Untypable Genotypes Research Articles

Real-world Effectiveness and Tolerability of Interferon-free Direct-acting Antiviral for 15,849 Patients with Chronic Hepatitis C: A Multinational Cohort Study.

As practice patterns and hepatitis C virus (HCV) genotypes (GT) vary geographically, a global real-world study from both East and West covering all GTs can help inform practice policy toward the 2030 HCV elimination goal. This study aimed to assess the effectiveness and tolerability of DAA treatment in routine clinical practice in a multinational cohort for patients infected with all HCV GTs, focusing on GT3 and GT6. We analyzed the sustained virological response (SVR12) of 15,849 chronic hepatitis C patients from 39 Real-World Evidence from the Asia Liver Consortium for HCV clinical sites in Asia Pacific, North America, and Europe between 07/01/2014-07/01/2021. The mean age was 62±13 years, with 49.6% male. The demographic breakdown was 91.1% Asian (52.9% Japanese, 25.7% Chinese/Taiwanese, 5.4% Korean, 3.3% Malaysian, and 2.9% Vietnamese), 6.4% White, 1.3% Hispanic/Latino, and 1% Black/African-American. Additionally, 34.8% had cirrhosis, 8.6% had hepatocellular carcinoma (HCC), and 24.9% were treatment-experienced (20.7% with interferon, 4.3% with direct-acting antivirals). The largest group was GT1 (10,246 [64.6%]), followed by GT2 (3,686 [23.2%]), GT3 (1,151 [7.2%]), GT6 (457 [2.8%]), GT4 (47 [0.3%]), GT5 (1 [0.006%]), and untyped GTs (261 [1.6%]). The overall SVR12 was 96.9%, with rates over 95% for GT1/2/3/6 but 91.5% for GT4. SVR12 for GT3 was 95.1% overall, 98.2% for GT3a, and 94.0% for GT3b. SVR12 was 98.3% overall for GT6, lower for patients with cirrhosis and treatment-experienced (TE) (93.8%) but ≥97.5% for treatment-naive patients regardless of cirrhosis status. On multivariable analysis, advanced age, prior treatment failure, cirrhosis, active HCC, and GT3/4 were independent predictors of lower SVR12, while being Asian was a significant predictor of achieving SVR12. In this diverse multinational real-world cohort of patients with various GTs, the overall cure rate was 96.9%, despite large numbers of patients with cirrhosis, HCC, TE, and GT3/6. SVR12 for GT3/6 with cirrhosis and TE was lower but still excellent (>91%).

KASP-IEva: an intelligent typing evaluation model for KASP primers.

KASP marker technology has been used in molecular marker-assisted breeding because of its high efficiency and flexibility, and an intelligent evaluation model of KASP marker primer typing results is essential to improve the efficiency of marker development on a large scale. To this end, this paper proposes a gene population delineation method based on NTC identification module and data distribution judgment module to improve the accuracy of K-Means clustering, and introduces a decision tree to construct the KASP-IEva primer typing evaluation model. The model firstly designs the NTC identification module and data distribution judgment module to extract four types of data, grouping and categorizing to achieve the improvement of the distinguishability of amplification product signals; secondly, the K-Means algorithm is used to aggregate and classify the data, to visualize the five aggregated clusters and to obtain the morphology location eigenvalues; lastly, the evaluation criteria for the typing effect level are constructed, and the logical decision tree is used to make conditional discrimination on the eigenvalues in order to realize the score prediction. The performance of the model was tested by the KASP marker typing test results of 2519 groups of cotton varieties, and the following conclusions were obtained: the model is able to visualize the aggregation and classification effects of the amplification products of NTC, pure genotypes, heterozygous genotypes, and untyped genotypes, enabling rapid and accurate KASP marker typing evaluation. Comparing and analyzing the model evaluation results with the expert evaluation results, the average accuracy rate of the four grades evaluated by the model was 87%, and the overall evaluation results showed an uneven distribution of the grades with significant differential characteristics. When evaluating 2519 KASP fractal maps, the expert evaluation consumes 15 hours, and the model evaluation only uses 8min27.45s, which makes the model intelligent evaluation significantly better than the expert evaluation from the perspective of time. The establishment of the model will further enhance the application of KASP markers in molecular marker-assisted breeding and provide technical support for the large-scale screening and identification of excellent genotypes.

Issues and Challenges of Imputation Techniques in Genome Wide Association Studies (GWAS): A Review

A genome-wide association study (GWAS) rapidly scans DNA markers in many individuals to find genetic links to diseases. New findings aid in disease detection, treatment and prevention. Imputation predicts untyped genotypes in genetic studies when data is missing due to quality, cost, or design issues. It’s a proven statistical technique for estimating unobserved genotypes by borrowing haplotype segments from a densely genotyped reference panel. This allows estimation and testing of associations at unassayed variants.Genotype imputation is vital in analyzing genome-wide association scans, helping geneticists evaluate evidence for association at untyped genetic markers. This summary outlines missing data issues and various imputation methods.

Genetic characteristics of archival noroviruses detected from the 1970s to the 1990s.

The genetic characterization of archival specimens is important for evaluating the evolutionary processes of noroviruses. Complete viral genome sequences, GVIII.1[GII.P28] and GIX.1[GII.P15], were determined from two archival specimens collected in Tokyo, Japan, in 1986 and 1995. In addition, complete VP1 and partial RdRp sequences of four samples collected between 1975 and 1983 were determined. Two viruses were classified as GI.5[P5] and GI.9[P9]; however, the viruses from the other two samples could not be assigned to any known genotypes using norovirus typing tools and phylogenetic analysis, suggesting that they might be untypable genotypes. Further evolutionary analysis of these viruses is warranted.

Epidemiological and Genotypical Features of HCV in District Upper Dir, Khyber Pakhtunkhwa, Pakistan

Objectives: Objective: The incidence of Hepatitis C Virus (HCV) infection is high in Pakistan. The current study evaluates HCV risk factors, epidemiology, and genotype distribution in the less-developed Upper Dir Region, Khyber Pakhtunkhwa Province (KPK), Pakistan. Methods: Blood samples from 500 outpatients from the Upper Dir Region were collected for initial screening using a strip-based immunochromatography test (ICT). In this study, ICT analysis followed Polymerase chain reaction (PCR) analysis for HCV-RNA confirmation. In addition, PCR positive samples were subject to genotypic description using genotype-specific primers. Results: ICT analysis revealed that out of 500 patients, 13 (2.6%) were HCV positive. PCR analysis identified that 10 (2.0%) of the 13 HCV-positive patients were HCV-RNA positive. The overall incidence of HCV in the study group was 2.6%. The rate of HCV incidence was found to be higher in males (10/339 participants, 3.0%), while a lower incidence was found in females (3 of 161 participants, 1.9%). The patients between 45 and 60 years had the highest incidence rate (6 of 53 participants, 11.3%, P<0.05); while the patients between 25 and 44 years had an incidence rate of 4.2% (7 of 166 participants). Different HCV subtypes were also detected; 1a (two participants), 2b (one participant), 3a (two participants) and 3b (two participants). Three of the patients had untypable (UT) HCV genotype variants. Conclusion: The study results suggest that; most of the cases were males and between 25 and 60-year-old. At the same time, several HCV subtypes are present in the inhabitants of the study area. Therefore, the presence of untypable genotypes may lead to diagnostic and therapeutic difficulties or failures. J Microbiol Infect Dis 2021; 11(4):218-224.

Detection of hepatitis through proteases and protease inhibitors genes expression and identification of HCV untypable genotype in Abbottabad, Pakistan.

Liver cancer is the 5th most common cancer caused mainly due to the late detection of hepatitis. Therefore, the early detection of hepatitis through genetic markers boosts effective and remedial management. In addition, to determine the occurrence of hepatitis C virus (HCV), genotyping is indispensable as majority of hepatitis cases remain undiagnosed. The current study was designed to find the gene expression of proteases and proteases inhibitors in different hepatitis patients and to determine HCV genotypes mainly focusing on untypeable genotypes of HCV in Abbottabad, Pakistan. PCR was conducted to find the expression of proteases and protease inhibitors genes in hepatitis patients and healthy individuals. HCV genotyping was done by PCR based method and untypeable genotypes were sequenced and verified using online tools. Controlled individuals showed normal expression of cystatin C and leptin, low expression of cathepsin B while high expression of other studied genes including cathepsin D and G, TPP1 and serpin B1 could be seen. Hepatitis A patients showed high expression of leptin while other genes showed low expression. Hepatitis B patients revealed considerable variations in the cathepsin and cystatin C gene expression. Therefore, low cystatin C (high cathepsin B) and/or high cystatin C (low cathepsin B) levels can be regarded as a potential marker for hepatitis B. Hepatitis C infected patients showed high gene expression of cystatin C and leptin, so they could be useful markers for the diagnostics and prediction of the severity of HCV infections. While genotyping findings showed that about 45% of total PCR positive samples (110) were found to be of 3a genotype followed by 3b in 18%, 1a in 13.6% and 1b in 10%. About 9% of infections turned out to be mixed infections, whereas only 4.5% were untraceable by our genotyping system. Sequencing of untypeable genotypes and applying online tools revealed that the described untypeable genotypes of HCV were in fact variants of 3a genotype. Furthermore, full length characterization of these variants could help to classify them into types and subtypes. Keywords: hepatitis; genotyping; genes expression; proteases and protease inhibitors; ML; NJ.

Molecular epidemiology and viral load analysis of hepatitis C virus genotypes from Sindh, Pakistan

The present study was aimed to assess the molecular epidemiology of Hepatitis C Virus (HCV) genotypes and viral load (VL) of HCV infected patients. A total of 261 anti-HCV positive patients were enrolled for VL analysis using COBAS AmpliPrep/COBAS TaqMan HCV and Abbott Real Time HCV Genotype II Assay Kit to determine the genotypes. The data demonstrated that 60.15% (n=157) samples were from females and 39.85% (n=104) from male patients. Serum markers such as Bilirubin, SGPT, PT, APTT, and α-fetoproteins were analyzed by commercial kits and Cobas-C 501, Sysmex Ca500 and Cobas-C 601 automatic analyzers as per manufacturer’s guidelines. All patients were above 15 years age and their mean age was 39 years. Male to female ratio was 0.66% (104/157). Age-wise ddistribution revealed that 31-45 years age group comprised majority of the patients, whereas lowest number belonged to age group 61 and above. HCV genotyping data revealed five different genotypes/subtypes. Genotype 3a was most frequent accounting 76.24% (n=199) followed by Genotype 1a (2.29%), 1b (2.68%), 2a (4.98%). Moreover, 13.79% (n=36) samples revealed untypable genotype. Subtype 1b had highest mean VL of 6.84 log10 IU/ml among all other genotypes. The data of serum markers analysis revealed a slight fluctuation but appeared under the normal range. However, Bilirubin was slightly lower in Genotype 1a as compared with other genotypes while elevated α-fetoprotein was found in patients infected with untypable genotypes. In summary, Genotype 3a was predominant genotype throughout Sindh and subtype 1b had highest viral load compared with subtypes 3a and 2a. Keywords: Genotyping; Hepatitis C; HCV; Prevalence; Serum markers http://dx.doi.org/10.19045/bspab.2021.100037

The rotavirus causing acute gastroenteritis in children of under 5-year of age in Indonesia 1972-2018: a review

The reason of this review is the absence of thorough information of rotavirus infection that had been the major cause of severe diarrhea in children of under 5-years of age in Indonesia, despite numerous publications elaborating rotavirus infection in some geographic areas of Indonesia. A review was conducted towards 52 published articles covering rotavirus research in Indonesia during period of 1972-2018.A thirty three selected articles were match with review criteria which comprises rotavirus positive rate, clinical features, and severity of rotavirus infection, as well as genotypes of the rotavirus. Rotavirus has been known as the major cause of severe diarrhea among children under 5 years of age world wide including in Indonesia. The rotavirus positive rates were range from 31.1 to 90.9%, which variably to different subject’s population, study criteria and methods, and time. Rotavirus can cause severe diarrhea with majority of infected children suffered from dehydration, vomiting, and fever. The first genotyping conducted in Indonesia in the early 1980s revealed the predominant genotypes were G3 and G4, followed by G2, and small proportion of G1 and mixed genotypes. However the following decades G1 and G2 were on the raise with G3 predominantly re-appeared on 2015. G9 was first identified in 2004, and occasionally detected until 2015. The P genotyping revealed P[4], P[6], and P[8] were the common genotypes detected. Mixed and untyped genotypes were also detected in various proportion. Rotavirus diarrhea is a vaccination preventable disease, after natural infection, the immune system will produce protective antibodies that will protect from infection of both homotypic and heterotypic, however homotypic infection will protect stronger. Therefore this review recommends continuous rotavirus genotypes surveillance in Indonesia.

Phylogenetic Characterization of the 5′ Untranslated Region of Untypable HCV Genotypes Circulating in Pakistan

Introduction: Commercial methods for HCV genotyping is challenged by the increased prevalence of untypable genotypes in Pakistan. Objective: The aim of the current study was to perform nucleotide sequencing of 5′ UTR region for genotyping of viral isolates circulating in Peshawar, Pakistan. Methods: The total number of commercially untypable samples were 94 in which 18 samples were sequenced for the characterization of 5′ UTR region. Post-sequencing analysis was performed for genotype identification (n = 18) and molecular phylogenetic analysis. Results: The current study reveals different genotypes, that is, 10/18 viral isolates were found to be genotype 3a (55.55%), 3 isolates (genotype 3b, 16.66%), 2 isolates (genotype 6h/6g, 11.11%), 2 (6g/d, 11.11%), and 1 sample (genotype 1c, 5.55%). In addition, genotype 3a is the dominant representative of HCV circulating in Pakistan and has been increasing across the country. Conclusion: The current study also reveals that genotype 6 (2 were genotype 6h/6g and 2 were 6g/d) is also circulating in Pakistan and not restricted to South China and Hong Kong.

Comparative Analysis of PCR and LIPA Method for HCV Genotypes Screening

Background: Hepatitis C virus (HCV) is a major health problem worldwide. About 6% of the population of Pakistan is suffering from HCV infection. HCV has a high mutation rate and consists of seven genotypes and sixty-seven subtypes. Genotype information of patients infected with HCV is significant for its treatment. Methods: In this study, 416 HCV serum samples were collected and HCV prevalence rate was studied in different districts of Punjab, Pakistan. Nested PCR and INNO LIPA HCV-II were used for HCV genotyping and their respective performance was evaluated. This study was conducted by the approval of Lahore Clinical Laboratory and Research Centre situated at Shadman, Lahore. Results: The highest prevalence of HCV was found in Shekhupura district followed by Bhakkar, Narowal and Okara districts, respectively. In Punjab, the most prevalent genotype was 3a (70.29%), followed by genotype 1 (5.47%), untypable genotypes (5.44%) and genotype 3a/3b (4.64%). Nested PCR was found to be more reliable than INNO LIPA-II. Nested PCR results were more accurate and only 5 samples remained untypable whereas 33 samples could not be typed by LIPA method. Conclusion: This study was focused on the comparative analysis of Nested PCR and LIPA method for screening HCV genotypes and their prevalence in different districts of Punjab, Pakistan. HCV genotyping is important since different genotypes require different therapeutic treatments. In Punjab, 3a is the most prevalent genotype followed by non-typable genotypes. LIPA is the most commonly used HCV genotype assay but this study found Nested PCR to be a highly sensitive and cost-effective method in this regard. This study can lead to the better selection of genotyping methods and treatment.

Prevalence of Hepatitis C Virus Genotypes in Pakistan: Current Scenario and Review of Literature.

Hepatitis C virus (HCV) is a major public health concern globally, resulting in liver-related complications. Approximately 6% population of Pakistan is infected with HCV. HCV is error prone, due to which it is classified into 7 genotypes and 67 subtypes. HCV genotype determination is critical for treatment and therapy response. In this study, 3,539 samples were collected from 2015 to 2019 from all over Punjab. RNA was extracted from samples using QIA Amp Viral RNA MINI kit (Qiagen, Germany) and viral genotyping was performed. Furthermore, a systemized literature search (2009-2018) was done to analyze the HCV genotype distribution pattern in Pakistan. In Punjab, genotype 3a (86.46%) is most prevalent, followed by untypable (7.17%) and genotype 1a (3.84%) and 3b (1.04%). Mixed genotype constitutes only 0.67% of total infections. Genotype 2a, 2b, 3c, and 4 were found to be rare. Data available from literature review when compiled showed that HCV genotype 3a (58.16%) was predominant in Pakistan, followed by genotypes 3b (9.05%), 2a (6.70%), 1a (6.22%), and 1b (2.39%). The frequency of mixed genotypes was found to be 4% and 12% of untypable HCV variants. This study highlights the HCV genotype distribution pattern in different regions of Pakistan. Therapy response and disease management depend on genotype, so HCV genotype determination is crucial. In Pakistan, the most prevalent genotype is 3a, followed by untypable genotype. Both interferon and sofosbuvir are effective against genotype 3a, but treatment with sofosbuvir has comparatively high sustained virological response, less adverse effects, and more tolerability.

Exposure to Infectious Healthcare Waste, Homosexuality, Unhygienic Dental Surgery and Circumcision Found Responsible for Alarmingly High Prevalence of Hepatitis B and C Among Vulnerable Children in Pakistan: A Multifaceted Countrywide Study

Background: Hepatitis B and C infections are major health concerns in Pakistan which is sixth largest country population wise with an estimated disease burden of over 20 million patients in the country. This multi-faceted country-wide study aimed at determining the prevalence status, treatment and preventive interventions of deadly hepatitis B and C among the most vulnerable underprivileged orphan children in Pakistan. Methodology: After a detailed survey, a total of 3980 male and female vulnerable orphan (fatherless) children and adolescents of 5-17 years of age with low socioeconomic status were included in the study. Rapid screening kits were used for the primary screening of anti-HCV antibody and anti-HBsAg. The positive samples in initial screening were confirmed by quantitative PCR of Hepatitis B Virus (HBV) DNA and Hepatitis C Virus (HCV) RNA after which the HCV genotyping was performed using multiplex genotyping PCR. Assessment of risk factors of HBV and HCV transmission was performed on a pretested structured questionnaire. Preventive education regarding hepatitis B and C was disseminated to participants and their guardians by Information, Education and Communication (IEC) material in local languages and interactive sessions. Results: A total of 353 (8.9%) children were found infected with either hepatitis B or C. Of these, 58 individuals (16.4%) were positive for HBV surface antigen while 295 (83.6%) were found positive for HCV antibodies. Nineteen out of 58 HBV DNA positive samples exhibited high viral load (over 2.0 million IU/ ml) in quantitative PCR. Of the 295 HCV antibody positive children, 87 were found positive for HCV RNA in their blood serum after PCR. During genotype analysis, an untypable HCV strain was found in highest frequency (33.3%) followed by genotype 2a (27.6%), 3a (20.6%), 1a (6.9%), 3b (5.7%), 1b (3.4%) and 2b (2.3%) of the positive cases of HCV. Genotypes were not correlated with the gender or geographic variation of the population. During the geographic distribution, Upper Sindh region, exhibited highest disease prevalence; Jacobabad was found to be the worst affected area by both hepatitis B (7.3%) and C (27.3%). In Khyber Pakhtunkhwa (KPK), Swat Valley exhibited HBV and HCV prevalence of 1.8% and 25.5%, Ghauspur 3.2% and 22.3%, Lower Dir 0.7% and 18.4%, Kandhkot 3% and 16.6%, Dera Murad Jamali 4.4% and 13.3% respectively. None of these infections were reported from Gilgit region in the Northern Areas. Dental surgery/procedure was the most common risk factor regarding the spread of hepatitis B and C in all provinces of Pakistan. Sexual exposure and male homosexuality were found significant only in KPK while exposure to infectious hospital waste was a significant risk factor in Sindh. Male circumcision by barbers with unsterilized razors was found a common risk factor in both KPK and Punjab. Awareness dissemination activities have raised the preventive knowledge of participants significantly. Conclusion: The current study reports a fairly high prevalence of hepatitis B and C in a vulnerable juvenile population of Pakistan. An untypable genotype of HCV found in high numbers opens new research dimensions for HCV control in Pakistan. Upper Sindh region and Swat Valley are the most disease prone areas. There is an urgent need to work on the screening, treatment and preventive education of all stake holders to eradicate Hepatitis B and C in order to minimize public sufferings. This study sets a disease eradication model for the countries with high endemicity, low HDI and fragile law and order situation. Funding Statement: This study was supported by Higher Education Commission (HEC), Pakistan; Helping Hand for Relief and Development (HHRD). Declaration of Interests: The authors hereby declare that they have no competing interests with any other author or reviewer. Ethics Approval Statement: The study was approved from the Directorate of Advance Studies and Research (DASR), University of Azad Jammu and Kashmir, Muzaffarabad, Pakistan. All the information of the participants was kept confidential and laboratory testing and results provision was free of cost.

Molecular Detection of Group A Rotavirus in under Five Years Old Children with Acute Diarrhoea Admitted to Yangon Children’s Hospital

Rotaviruses are regarded as the most common cause of viral gastroenteritis and are responsible for considerable morbidity and mortality among children especially under five years of age worldwide. In developing countries like Myanmar, where diarrhoea is in the priority childhood disease, rotavirus surveillance and detection of rotavirus genotypes are utmost important. A hospital-based, cross-sectional descriptive study was conducted at Yangon Children‟s Hospital among under five children admitted for acute diarrhoea from January to October 2016. This study includes detection of Group A rotavirus antigen by commercial enzyme-linked immunosorbent assay (ELISA) and genotyping by multiplex RT-PCR. From a total of 488 collected samples, rotavirus antigen was detected in 219 samples (45%). Rotavirus diarrhoea was most common among the age of 6-11 months (38.8%) followed by 12-23 months (37.9%). The results showed that boys were more commonly affected than girls. Detection of rotavirus positivity was peak in February (57.6 %). Out of 219 stool samples with positive ELISA result, 40 stool samples with high optical density value were proceeded for further determination of G and P genotypes. Regarding distribution of G genotypes, the most common G genotype was G9 which comprised 45%, and that of P genotype was P[8] which comprised 92.5%. Regarding combination of G and P genotypes, the most frequent combination is G9P[8], and it constituted 42.5%. Untypable genotypes were seen in 30% of G and 2.5% of P typing. As rotavirus infection can be prevented by vaccine, WHO recommended that rotavirus vaccination should be included in national immunization program especially in countries where prevalence of rotavirus is high. The distribution of G and P genotypes is important in consideration of appropriate vaccine in pre-vaccination and evaluation of effectiveness of vaccine in post-vaccination period. Therefore, the information on currently circulating genotypes of rotavirus in this study will serve as valuable data for vaccination programme.

Increasing Prevalence of Untypable and Mixed Genotypes of Hepatitis C Virus in Pakistan: Latest Trends in 2018.

Hepatitis C virus (HCV) genotyping is a critical parameter that acts as predictor of treatment response. According to previously reported findings, about 11 million population of Pakistan are HCV infected. Accumulating data suggest that genotype is the most prevalent genotype and mixed and untypable genotypes are the least prevalent genotypes of HCV. We observed that overall prevalence of mixed genotype (5.03%) and untypable genotype (3.3%) of HCV is on constant rise. This study highlights that the emergence of novel quasispecies could be reason of treatment failure in patients receiving direct-acting antiviral drugs.

Prevalence and Treatment of Untypable HCV Variants in Different Districts of Punjab, Pakistan.

In Pakistan, around 11 million people are infected with, at least, six circulating genotypes and subtypes of hepatitis C virus (HCV). The viral burden is still on rise. HCV genotype determination is necessary to devise appropriate antiviral therapy. Previous reports highlight the prevalence circulating untypable (in diagnostic perspectives) HCV subtype in local Pakistani patients. Present study was designed to investigate the epidemiological distribution of genotypes in different districts of Punjab, Pakistan and focuses on prevalence of diagnostically untypable subtype and its possible antiviral therapy. A total of 8,353 HCV patients were included through 2011 to 2016 for determination of HCV prevalent genotypes in the local community. HCV genotyping was carried out using Ohno et al. method, and five cases of diagnostically untypable patients were treated with interferon-based antiviral regimen to find the possible treatment strategy. Of the 8,353 patients, the genotype 3a (n = 6,650, 79.6%) was most significantly prevalent throughout the study period followed by undetermined genotype that is, untypable (n = 1,377, 16.5%). The presence of untypable variants suggested that there are some novel or quasi species prevailing among HCV patients in local community. Five HCV patients infected with untypable genotype had been treated effectively with interferon alpha plus ribavirin therapy for 24 weeks at standard doses and achieved sustained virological response. This study highlighted an important aspect of HCV treatment for local Pakistani HCV patients. Untypable HCV variants could be treated with interferon-based antiviral regimens such as 3a genotype. The study raises the need of characterization of diagnostically untypable HCV variant. On the basis of these results, it can be proposed that until the availability of pangenotypic direct-acting antiviral for HCV treatment in Pakistan, interferon-based antiviral regimens will be of choice for majority of patients infected with 3a or untypable genotypes.

Prevalence of hepatitis-C virus genotypes and potential transmission risks in Malakand Khyber Pakhtunkhwa, Pakistan

BackgroundHepatitis C virus (HCV) is a leading cause of chronic liver disease and frequently progresses towards liver cirrhosis and Hepatocellular Carcinoma (HCC). This study aimed to determine the prevalence of HCV genotypes and their association with possible transmission risks in the general population of Malakand Division.MethodologySum of 570 serum samples were collected during March 2011 to January 2012 from suspected patients visited to different hospitals of Malakand. The suspected sera were tested using qualitative PCR and were then subjected to molecular genotype specific assay. Quantitative PCR was also performed for determination of pre-treatment viral load in confirmed positive patients.ResultsOut of 570 serum samples 316 sera were seen positive while 254 sera were found negative using qualitative PCR. The positive samples were then subjected to genotyping assay out of 316, type-specific PCR fragments were seen in 271 sera while 45 samples were found untypable genotypes. Genotype 3a was seen as a predominant genotype (63.3%) with a standard error of ±2.7%. Cramer’s V statistic and Liklihood-Ratio statistical procedures are used to measure the strength and to test the association, respectively, between the dependent variable, genotype, and explanatory variables (e.g. gender, risk, age and area/districts). The dependent variable, genotype, is observed statistically significant association with variable risk factors. This implies that the genotype is highly dependent on how the patient was infected. In contrast, the other covariates, for example, gender, age, and district (area) no statistical significant association are observed. The association between gender-age indicates that the mean age of female was older by 10.5 ± 2.3 years with 95% confidence level using t-statistic.ConclusionIt was concluded from the present study that the predominant genotype was 3a in the infected population of Malakand. This study also highlights the high prevalence rate of untypable genotypes which an important issue of health care setup in Malakand and create complications in therapy of infected patients. Major mode of HCV transmission is multiple uses and re-uses of needles/injections.Trial registrationISRCTN ISRCTN73824458. Registered: 28 September 2014

Hepatitis C Virus and Interferon-Free Antiviral Therapeutics Revolution: Implications for Pakistan.

Hepatitis C virus (HCV) is a major health concern worldwide as a leading cause of liver-related mortalities and morbidities. Pakistan ranks second among countries with endemic HCV infection; ∼11 million cases are reported so far. HCV burden is continuously rising in Pakistan, mainly because of unsafe blood transfusions, surgical procedures, dental procedures, untrained clinicians, reuse of syringes, barbers, and ear/nose piercing tools. Lack of awareness about HCV transmission routes among the general and high-risk population is a major hurdle in disease management. HCV prevalence in the general population and healthy blood donors ranges from 3.13% to 23.83% and from 1.05% to 20.8%, respectively; whereas in the high-risk groups, HCV prevalence is up to 66%. Genotype 3 is most prevalent in Pakistan followed by genotypes 1 and 2 along with an alarming number of untypable viral genotypes in the local community. Mainly interferon-based antiviral regimens are used in Pakistan and are quite effective, because the major prevalent genotype (genotype 3) showed the best sustained virological response (SVR) with it. But a large number of individuals did not show SVR either because of infection with nonresponder genotypes or because of side effects. Due to these reasons, there was a need for interferon-free direct acting antivirals (DAAs). Recently, Sovaldi (Sofosbuvir: NS5B inhibitor) is approved on a heavy discounted rate for Pakistan; it is currently in effective use and showed good SVR. Sovaldi plus ribavirin is used alone or along with interferon to treat different viral genotypes. Sovaldi will be the future treatment regime for Pakistan, because genotype 2 and genotype 3 infected individuals achieve the best SVR with it. For the treatment of other prevalent viral genotypes, approval of some other DAAs such as Ledipasvir on discounted price is required for better disease management.

Detection of Merkel Cell Polyomavirus and Human Papillomavirus in Esophageal Squamous Cell Carcinomas and Non-Cancerous Esophageal Samples in Northern Iran.

Human papillomavirus (HPV) infection is one of the hypothesized causes of esophageal squamous cell carcinoma (ESCC), but the etiological association remains uncertain. It was postulated that other infectious agents together with HPV may increase the risk of ESCC. The current investigation aimed to explore the presence of a new human tumor virus, Merkel cell polyomavirus (MCPyV), together with HPV in ESCC tumors and non-cancerous esophageal samples in northern Iran. In total, 96 esophageal samples (51 with ESCC, and 45 without esophageal malignancy) were examined. HPV DNA was detected in esophageal specimens of 16 out of the 51 ESCC cases (31.4%) and 20 out of the 45 non-cancerous samples (44.4%). Untypable HPV genotypes were recognized in high rates in cancerous (75.0%) and non-cancerous (55.0%) esophageal specimens. MCPyV DNA was detected in esophageal specimens of 23 out of the 51 ESCC cases (45.1%) and 16 out of the 45 non-cancerous samples (35.6%). The mean MCPyV DNA copy number was 1.0×10(-5)±2.4×10(-5) and 6.0×10(-6)±1.3×10(-5) per cell in ESCC cases and non-cancerous samples, respectively. There was no statistically significant difference between cancerous and non-cancerous samples regarding mean MCPyV DNA load (P=0.353). A bayesian logistic regression model adjusted to the location of esophageal specimen and MCPyV infection, revealed a significant association between HPV and odds of ESCC (OR, 2.45; 95% CI: 1.01-6.16). This study provides the evidence of the detection of the MCPyV DNA at a low viral copy number in cancerous and non- cancerous esophageal samples.

In-season and out-of-season variation of rotavirus genotype distribution and age of infection across 12 European countries before the introduction of routine vaccination, 2007/08 to 2012/13

The EuroRotaNet surveillance network has conducted rotavirus genotype surveillance since 2007 in 16 European countries. Using epidemiological and microbiological data from 39,786 genotyped rotavirus-positive specimens collected between September 2007 and August 2013, we assessed genotype distribution and age distribution of rotavirus gastroenteritis (RVGE) cases in and out of peak season in 12 countries which were yet to implement routine rotavirus vaccination. In multinomial multivariate logistic regression, adjusting for year, country and age, the odds of infection caused by genotype-constellation 2 DS-1-like stains (adjusted multinomial odds ratio (aM-OR) = 1.25; 95% confidence interval (CI): 1.13-1.37; p < 0.001), mixed or untypable genotypes (aM-OR = 1.55; 95% CI: 1.40-1.72; p < 0.001) and less common genotypes (aM-OR = 2.11; 95% CI:1.78-2.51; p < 0.001) increased out of season relative to G1P[8]. Age varied significantly between seasons; the proportion of RVGE cases younger than 12 months in the United Kingdom increased from 34% in season to 39% out of season (aM-OR = 1.66; 95% CI: 1.20-2.30), and the proportion five years and older increased from 9% in season to 17% out of season (aM-OR = 2.53; 95% CI: 1.67-3.82). This study provides further understanding of the rotavirus ecology before vaccine introduction, which will help interpret epidemiological changes in countries introducing or expanding rotavirus vaccination programmes.

Abstract 33: A Meta-analysis Of Three Genome-wide Association Studies Identifies A Novel Susceptibility Locus For Kawasaki Disease.

Background: Although genome-wide association studies (GWAS) have conclusively identified several susceptibility genes / loci for Kawasaki disease (KD), a large part of the genetic etiology of this disease have not been unraveled and, above all, its marked predilection for East Asian populations have not been explained. Objective: To identify genetic variants commonly associated with KD in the East Asian populations, we conducted a meta-analysis of three GWASes from Japan, Korea and Taiwan. Methods: In the GWAS analyses, we genotyped 6322 subjects (1236 cases and 5086 controls) using either Illumina 550K or Affymetrix SNP 6.0 platforms and then imputed untyped genotypes using Impute2 or minimac software with 1000 Genomes Project’s East Asian population (JPT, CHB and CHS) reference haplotype data. We then performed a meta-analysis using a weighted-average method with inverse-variance weights and selected representative SNPs in 49 top associated loci, which were then genotyped in 4798 independent subjects (2151 cases and 2747 controls). Finally, we combined the data for the three GWASes and follow up studies in a meta-analysis. Results: SNPs within previously identified susceptibility loci showed significant association in the meta-analysis of the GWASes (ITPKC: rs28493229, P = 3.07 x 10-9; CASP3: rs2720377, P = 2.66 x 10-9; BLK: rs2736340, P = 1.23 x 10-16; CD40: rs1883832, P = 1.76 x 10-8; HLA class2: rs189914842, P = 4.57 x 10-11). In a meta-analysis of the three GWASes and follow-up studies, we observed a genome-wide significant level of association at a SNP in a chromosomal region different from the six known loci (P = 6.49 x 10-9). Conclusion: The meta-analysis of three GWAses and follow-up studies successfully identified a new SNP significantly associated with KD. Further investigation of the region where the SNP is located toward specification of the susceptibility gene, the responsible variant, as well as its effect on gene function is warranted. Acknowledgement: T.A.J., J.Y.W. and D.Y. equally contributed to this work and J.K.L., Y.T.C., and Y.O. are co-directing this project.