Microbial Community Distribution and Core Microbiome in Successive Wound Grades of Individuals with Diabetic Foot Ulcers.

Diabetic foot ulcer (DFU) is a major complication of diabetes with high morbidity and mortality rates. The pathogenesis of DFUs is governed by a complex milieu of environmental and host factors. The empirical treatment is initially based on wound severity since culturing and profiling the antibiotic sensitivity of wound-associated microbes is time-consuming. Hence, a thorough and rapid analysis of the microbial landscape is a major requirement toward devising evidence-based interventions. Toward this, 122 wound (100 diabetic and 22 nondiabetic) samples were sampled for their bacterial community structure using both culture-based and next-generation 16S rRNA-based metagenomics approach. Both the approaches showed that the Gram-negative microbes were more abundant in the wound microbiome. The core microbiome consisted of bacterial genera, including Alcaligenes, Pseudomonas, Burkholderia, and Corynebacterium in decreasing order of average relative abundance. Despite the heterogenous nature and extensive sharing of microbes, an inherent community structure was apparent, as revealed by a cluster analysis based on Euclidean distances. Facultative anaerobes (26.5%) were predominant in Wagner grade 5, while strict anaerobes were abundant in Wagner grade 1 (26%). A nonmetric dimensional scaling analysis could not clearly discriminate samples based on HbA1c levels. Sequencing approach revealed the presence of major culturable species even in samples with no bacterial growth in culture-based approach. Our study indicates that (i) the composition of core microbial community varies with wound severity, (ii) polymicrobial species distribution is individual specific, and (iii) antibiotic susceptibility varies with individuals. Our study suggests the need to evolve better-personalized care for better wound management therapies.IMPORTANCE Chronic nonhealing diabetic foot ulcers (DFUs) are a serious complication of diabetes and are further exacerbated by bacterial colonization. The microbial burden in the wound of each individual displays diverse morphological and physiological characteristics with unique patterns of host-pathogen interactions, antibiotic resistance, and virulence. Treatment involves empirical decisions until definitive results on the causative wound pathogens and their antibiotic susceptibility profiles are available. Hence, there is a need for rapid and accurate detection of these polymicrobial communities for effective wound management. Deciphering microbial communities will aid clinicians to tailor their treatment specifically to the microbes prevalent in the DFU at the time of assessment. This may reduce DFUs associated morbidity and mortality while impeding the rise of multidrug-resistant microbes.

Relevance and actionable mutational spectrum in oral squamous cell carcinoma.

BACKGROUND: Screening for lesions in the oral cavity is critical for early diagnosis of oral squamous cell carcinoma (OSCC). Targeted next generation sequencing-based (NGS) mutation analysis of cancer driver genes becomes a reality for personalized medicine and cancer therapeutics. MATERIALS AND METHODS: In the present study, we have performed a targeted NGS-based mutation analysis of 50 known oncogenes and tumor suppressor genes in clinically diagnosed potentially malignant lesions and tissues of OSCC. NGS-based analysis of DNA obtained from biopsies of histopathologically confirmed cases of potentially malignant lesions and OSCC specimens were performed using Ion AmpliSeq™ Cancer Hotspot Panel V2 using the Ion Proton™ Sequencer System, followed by data analysis using Ion Reporter™ and Torrent Suite™ software. RESULTS: NGS analysis indicated a total of 69 mutations present in 25 genes in potentially malignant lesions and OSCC specimens. We identified recurrent mutations in known OSCC driver genes ATM (11%), TP53 (55%), HRAS (16%), SMAD4 (13%), PIK3CA (16%), and ERBB4 (11%) in potentially malignant lesions and OSCC specimens. Driver mutation analysis identified recurrent TP53 and HRAS driver mutations in our OSCC specimens. CONCLUSION: Data generated from our study may enable an application of targeted NGS analysis of driver mutations for better therapeutic choice and improved outcomes for OSCC subjects when combined with clinical diagnosis.

Enumeration of deregulated miRNAs in liquid and tissue biopsies of cervical cancer.

OBJECTIVE: The altered miRNAs expression in cervical cancer tissue can be a critical player during tumorigenesis, may contribute to tumor cell heterogeneity and may determine distinct phenotypes within the tumor. Recent studies have highlighted the role of circulating miRNAs as a minimally-invasive biomarker and its potential as biosignature to complement routine tissue-based procedures. METHODS: In order to determine whether miRNAs in serum can indicate changes in cervical tissue specimens, we performed small RNA sequencing and selected miRNAs were validated using qRT-PCR in serum and tissue specimens (n = 115). Further, luciferase assay were performed to investigate the interactions between hsa-miR-409-3p and hsa-miR-454-3p binding sites on 3'UTR region of MTF2 and ST18 respectively. RESULTS: We have identified a total of 14 differentially expressed miRNAs common in serum and tissue specimens. Among them, hsa-miR-17-5p, hsa-miR-32-5p and hsa-miR-454-3p were upregulated while, hsa-miR-409-3p was downregulated in serum and tissue of cervical cancer subjects. Our in-silico small RNA sequencing data analysis identified isomiRs and classified miRNA into clusters and subtypes (exonic, intronic and intergenic) with respect to the expression status in serum and tissue specimens. Expression level of hsa-miR-409-3p and hsa-miR-454-3p were inversely correlated with their target genes MTF2 and ST18 levels respectively in human cervical cancer specimens. Luciferase assay demonstrated that hsa-miR-409-3p and hsa-miR-454-3p functionally interacts with 3'-UTR of MTF2 and ST18 respectively to decrease their activity. CONCLUSION: Our results support the significant role of circulating miRNAs in disease dissemination and their potential utility as biosignatures of clinical relevance.

Targeted sequencing-based analyses of candidate gene variants in ulcerative colitis-associated colorectal neoplasia.

BACKGROUND: Long-standing ulcerative colitis (UC) leading to colorectal cancer (CRC) is one of the most serious and life-threatening consequences acknowledged globally. Ulcerative colitis-associated colorectal carcinogenesis showed distinct molecular alterations when compared with sporadic colorectal carcinoma. METHODS: Targeted sequencing of 409 genes in tissue samples of 18 long-standing UC subjects at high risk of colorectal carcinoma (UCHR) was performed to identify somatic driver mutations, which may be involved in the molecular changes during the transformation of non-dysplastic mucosa to high-grade dysplasia. Findings from the study are also compared with previously published genome wide and exome sequencing data in inflammatory bowel disease-associated and sporadic colorectal carcinoma. RESULTS: Next-generation sequencing analysis identified 1107 mutations in 275 genes in UCHR subjects. In addition to TP53 (17%) and KRAS (22%) mutations, recurrent mutations in APC (33%), ACVR2A (61%), ARID1A (44%), RAF1 (39%) and MTOR (61%) were observed in UCHR subjects. In addition, APC, FGFR3, FGFR2 and PIK3CA driver mutations were identified in UCHR subjects. Recurrent mutations in ARID1A (44%), SMARCA4 (17%), MLL2 (44%), MLL3 (67%), SETD2 (17%) and TET2 (50%) genes involved in histone modification and chromatin remodelling were identified in UCHR subjects. CONCLUSIONS: Our study identifies new oncogenic driver mutations which may be involved in the transition of non-dysplastic cells to dysplastic phenotype in the subjects with long-standing UC with high risk of progression into colorectal neoplasia.

Aberrant gene-specific DNA methylation signature analysis in cervical cancer.

Multicomponent molecular modifications such as DNA methylation may offer sensitive and specific cervical intraepithelial neoplasia and cervical cancer biomarkers. In this study, we tested cervical tissues at various stages of tumor progression for 5-methylcytosine and 5-hydroxymethylcytosine levels and also DNA promoter methylation profile of a panel of genes for its diagnostic potential. In total, 5-methylcytosine, 5-hydroxymethylcytosine, and promoter methylation of 33 genes were evaluated by reversed-phase high-performance liquid chromatography, enzyme-linked immunosorbent assay based technique, and bisulfate-based next generation sequencing. The 5-methylcytosine and 5-hydroxymethylcytosine contents were significantly reduced in squamous cell carcinoma and receiver operating characteristic curve analysis showed a significant difference in (1) 5-methylcytosine between normal and squamous cell carcinoma tissues (area under the curve = 0.946) and (2) 5-hydroxymethylcytosine levels among normal, squamous intraepithelial lesions and squamous cell carcinoma. Analyses of our next generation sequencing results and data from five independent published studies consisting of 191 normal, 10 low-grade squamous intraepithelial lesions, 21 high-grade squamous intraepithelial lesions, and 335 malignant tissues identified a panel of nine genes ( ARHGAP6, DAPK1, HAND2, NKX2-2, NNAT, PCDH10, PROX1, PITX2, and RAB6C) which could effectively discriminate among the various groups with sensitivity and specificity of 80%-100% (p < 0.05). Furthermore, 12 gene promoters (ARHGAP6, HAND2, LHX9, HEY2, NKX2-2, PCDH10, PITX2, PROX1, TBX3, IKBKG, RAB6C, and DAPK1) were also methylated in one or more of the cervical cancer cell lines tested. The global and gene-specific methylation of the panel of genes identified in our study may serve as useful biomarkers for the early detection and clinical management of cervical cancer.

Regulation and tumor-suppressive function of the miR-379/miR-656 (C14MC) cluster in cervical cancer.

Cervical cancer (CC) is a key contributor to cancer-related mortality in several countries. The identification of molecular markers and the underlying mechanism may help improve CC management. We studied the regulation and biological function of the chromosome 14 microRNA cluster (C14MC; miR-379/miR-656) in CC. Most C14MC members exhibited considerably lower expression in CC tissues and cell lines in The Cancer Genome Atlas (TCGA) cervical squamous cell carcinoma and endocervical adenocarcinoma patient cohorts. Bisulfite Sanger sequencing revealed hypermethylation of the C14MC promoter in CC tissues and cell lines. 5-aza-2 deoxy cytidine treatment reactivated expression of the C14MC members. We demonstrated that C14MC is a methylation-regulated miRNA cluster via artificial methylation and luciferase reporter assays. C14MC downregulation correlated with poor overall survival and may promote metastasis. C14MC activation via the lentiviral-based CRISPRa approach inhibited growth, proliferation, migration, and invasion; enhanced G2/M arrest; and induced senescence. Post-transcriptional regulatory network analysis of C14MC transcriptomic data revealed enrichment of key cancer-related pathways, such as metabolism, the cell cycle, and phosphatidylinositol 3-kinase (PI3K)-AKT signaling. Reduced cell proliferation, growth, migration, invasion, and senescence correlated with the downregulation of active AKT, MYC, and cyclin E1 (CCNE1) and the overexpression of p16, p21, and p27. We showed that C14MC miRNA activation increases reactive oxygen species (ROS) levels, intracellular Ca2+ levels, and lipid peroxidation rates, and inhibits epithelial-mesenchymal transition (EMT). C14MC targets pyruvate dehydrogenase kinase-3 (PDK3) according to the luciferase reporter assay. PDK3 is overexpressed in CC and is inversely correlated with C14MC. Both miR-494-mimic transfection and C14MC activation inhibited PDK3 expression. Reduced glucose uptake and lactate production, and upregulation of PDK3 upon C14MC activation suggest the potential role of these proteins in metabolic reprogramming. Finally, we showed that C14MC activation may inhibit EMT signaling. Thus, C14MC is a tumor-suppressive and methylation-regulated miRNA cluster in CC. Reactivation of C14MC can be useful in the management of CC.

Salivary DNA methylation markers for cancer of oral cavity.

PURPOSE: Aberrant DNA methylation plays a crucial role in oral carcinogenesis. Our previous study demonstrated hypermethylation of DAPK1, LRPPRC, RAB6C, and ZNF471 promoters in patients with tongue squamous cell carcinoma compared with normal samples. Methylation profiling using salivary DNA is considered a non-invasive alternative to tissue samples. Hence, the present study tested the DNA methylation status of these four promoters as indicators of oral cancer progression. METHODS: We performed the bisulfite-based targeted next-generation sequencing of four candidate genes in saliva and tissue DNA from normal, premalignant, and squamous cell carcinoma subjects. The clinicopathological association, diagnostic, and prognostic utility of aberrant DNA methylation were evaluated using the TCGA-HNSCC dataset. Using the Xgboost algorithm and logistic regression, CpG sites were prioritized, and Receiver Operating Characteristic was generated. By Log-rank test and Kaplan-Meier (KM) curves, an association between methylation and overall survival (OS), disease-free interval (DFI), and progression-free interval (PFI) were computed. RESULTS: We identified all four genes as significantly hypermethylated in premalignant and malignant samples compared with normal samples. The methylation levels were comparable between saliva and tissue samples with an r-value of 0.6297 to 0.8023 and 0.7823 to 0.9419 between premalignant tissue vs. saliva and OC vs. saliva, respectively. We identified an inverse correlation between DAPK1, LRPPRC, RAB6C, and ZNF471 promoter methylation with their expression. A classifier of 8 differentially methylated CpG sites belonging to DAPK1, RAB6C, and ZNF471 promoters was constructed, showing an AUC of 0.984 to differentiate tumors from normal samples. The differential methylation status of DAPK1, LRPPRC, and ZNF71 promoters was prognostically important. Abnormal expression of all four genes was associated with immune infiltration. CONCLUSIONS: Thus, methylation analysis of these candidate CpG sites from saliva can be helpful as a non-invasive tool for the clinical management of OC.

Cluster miRNAs and cancer: Diagnostic, prognostic and therapeutic opportunities.

MiRNAs are class of noncoding RNA important for gene expression regulation in many plants, animals and viruses. MiRNA clusters contain a set of two or more miRNA encoding genes, transcribed together as polycistronic miRNAs. Currently, there are approximately 159 miRNA clusters reported in the human genome consisting of miRNAs ranging from two or more miRNA genes. A large proportion of clustered miRNAs resides in and around the fragile sites or cancer associated genomic hotspots and plays an important role in carcinogenesis. Altered expression of miRNA cluster can be pro-tumorigenic or anti-tumorigenic and can be targeted for clinical management of cancer. Over the past few years, manipulation of miRNA clusters expression is attempted for experimental purpose as well as for diagnostic, prognostic and therapeutic applications in cancer. Re-expression of miRNAs by epigenetic therapy, genome editing such as clustered regulatory interspaced short palindromic repeats (CRISPR) and miRNA mowers showed promising results in cancer therapy. In this review, we focused on the potential of miRNA clusters as a biomarker for diagnosis, prognosis, targeted therapy as well as strategies for modulating their expression in a therapeutic context. This article is categorized under: Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs RNA Processing > Processing of Small RNAs RNA in Disease and Development > RNA in Disease Regulatory RNAs/RNAi/Riboswitches > Biogenesis of Effector Small RNAs.

Role of miRNA clusters in epithelial to mesenchymal transition in cancer.

Epithelial to mesenchymal transition (EMT) is a multistep biological process in which epithelial cells acquire characteristics of mesenchymal cells. Inappropriate activation of EMT contributes to the acquisition of pro-metastatic characteristics and cancer progression. EMT process involves the downregulation of epithelial markers (EpCAM, CDH1) and upregulation of mesenchymal markers (VIM, CDH2) and EMT-transcription factors (ZEB1/2, TWIST1/2, SNAI1, SLUG). MicroRNAs, a class of non-coding RNA post-transcriptionally govern gene expression by binding to the target mRNAs. A large proportion of miRNAs occur as miRNA clusters consisting of two or more miRNA coding genes. MiRNA clusters are reported to regulate diverse biological functions, including EMT. This comprehensive review discusses the role of miRNA clusters in EMT.

Prognostic role of 14q32.31 miRNA cluster in various carcinomas: a systematic review and meta-analysis.

Deregulated miR-379/miR-656 cluster expression is considered as important for carcinogenesis and can be used as a potential prognostic marker. Hence, the meta-analysis was conducted to test the utility of miR-379/miR-656 cluster as a prognostic marker in various cancers. A literature search was performed using Web of Science, PubMed and Cochrane Library to obtain relevant studies and were subjected to various subgroup and bioinformatics analyses. Selected twenty-three studies contained 13 cancer types comprising of 3294 patients from 7 nations. Univariate and multivariate data showed an association of high expression of miRNAs with the poor prognosis of cancer patients (p < 0.001). The subgroup analysis showed that lung cancer, breast cancer and papillary renal cell carcinoma (p < 0.001) have a negative association with the survival of patients. Our study is the first meta-analysis showing the association of miR-379/miR-656 cluster expression and overall survival, suggesting its potential as a prognostic indicator in multiple cancers.

Characterizing methylation regulated miRNA in carcinoma of the human uterine cervix.

Gene regulatory mechanisms determine the multistep carcinogenesis process. Two aspects of epigenetics are microRNA (miRNAs) and DNA methylation that regulate distinct biological mechanisms such as metastasis, apoptosis cell proliferation and induction of senescence. Although critical, the interplay between these two epigenetic mechanisms is yet to be completely understood, particularly in cervical cancer. To study the DNA methylation regulation of miRNAs and its potential role in cervical cancer, we investigated the differential methylation pattern of two candidate miRNAs (miR-375 and miR-196a-1) during cervical cancer progression against normal cervical epithelium (NCE) by bisulfite DNA sequencing. miR-375 and miR-196a-1 were hypermethylated in Squamous Cell Carcinoma (SCC) against NCE and Cervical Intra-Epithelial Neoplasia (CIN) (p < 0.05). Treatment with demethylating agent reactivated the miR-375 and miR-196a-1 expression in SiHa, HeLa and CaSki cells. In vitro artificial methylation by M.SssI followed by dual luciferase assay confirmed miR-375 and miR-196a-1 as methylation regulated miRNAs (P < 0.05). miR-375 and miR-196a-1 expression levels were negatively correlated with methylation levels in clinical specimens. We further identified Replication Factor C Subunit 3 (RFC3) and High Mobility Group AT-Hook 1 (HMGA1) as targets of miR-375 and miR-196a-1 respectively by dual luciferase reporter assay. Our analysis indicates that miR-375 and miR-196a-1 are DNA methylation regulated miRNAs whose deregulation may facilitate pathophysiology of cervical cancer.

Whole mitochondria genome mutational spectrum in occupationally exposed lead subjects.

Lead is a public health hazard substance affecting millions of people worldwide especially those who are occupationally exposed. Our study aimed to investigate the effect of occupational lead exposure on mitochondria DNA (mtDNA). By sequencing the whole mitochondria genome, we identified 25 unique variants in lead exposed subjects affecting 10 protein coding genes in the order of MT-ND1, MT-ND2, MT-CO2, MT-ATP8, MT-ATP6, MT-CO3, MT-ND3, MT-ND4, MT-ND5, and MT-CYB. Mitochondria functional analysis revealed that exposure to lead can reduce reactive oxygen species (ROS) levels, alter mitochondria membrane potential (MMP) and increase mitochondrial mass (MM). This was further supported by mtDNA copy number analysis which was increased in lead exposed individuals compared to unexposed control group indicating the compensatory mechanism that lead has in stabilizing the mitochondria. This is the first report of mtDNA mutation and copy number analysis in occupationally lead exposed subjects where we identified mtDNA mutation signature associated with lead exposure thus providing evidence for altered molecular mechanism to compensate mitochondrial oxidative stress.

A compilation of Web-based research tools for miRNA analysis.

Since the discovery of microRNAs (miRNAs), a class of noncoding RNAs that regulate the gene expression posttranscriptionally in sequence-specific manner, there has been a release of number of tools useful for both basic and advanced applications. This is because of the significance of miRNAs in many pathophysiological conditions including cancer. Numerous bioinformatics tools that have been developed for miRNA analysis have their utility for detection, expression, function, target prediction and many other related features. This review provides a comprehensive assessment of web-based tools for the miRNA analysis that does not require prior knowledge of any computing languages.

Hypermethylation of Death-Associated Protein Kinase (DAPK1) and its association with oral carcinogenesis - An experimental and meta-analysis study.

OBJECTIVES: The value of abnormal DNA methylation of DAPK1 promoter and its association with various cancers have been suggested in the literature. To establish the significance of DNA methylation of DAPK1 promoter in oral squamous cell carcinoma (OSCC), we a) performed a case-control study, b) evaluated published data for its utility in the diagnosis and prognosis of OSCC and c) identified the association of DAPK1 gene expression with promoter DNA methylation status. DESIGN: Bisulfite gene sequencing of DAPK1 promoter region was performed on non-malignant and malignant oral samples. Further, using a systematic search, 330 publications were retrieved from PubMed, Scopus, and Google Scholar and 11 relevant articles were identified. RESULTS: Significant association of DAPK1 promoter methylation with OSCC (p<0.0001) was observed in the case-control study. The studies chosen for meta-analysis showed prognostic and predictive significance of DAPK1 gene promoter, despite defined inconsistencies in few studies. Overall, we obtained a statistically significant (p-value<0.001) association for both sensitivity and specificity of DAPK1 DNA promoter methylation in oral cancer cases, without publication bias. CONCLUSION: DNA hypermethylation of DAPK1 gene promoter is a promising biomarker for OSCC prediction/prognostics and suggests further validation in large distinct cohorts to facilitate translation to clinics.

DNA methylation detection at single base resolution using targeted next generation bisulfite sequencing and cross validation using capillary sequencing.

With a purpose of accurate and simultaneous determination of DNA methylation from multiple loci in multiple samples, here, we are demonstrating a method to aid rapid DNA methylation detection of genomic sequences. Using genomic DNA of peripheral blood from 14 healthy individuals, DNA methylation in 465 CpG sites from 12 loci of genes (ADAM22, ATF2, BCR, CD83, CREBBP, IL12B, IL17RA, MAP2K2, RBM38, TGFBR2, TGFBR3, and WNT5A) was analysed by targeted next generation bisulfite sequencing. Analysed region for three genes, BCR, IL17RA and RBM38 showed an absolute mean DNA methylation of 25.6%, 89.2% and 38.9% respectively. Other nine gene loci were unmethylated and exhibited <10% absolute mean DNA methylation. Two genes, IL17RA and RBM38 were technically validated using direct capillary sequencing and results were comparable with positive correlation (P=0.0088 & P<0.0001 respectively) in the CpG sites for DNA methylation. All CpG sites analysed from RBM38 genes locus displayed 95% limits of agreement for DNA methylation measurements from the two methods. The present approach provides a fast and reliable DNA methylation quantitative data at single base resolution with good coverage of the CpG sites under analysis in multiple loci and samples simultaneously. Use of targeted next generation bisulfite sequencing may provide an opportunity to explore genes in the discovery panel for biomarker identification and facilitate functional validation.

Mitochondrial biology: From molecules to diseases.

As an integral part of the cell, mitochondria play a pivotal role in the regulation of energy metabolism, signaling pathways, cell differentiation, cell proliferation and cell death. Mitochondrion with its own genetic material has characteristics distinct from those of the nuclear counterpart and its dysregulation is associated with a myriad of diseases. The discovery of interplay between the nuclear and mitochondrial genes, and various post-transcriptional modifications associated with their products has added excitement in the field. This has led to a better understanding of the basic mitochondrial function in normal and disease states, and is important for diagnosis and prognosis of a large number of disorders. The Fourth Annual Conference of Society for Mitochondrial Research and Medicine - India (SMRM) was titled "Mitochondrial Biology: from Molecules to Disease". The conference was organized by K. Satyamoorthy and K. Thangaraj at School of Life Sciences, Manipal University, Manipal, India, during 8-9 December, 2014. The aim of the conference was to bring researchers and clinicians to a common platform; create an opportunity for networking between laboratories; and to discuss about the recent development in mitochondrial biology, diagnosis, and therapy. This review summarizes the key outcomes of the conference.

Prevalence of human papillomavirus types and phylogenetic analysis of HPV-16 L1 variants from Southern India.

BACKGROUND: The human papillomavirus (HPV) and its variants show wide geographical distribution and have been reported to cause cervical lesions. With cervical neoplasia as the leading cancer in Indian women, the aim of the present study was to evaluate the multiple infection HPV type distribution and variant genotypes in cervical samples from the coastal Karnataka region, India. MATERIALS AND METHODS: A total of 212 samples were screened by nested polymerase chain reaction using PGMY9/11 and GP5+/6+ primers. HPV positive samples were sequenced to identify the types and a phylogenetic tree was constructed using the neighbor-joining method. RESULTS: Sequence analysis identified a total of 14 HPV types distributed in 20%, 73.3% and 82.5% of non-malignant, pre-malignant [low grade squamous intraepithelial lesion (LSIL) and high grade squamous intraepithelial lesion (HSIL)] and cervical cancer samples. The distribution of high risk HPV in cancer samples was HPV 16, 76.4%, HPV18, 11.7%, HPV81, 2.9%, HPV31, 1.4%, HPV35, 1.4% and HPV 45, 1.4%. Multiple infections were observed in 11.8% of tumor samples with HPV 16 contributing to 62.5% of cases. In non-malignant samples, 20% of HPV positive samples were detected with HPV16, 82.3%, HPV33, 5.8% and HPV58, 5.8% and very low incidence of multiple infections. Comparative phylogenetic analysis of HPV variants identified 9 HPV sequences as new papillomavirus species, predominantly classified as European lineage type. CONCLUSIONS: The findings for HPV infections associated with progression of cervical cancer in coastal Karnataka region and HPV variant analysis provide baseline data for prevention and HPV vaccination programs.