronic hepatitis, cirrhosis, and liver cancer and is the primary cause for liver transplantation worldwide. HCV genotype 6 (HCV-6) is restricted to South China, South-East Asia, and it is also occasionally found in migrant patients from endemic countries. HCV-6 has considerable genetic diversity with 23 subtypes (a to w). Although direct sequencing followed by phylogenetic analysis is the gold standard for HCV-6 genotyping and subtyping, there are also now rapid genotyping tests available such as the reverse hybridization line probe assay (INNO-LiPA II; Innogenetics, Zwijnaarde, Belgium). HCV-6 patients present with similar clinical manifestations as patients infected with other genotypes. Based on current evidence, the optimal treatment duration of HCV-6 with pegylated interferon/ribavirin should be 48 wk, although a shortened treatment duration of 24 wk could be sufficient in patients with low pretreatment viral load who achieve rapid virological response. In addition, the development of direct-acting antiviral agents is ongoing, and they give high response rate when combined with standard therapy. Herein, we review the epidemiology, classification, diagnosis and treatment as it pertain to HCV-6.

Prevalence of Human Papillomavirus (HPV) Genotypes and Multiple Infections in Routine Cervical Cancer Screening in a Spanish Regional Population

Background: Infection with human papillomavirus (HPV) has been identified as the primary cause of cervical cancer. Persistent infection with high risk-HPV is the required factor for the development of cervical cancer. Objective: The aim of this study was to know the prevalence and distribution of cervical HPV infection in women attending routine cervical cancer screening. Study design: Liquid-based cytology (LBC) of cervical specimens was processed for HPV testing by INNO-LiPA HPV genotyping Extra Reverse Hybridization Line Probe Assay kit. Results: The cytological results of 115 LBCs were: 11(9.6%) negative for intraepithelial lesion or malignancy (NILM), 32(27.8%) atypical squamous cells of undetermined significance (ASC-US), 62(53.9%) low-grade squamous intraepithelial lesion (LSIL) and 10(8.7%) high-grade squamous intraepithelial lesion (HSIL). No cases of cervical cancer were detected. The mean age was 33.78 years ± 10.75 years (range 17-62 years). The overall prevalence DNA HPV detection was 81.7% (94/115), with a large proportion of multiple HPV infection (55.0%). HPV16 was the most common type detected (8.7%) followed by HPV51 (7.8%), HPV52 (5.2%), and HPV66 (2.6%). We found a high proportion of HPV positive cases in all cytological categories, including NLIM cases. Conclusions: These results indicate that prevalence of HPV infection in women attending routine cervical cancer screening is high. Our results confirm the importance of identifying the types of HPV that affect this community to design more effective prevention strategies and thus contribute to the fight against cervical cancer and its precursor lesions.

Human papillomavirus prevalence and type distribution in invasive cervical cancer in sub‐Saharan Africa

In sub-Saharan Africa, invasive cervical cancer (ICC) incidence and mortality are among the highest in the world. This cross-sectional epidemiological study assessed human papillomavirus (HPV) prevalence and type distribution in women with ICC in Ghana, Nigeria, and South Africa. Cervical biopsy specimens were obtained from women aged ≥ 21 years with lesions clinically suggestive of ICC. Histopathological diagnosis of ICC was determined by light microscopy examination of hematoxylin and eosin stained sections of paraffin-embedded cervical specimens; samples with a confirmed histopathological diagnosis underwent HPV DNA testing by polymerase chain reaction. HPV-positive specimens were typed by reverse hybridization line probe assay. Between October 2007 and March 2010, cervical specimens from 659 women were collected (167 in Ghana, 192 in Nigeria and 300 in South Africa); 570 cases were histologically confirmed as ICC. The tumor type was identified in 551/570 women with ICC; squamous cell carcinoma was observed in 476/570 (83.5%) cases. The HPV-positivity rate in ICC cases was 90.4% (515/570). In ICC cases with single HPV infection (447/515 [86.8%]), the most commonly detected HPV types were HPV16 (51.2%), HPV18 (17.2%), HPV35 (8.7%), HPV45 (7.4%), HPV33 (4.0%) and HPV52 (2.2%). The prevalence of single and multiple HPV infections seemed higher among HIV-positive women and HPV type distribution appeared to differ according to tumor type and HIV status. In conclusion, HPV16, 18, 45 and 35 were the most common HPV types in sub-Saharan African women with ICC and HPV infections were more common in HIV-positive women.

Worldwide human papillomavirus genotype attribution in over 2000 cases of intraepithelial and invasive lesions of the vulva

BackgroundHuman papillomavirus (HPV) contribution in vulvar intraepithelial lesions (VIN) and invasive vulvar cancer (IVC) is not clearly established. This study provides novel data on HPV markers in a large series of VIN and IVC lesions. MethodsHistologically confirmed VIN and IVC from 39 countries were assembled at the Catalan Institute of Oncology (ICO). HPV-DNA detection was done by polymerase chain reaction using SPF-10 broad-spectrum primers and genotyping by reverse hybridisation line probe assay (LiPA25) (version 1). IVC cases were tested for p16INK4a by immunohistochemistry (CINtec histology kit, ROCHE).An IVC was considered HPV driven if both HPV-DNA and p16INK4a overexpression were observed simultaneously. Data analyses included algorithms allocating multiple infections to calculate type-specific contribution and logistic regression models to estimate adjusted prevalence (AP) and its 95% confidence intervals (CI). ResultsOf 2296 cases, 587 were VIN and 1709 IVC. HPV-DNA was detected in 86.7% and 28.6% of the cases respectively. Amongst IVC cases, 25.1% were both HPV-DNA and p16INK4a positive. IVC cases were largely keratinising squamous cell carcinoma (KSCC) (N=1234). Overall prevalence of HPV related IVC cases was highest in younger women for any histological subtype. SCC with warty or basaloid features (SCC_WB) (N=326) were more likely to be HPV and p16INK4a positive (AP=69.5%, CI=63.6–74.8) versus KSCC (AP=11.5%, CI=9.7–13.5). HPV 16 was the commonest type (72.5%) followed by HPV 33 (6.5%) and HPV 18 (4.6%). Enrichment from VIN to IVC was significantly high for HPV 45 (8.5-fold). ConclusionCombined data from HPV-DNA and p16INK4a testing are likely to represent a closer estimate of the real fraction of IVC induced by HPV. Our results indicate that HPV contribution in invasive vulvar cancer has probably been overestimated. HPV 16 remains the major player worldwide.

Human papillomavirus (HPV)-associated early stage non-small cell lung cancer (NSCLC).

7560 Background: HPV is an established risk factor for cervical and oropharyngeal cancer. It has been suggested as a potential risk factor for lung cancer based on studies conducted mostly in Asian patients with advanced NSCLC. We characterize potential role of HPV in a North American patient population with early stage NSCLC. Methods: We analyzed surgically resected samples of NSCLC patients diagnosed between 2002-2007. HPV status was determined by polymerase chain reaction (PCR) using the INNO-LiPA genotyping Extra Amplification and Genotyping Extra kits, followed by reverse hybridization line probe assay to identify specific HPV serotypes. Differences between HPV+ and HPV- patients were assessed by Chi-square, Fisher’s exact, and Wilcoxon rank-sum tests. Survival was estimated by the Kaplan-Meier method and differences between HPV+ and HPV- patients were assessed by Log-rank test. Multivariable logistic regression modeling was employed to select predictors of HPV+ NSCLC. The significance levels were set at 0.05 for all tests. Results: Paraffin-embedded tumor samples from 208 patients were analyzed; M/F (49%/51%); stage I: 80%; II: 11%; III: 9%; IV: &lt;1%; Caucasians 95.6% and African-Americans (AA) 4.4%. There were 32 (14.9%) HPV+ cases including 10 cases with HPV 16/18. Ethnicity was significantly associated with HPV status (p-value=0.033). AA patients are more likely to be HPV+ (OR: 7.373; p=0.01) and more likely to harbor high risk serotypes 16/18 (OR: 10.8; p=0.05). Regression modeling identified AA ethnicity, adenocarcinoma (ADC) histology and current smoking (parameter estimates of 2.04, -2.34 and -2.82 respectively) as predictors of HPV+ NSCLC. Smoking status and histology showed significant interaction in predicting HPV+ tumors: OR: 0.06; p=0.0023 for smokers with squamous cancer; 2.52; p=0.24 for smokers with ADC and 10.339; p=0.0293 for smokers with adenosquamous cancer. Median disease free survival (NR; p=0.42) and median overall survival (71 months; vs. 55 months) were not significantly different between HPV+ and HPV- patients. Conclusions: HPV positivity is observed in 15% of early stage NSCLC with strong association with AA ethnicity, adenosquamous histology and non-smoking status.

Role of CCR5Δ32 mutation in protecting patients with Schistosoma mansoni infection against hepatitis C viral infection or progression.

Schistosomiasis has been incriminated in the significant increase in hepatitis C virus (HCV) infections, although the association has not been adequately explained. We hypothesized that the CCR5Δ32 mutation may be involved in the high prevalence of HCV with schistosomiasis. The aim was to explore the association between the CCR5Δ32 mutation in schistosomiasis patients and protection against HCV infection or progression. We compared 220 schistosomiasis patients (S group) and 190 patients with HCV and schistosomiasis (HCV/S group) for the presence of the CCR5Δ32 mutation. Clinical, biochemical, and radiological assessments were done. HCV infection was diagnosed with anti-HCV antibodies and a recombinant HCV antigen-based rapid immunochromatographic test, and confirmed by HCV reverse transcriptase PCR. HCV genotyping was done by reverse hybridization line probe assay. Schistosomiasis was diagnosed by FAST-ELISA and indirect hemagglutination for Schistosoma mansoni antibodies, and stool analysis for ova. Polymorphisms of the CCR5 receptor gene were assessed by PCR-based genotyping of the 32-bp deletion at the CCR5 locus in whole blood. Of HCV/S patients, 91.6 vs. 91.8 % of S patients had CCR5 WT/WT homozygosity (nonmutants). Heterozygous and homozygous CCR5Δ32 mutation patterns (CCR5Δ32/WT and CCR5Δ32/Δ32) were distributed similarly in the HCV/S and S groups (6.8 vs. 7.2 % and 0.53 vs. 0.90 %, respectively; p > 0.05, OR = 0.97). Genotype 4 was the predominant viral genotype (93 % of cases). No differences were observed in CCR5 gene patterns according to viral genotype, viral RNA count, or ALT level. However, CCR5Δ32 mutants (homozygous and heterozygous) had a lower rate of severe hepatic fibrosis vs. nonmutants (27 vs. 42 %, p = 0.101, OR = 0.51). Moreover, 53.4 % of CCR5Δ32/WT mutants showed spontaneous viral clearance vs. 26.2 % of nonmutants (p = 0.000, OR = 4.1). In conclusion, no association was detected between the CCR5Δ32 mutation and HCV disease susceptibility in schistosomiasis patients. However, patients with the CCR5Δ32 mutation and HCV infection were less prone to severe hepatic fibrosis and more likely to have spontaneous viral clearance than patients with the nonmutant genotype.