Semi-nested Multiplex PCR Research Articles

Molecular detection and genotyping of HBV from HBsAg positive patients in a tertiary hospital in Nigeria.

Nigeria remains one of the countries with a high hepatitis B virus (HBV) burden in Africa. Reports have indicated the presence of mixed HBV genotypes in Nigeria; however, there is still paucity of data regarding mixed genotype infections particularly in the Southern part of the country. Our aim is to determine the HBV genotype distribution among HBsAg-positive gastroenterology patients at the University College Hospital Ibadan, Nigeria. Serum samples were screened for HBsAg by ELISA, and positive samples were genotyped by semi-nested multiplex PCR for HBV genotypes A, B, C, D, E and F. Data generated were analyzed in R-studio. A total of 81/90 (90%) of HBsAg-positive samples were successfully genotyped, and genotype A was most prevalent with 15.7%, while genotypes B and E were the least with 1.2% each. Genotypes A/C infection was the highest among mixed infections with 40% prevalence, while genotypes A/D were the least prevalent mixed infection with 4.8%. We advocate for a comprehensive genotype analysis in larger cohorts across Nigeria, to give a more comprehensive understanding of the distribution and prevalence of different HBV genotypes population wide.

Comparative analysis of the RVA VP7 and VP4 antigenic epitopes circulating in Iran and the Rotarix and RotaTeq vaccines

Analyzing the lineages and detecting antigenic variation in immunogenic motifs of Group A Rotavirus (RVA) variants is crucial because it can impact vaccine efficacy. This study investigated the circulating lineages of VP4 and VP7 proteins of human RVA isolates and their phylogeny in ≤24-month-old symptomatic, rotavirus-positive children with transudative diarrhea within 48 h of admission to Mofid Children's Hospital between December 2020 and March 2022 in Tehran, Iran. Antigen detection was performed by ELISA, RNA extraction, and semi-nested multiplex PCR for G/P genotypes, followed by sequencing and bioinformatic analysis using multiple sequence alignments in MEGA and phylogenetic analysis by Geneious Prime. The similarity of VP7 and VP4 amino acids with the RotaTeq and Rotarix vaccine strains for cytotoxic T cell and antigenic epitopes was evaluated using the UCSF Chimera Molecular Modeling System. Overall, 27.3 % of the samples were RVA positive, showing untypeable (2.5 %), single (76.9 %), and mixed (20.5 %) genotypic characteristics. The strains clustered in the G1/II, G2/IV, G3/I, G4/I, G9/III, P[8]/III, P[4]/V, and P[6]/I lineages. Comparative analysis of VP7 antigenic epitopes showed that the G1/II strains were completely conserved, while the G2/IV, G3/I, G4/I, G6, G9/III strains contained 2, 3–5, 2, 4 and 9 amino acid substitutions, respectively. The P[8]/III genotypes differed by 3 amino acids, while the P[6]/I genotype had the most substitutions. CTL epitopes were completely conserved in G3/I strains, but other genotypes differed by 1–4 amino acids compared to the vaccine strains. Given the diversity of circulating RVA genotypes and the observed mutations in neutralizing and CTL epitopes, immune escape by some of the strains is likely in Iran. This finding underscores the importance of evaluating the effect of rotavirus vaccines on local genotypes and related lineages before implementing a vaccination program.

Detection of hepatitis B virus genotypes in a group of hepatitis B virus-infected patients in central and northern Sri Lanka.

Introduction. Hepatitis B infection causes a spectrum of clinical diseases varying from asymptomatic infection to severe or fulminant acute hepatitis, chronic liver disease, cirrhosis and hepatocellular carcinoma. Hepatitis B virus (HBV) genotypes appear to influence transmission dynamics, clinical outcomes and responses to antiviral therapy. However, hepatitis B genotyping has been poorly investigated in Sri Lanka. This study intended to determine hepatitis B genotypes in a group of HBV-infected people in central and northern Sri Lanka. Methodology. The study was a laboratory-based descriptive cross-sectional study. Initial detection of HBV DNA in 100 EDTA blood samples was done by using a commercially validated quantitative real-time PCR kit. Hepatitis B genotyping was performed by in-house conventional semi-nested multiplex PCR using genotype-specific primers (for genotypes A-F). The serological profile was determined using a commercially validated ELISA/chemiluminescence immunoassay. The results were evaluated for genotype prevalence, viral load association and hepatitis B e antigen (HBeAg) expression in the study population. Results and conclusion. The study detected that genotype C (n=38) is most prevalent and infections with multiple genotypes (n=52, 52%) were commoner than mono-genotype (n=23, 23%) infections. In total, 25% of patients had no detectable genotype among genotypes A-F. The mean viral load in asymptomatic patients with a single genotype was 3.28 log10 copies ml-1 and in multiple genotypes was 4.18 log10 copies ml-1 before treatment. Statistical significance was not detected in mean viral loads and HBeAg expression in these two groups. In the future, chronic HBV infection may be effectively treated and managed according to the infected genotype.

549. The Application of Nanopore Sequencing Technology to Characterize Rotavirus Genotypes from Young Children with Diarrhea in the United Arab Emirates

Abstract Background In the United Arab Emirates, rotavirus A (RVA) remains a major cause of gastroenteritis in young children despite the universal use of rotavirus vaccines. Monitoring the genetic diversity of circulating strains is essential to better understand the disease burden. Although semi-nested multiplex-PCR is a popular method for RVA genotyping, some clinically significant strains may not be accurately identified by this method. Nanopore sequencing is a third-generation single-molecule sequencing technology that allows for real-time identification of viral and bacterial isolates at competitive costs. This study aims to investigate the use of nanopore sequencing and multiplex-PCR methods to identify RVA genotypes. Methods Thirty-three RVA isolates, obtained from stool specimens collected from children less than 5 years old who presented with diarrhea between December 2017 and April 2019, were used for genotyping by PCR and nanopore sequencing. Sixteen isolates were genotyped by Taqman-based qRT-PCR based on VP7 (G) and VP4 (P) genes and genotypes were confirmed by nanopore sequencing in thirteen isolates; three isolates were excluded from nanopore sequencing due to poor RNA quality. Another fifteen isolates were genotyped by the nanopore method alone and two samples were excluded due to poor RNA quality. Results The majority of isolates genotyped by qRT-PCR (9 out of 16) were found to harbor VP7 gene of G9 genotype, whereas 5 isolates were of G3 genotype. G12 genotype was rare (2 isolates). For VP4 (P) genotypes, P8 was the most prevalent (8 isolates), followed by P4 (7 isolates), and P6 (1 isolate). When combinations of VP7 and VP4 genotypes are considered, G9P[8] and G9P[4] were the most common genotypes. Nanopore sequencing of 28 isolates revealed G3P8 (29%) as the most prevalent G-P combination, followed by G8P[8] (18%). G9P[8] and G2P[4] were present in 14% of samples. G12P[6] was present in 7% of tested samples. Further, G9P[4], G3P[4], G1P[4], and G4P[2] were detected in 4% of total specimens. One sample was non-typable. Conclusion Nanopore sequencing detected various RVA strains among the studied samples including the recently emerging strain, G8P[8]. Using this method in surveillance studies may advance our understanding of the origin, genetic recombination, and burden of disease. Disclosures All Authors: No reported disclosures.

Human rotavirus VP4 and VP7 genetic diversity and detection of GII norovirus in Ibadan as Nigeria introduces rotavirus vaccine.

ObjectiveThis cross-sectional study investigated the circulating strains of rotavirus and screened for noravirus in Ibadan, Nigeria as the country introduces the rotavirus vaccine into its national immunization program.MethodsSixty-five stool samples were collected from children younger than 5 years with clinically diagnosed diarrhea and screened for the presence of rotavirus and norovirus using RT-PCR. Rotavirus-positive samples were further analyzed to determine the G and P genotypes using semi-nested multiplex PCR.ResultsThe rates of rotavirus and norovirus positivity were 30.8% and 10.8%, respectively, whereas the rate of rotavirus and norovirus mixed infection was 4.6%. G1 was the predominant VP7 genotype, followed by G2, G9, and G1G2G9, whereas the predominant VP4 genotype was P[4], followed by P[6], P[8], and P[9]. The mixed P types P[4]P[8] and P[4]P[6] were also detected. G1P[4] was the most common VP4 and VP7 combination, followed by G2P[4], G1[P6], G1P[8], G2P[6], G2P[9], G9P[6], G2G9P[4], G2P[4]P[6], G1P[4]P[8], G2G9P[8], G1G2G9P[8], and G1[non-typable] P[non-typable], which were detected in at least 5% of the samples. Four samples had a combination of non-typable G and P types.ConclusionsIt is essential to monitor the circulation of virus strains prior to and during the implementation of the immunization program.

Molecular investigation of the dengue outbreak in Karnataka, South India, reveals co-circulation of all four dengue virus serotypes.

The growing incidence of dengue outbreaks in the state of Karnataka prompted us to study the circulating dengue virus (DENV) and their proportion among the suspected cases of dengue patients during the disease outbreak at Mysuru district of Southern India. The presence of the DENV in a patient's serum sample was identified by RT-PCR using previously published primer pairs targeting CprM gene. DENV serotyping was carried out by semi-nested multiplex PCR using serotype-specific primers and nucleotide sequencing. Three hundred fifty-five samples of serum from suspected dengue cases were collected, and 203 samples (57.18%) were found positives. In 2016, DENV-4 (97.87%) was found to be the most dominant DENV serotype either alone or as co-infection, followed by DENV-2 (8.51%) and DENV-3 (4.25%). In 47 positive cases, co-infection with more than one serotype was detected in 4 cases (8.51%). The analysis of the dengue cases in 2017, DENV-4 was dominating serotype (33.97%), followed by the emergence of DENV-2 (32.05%), DENV-3 (25.64%), and DENV-1 (25.00%). Our study also reports the circulation of all four DENV serotypes in the Mysuru district of Southern India, with concurrent infections rate of 16.66% in 2017. The present study provides information regarding the genetic variation among the circulating DENV serotype in an Indian state of Karnataka. The need for the studying genetic diversity of DENV will be useful during the continuous monitoring for disease burden as well as the development of appropriate prophylactic measures to control the spread of dengue infection.

694. Unexpected Diversity of Rotavirus Genotypes in Pediatric Population

BackgroundRotavirus (RV) is the leading cause of viral gastroenteritis in children. Although RV genotypes differ geographically and temporally, five are the predominant genotypes circulating worldwide. Aim of our study was to monitor possible changes in distribution of Rotavirus genotypes circulating in Greek pediatric population during the post vaccine era.MethodsDemographic data and fecal samples were collected from children ≤15 years old with symptoms of acute gastroenteritis who visited emergency units of Pediatric Hospitals in Greece from September 2016 to August 2019. Samples were tested for RV Group A antigen with rapid immunochromatographic assay. Positive samples were further G and P typed employing RT-PCR, semi-nested multiplex PCR and Sanger sequencing of the VP7 and VP4 genes.ResultsA total of 660 children participated in the study with median age 31±29 months. Males outnumbered females (59%). Most of them lived in urban cities (85%). RV genotyping distribution was G4P[8] (41%), G1P[8] (22%), G2P[4] (14%), G9P[8] (8%), G9P[4] (5,5%), G12P[8] (2%) and G3P[8] (1,8%). Unusual and mixed genotypes were identified in 3,2% and 2,5% of the samples respectively. During 2016-2017 and 2017-2018, G4P[8] was the predominant genotype in 67% and 51% of the annual samples. However, in 2018-2019 the most common genotypes were G9P[8] and G9P[4] (33% in total) followed by G2P[4] (27%). Interestingly, the genotype G9P[4] was not detected at all in the first two years of the study.ConclusionThis study indicates diversity of the predominant RV genotypes in Greek children during 2016-2019. The emergence of G9 as the most common genotype as well as the significant detection of uncommon ones highlight the importance of continuous surveillance of RV genotyping during the post vaccine period.Disclosures All Authors: No reported disclosures

Detection and characterization of Plasmodium spp. by semi-nested multiplex PCR both in mosquito vectors and in humans residing in historically endemic areas of Paraguay

In Paraguay, no cases of Malaria have been recorded since 2011. Microscopy and the SnM-PCR technique were implemented to detect and characterize Plasmodium spp. both in mosquitoes and in humans residing in historically endemic areas of Paraguay, to evaluate the possibility of finding asymptomatic cases and/or Plasmodium parasites circulating in anophelines. Between 2013 and 2015, 361 human blood samples were collected on filter paper, and between 2016 and 2017, 938 female Anopheles mosquitoes were captured in 15 Paraguayan localities. All the diagnostic techniques showed negative results. We observed no asymptomatic case or Plasmodium circulating in vectors.

Evaluation of Asymptomatic Malaria Status in Eastern of Afghanistan Using High Resolution Melting Analysis

Malaria is threatening more than half of Afghanistan population. Asymptomatic malaria is notable problem against malaria controlling strategies. In this study we evaluated the asymptomatic malaria status in Nangarhar Province, Afghanistan in 2017. Overall, 296 finger blood samples were taken on DNA Banking Cards and microscopic slides from asymptomatic individuals in Jalalabad city. We used a novel post real time PCR high resolution melting analysis beside microscopy and semi-nested multiplex PCR to evaluate status of asymptomatic malaria in this city. The prevalence of asymptomatic malaria in Jalalabad city was determined 1.7% (5/296), 7.43% (22/296) and 7.78% (26/296) by microscopy, Seminested multiplex PCR and qRT-PCR-HRM, respectively. Out of 26 positive cases were detected by qRT-PCR-HRM, 21, 1 and 4 cases were detected P. falciparum, P. vivax and mixed infection of P. falciparum and P. vivax, respectively. Our data indicating on existence of significant number of asymptomatic reservoirs that assists in prolonged endemicity of the disease. On the other hand, the molecular methods are better alternatives for microscopy especially for monitoring of asymptomatic cases of malaria.

Group A Rotavirus Genotypes among Calf Herds in Southwest Nigeria and Implications for Human Rotavirus Vaccines’ Efficiency

Rotaviruses have been widely reported to be associated with diarrhea in humans but fewer studies abound on other mammalian species. This prospective study was conducted to detect and characterize Rotaviruses from freely ranged migratory herds of cattle in Ekiti and Ondo states, Nigeria with a view to further expanding knowledge on rotaviruses, possible animal-human interspecies transmission and impacts on vaccine efficiency. By convenience sampling, between September 2014 and February 2015, stool samples from 120 calves, comprising settled and migratory herds of cattle were obtained and examined for group A rotaviruses using Certest® Quadruple Enzyme Immuno Assay. Rotavirus genomes were isolated by extraction from the positive samples, reverse transcribed and amplified by One-Step reverse transcription polymerase chain reaction (RT-PCR), and genotyped by semi-nested multiplex PCR. Representative PCR products of the genotyped samples were purified and sequenced using Sanger method. The generated query sequences were queried to the GenBank to retrieve similar sequences for pairwise alignment by ClustalW. Phylogenetic analyses by Neighbour-joining method were conducted at 1000 bootstrap replicates to obtain phylograms using MEGA 6 software. Fifteen samples (15/120: 12.5%) were positive for rotavirus. No statistically significant association existed between rotavirus infection and gender of the calves (χ2 = 0, df = 1, p = 1). Eight samples (8/15: 53.3%) were successfully genotyped where the G1, G5, G9, G10, G12, and P[6] were detected. Nucleotide Sequencing of the VP7 and VP4 genes of the genotyped samples confirmed strains G1P[6], G10P[6], and G12P[6] with 58% - 100% nucleotide identity within these viruses. Phylogenetic analysis revealed 2 possible transmissions from India and Honduras. Bovine rotaviruses were detected in freely ranged and settled calf herds in southwestern Nigeria at a rate of 12.5%. The strains identified were analysed to be group A rotavirus strains with potential interspecies transmission from human to calves and from bovine to humans. The detected mixed strains could eventually impact negatively on the effectiveness of available rotavirus vaccines over the prevailing serotypes in human infections.

Improvement of Rotavirus Genotyping Method by Using the Semi-Nested Multiplex-PCR With New Primer Set.

Rotavirus A (RVA) is a major cause of gastroenteritis in infants and young children. After vaccine introduction, RVA surveillance has become more important for monitoring changes in genotype distribution, and the semi-nested multiplex-PCR is a popular method for RVA genotyping. In particular, the VP7 primer set reported by Gouvea and colleagues in 1990 is still widely used worldwide as the recommended WHO primer set in regional and national reference RVA surveillance laboratories. However, this primer set yielded some mistakes with recent epidemic strains. The newly emerged equine-like G3 strains were mistyped as G1, G8 strains were mistyped as G3, the G9 lineage 3 strains showed very weak band, and the G9 lineage 6 strains showed a G9-specific band and a non-specific band. Gouvea’s standard protocol has become relatively unreliable for identifying genotypes correctly. To overcome this limitation, we redesigned the primer set to include recent epidemic strains. Our new primer set enabled us to correctly identify the VP7 genotypes of representative epidemic strains by agarose gel electrophoresis (G1, G2, human typical G3, equine-like G3, G4, G8, G9, and G12). We believe that the multiplex-PCR method with our new primer set is a useful and valuable tool for surveillance of RVA epidemics.

Human parvovirus B19 genotype 1 in suspected dengue patients of Tefé, Amazonas State, Brazil.

Human parvovirus B19 (B19V) is a common pathogen, which on infection causes variety of clinical conditions from benign self-limiting exanthematous disease and other similar pathologies to fetal death. We collected 341 serum samples between the first and fourth day after the onset of symptoms from all patients suspected of dengue fever who were attended at Regional Hospital of Tefé. Initially, patients were screened for malaria by blood smear test and negative samples were sent to Fundação de Medicina Tropical Doutor Heitor Vieira Dourado (FMT-HVD) situated in Manaus (AM) for dengue testing using semi-nested multiplex PCR. Further, we investigated 44 malaria and dengue-negative samples of children for B19V DNA by nested-PCR. Positive samples were analyzed by BLAST against entire public non-redundant nucleotide database and genotyped by phylogenetic analyses using neighbor-joining clustering method. Eight samples (18.2%) were found to be PCR positive. Fever, headache, ocular pain, and/or muscle pain were reported as the most frequent symptoms by the patients and none were diagnosed with rash at the time of sample collection. Phylogenetic analysis of major capsid protein 2 (VP2) and VP3 coding region showed high similarity with B19V genotype 1. Our results reveal the spread of B19V genotype 1 in Tefé. Moreover, our results emphasize the significance of laboratorial differential diagnosis using molecular techniques in patients with acute febrile, and thereby aid the health surveillance system in improving patient care even in the remote areas of Amazon.

Detection and molecular characterization of uncommon rotavirus group A genotype G12 among hospitalized children in Chennai.

Arun P, Krishnasami K, Gunasekeran P, Fathima G, Padmanabhan V. Detection and molecular characterization of uncommon rotavirus group A genotype G12 among hospitalized children in Chennai. Turk J Pediatr 2019; 61: 209-216. Human group A (RVAs) possess genetic diversity and often newer RVA strains have been reported frequently with different G and P combinations worldwide. As the disease burden is more common in low income countries including India, monitoring and detection of the circulating strains of rotavirus needs to be monitored to understand the genetic diversity of the strains which evolve over time. This study was an attempt to provide data on rotavirus gastroenteritis prevalence in and around Chennai Tamilnadu-South India during the pre-vaccination era. Stool samples were collected and tested from 401 children less than five years of age admitted to hospitals with acute gastroenteritis. The samples were subjected to amplification for VP7 and VP4 genes by using consensus primers, followed by semi-nested type-specific multiplex PCR. Phylogenetic analysis of VP7 genes of G12 were carried out and the variations between strains isolated globally were documented. Rotavirus was detected in 167 (41.64%) samples and five were found to be the uncommon G12P[6] genotype (2.99%) tested by RT-PCR followed by G1P[8], G9P[4], G9 with P untypable and G3P[8] and combinations of G2G9P[6] and G9G2P[11] with few strains untypable. In this study we highlight the occurrence of uncommon G12P[6] strain of rotavirus infection in the community. Since rotavirus is transmitted through oral-fecal route and monitoring of environmental cleanliness is mandatory to cease the spread of this deadliest viral agent to achieve our MDG-IV. Covering single genotype will have to be modified with respect to the circulating stains.

Comparison of three diagnostic methods (microscopy, RDT, and PCR) for the detection of malaria parasites in representative samples from Equatorial Guinea

BackgroundMalaria in Equatorial Guinea remains a major public health problem. The country is a holo-endemic area with a year-round transmission pattern. In 2016, the prevalence of malaria was 12.09% and malaria caused 15% of deaths among children under 5 years. In the Continental Region, 95.2% of malaria infections were Plasmodium falciparum, 9.5% Plasmodium vivax, and eight cases mixed infection in 2011. The main strategy for malaria control is quick and accurate diagnosis followed by effective treatment. Early and accurate diagnosis of malaria is essential for both effective disease management and malaria surveillance. The quality of malaria diagnosis is important in all settings, as misdiagnosis can result in significant morbidity and mortality. Microscopy and RDTs are the primary choices for diagnosing malaria in the field. However, false-negative results may delay treatment and increase the number of persons capable of infecting mosquitoes in the community. The present study analysed the performance of microscopy and RDTs, the two main techniques used in Equatorial Guinea for the diagnosis of malaria, compared to semi-nested multiplex PCR (SnM-PCR).ResultsA total of 1724 samples tested by microscopy, RDT, and SnM-PCR were analysed. Among the negative samples detected by microscopy, 335 (19.4%) were false negatives. On the other hand, the negative samples detected by RDT, 128 (13.3%) were false negatives based on PCR. This finding is important, especially since it is a group of patients who did not receive antimalarial treatment.ConclusionsOwing to the high number of false negatives in microscopy, it is necessary to reinforce training in microscopy, the “Gold Standard” in endemic areas. A network of reference centres could potentially support ongoing diagnostic and control efforts made by malaria control programmes in the long term, as the National Centre of Tropical Medicine currently supports the National Programme against Malaria of Equatorial Guinea to perform all of the molecular studies necessary for disease control. Taking into account the results obtained with the RDTs, an exhaustive study of the deletion of the hrp2 gene must be done in EG to help choose the correct RDT for this area.

Improved reverse transcription-polymerase chain reaction assay for the detection of flaviviruses with semi-nested primers for discrimination between dengue virus serotypes and Zika virus.

BACKGROUND The genus Flavivirus includes a variety of medically important viruses, including dengue virus (DENV) and Zika virus (ZIKV), which are most prevalent in Brazil. Because the clinical profile of patients affected by different DENV serotypes or ZIKV may be similar, the development of new methods that facilitate a rapid and accurate diagnosis is crucial.OBJECTIVES The current study aimed to develop an improved reverse transcription-polymerase chain reaction (RT-PCR) protocol for universal detection of flaviviruses by using semi-nested primers that discriminate between DENV serotypes and ZIKV.METHODS The bioinformatics workflow adopted for primer design included: (1) alignment of 1,442 flavivirus genome sequences, (2) characterisation of 27 conserved regions, (3) generation of a primer set comprising 77 universal primers, and (4) selection of primer pairs with greatest coverage and specificity. Following primer design, the reaction was validated in vitro. The same approach was applied to the design of primers specific for DENV and ZIKV, using a species-specific sequence database.FINDINGS The new assay amplified an 800-806 nt variable region of the NS5 gene and allowed discrimination of virtually all flavivirus species using reference-sequence comparison. The 800-806 nt fragment was validated as a template for a semi-nested multiplex PCR using five additional primers for the detection of DENV and ZIKV. These primers were designed to generate amplicons of different sizes, allowing differentiation of the four serotypes of DENV, and ZIKV using agarose gel electrophoresis.MAIN CONCLUSIONS The bioinformatics pipeline allowed efficient primer design, making it possible to identify the best targets within the coding region of the NS5 protein. The multiplex system proved effective in differentiation of DENV1-4 and ZIKV on a 2% agarose gel. The possibility of discriminating DENV serotypes and ZIKV in the same reaction provided a faster result consuming less sample. In addition, this simplified approach ensured the reduction of the cost per analysis.

Prevalence and diversity of rotavirus A genotypes cirulating in Turkey during a 2-year sentinel surveillance period, 2014-2016.

Human rotavirus A (RVA) is the main etiological agent of watery diarrhea among children under 5 years of age worldwide. The aims of this study were to investigate the prevalence and diversity of RVA genotypes circulating in Turkey during a 2-year sentinel surveillance study. A total of 1639 rotavirus antigen-positive stool samples were obtained from children younger than 5 years of age hospitalized with acute gastroenteritis. Rotavirus G and P genotypes were determined by reverse transcription polymerase chain reaction (RT-PCR) with consensus primers for the VP7 and VP4 genes, followed by semi-nested type-specific multiplex PCR. Rotavirus RNA was detected in 1396 (85.3%) of the samples tested. The highest detection rate (38.2%) was obtained among children in the 0-12 months age group, followed by children in the 13-24 months age group (36.2%). The most prevalent genotype was G1P[8] (24.6%) followed by G3P[8] (19.6%), G9P[8] (12.2%), G2P[4] (9.5%), G2P[8] (6.5%), and G4P[8] (4.8%). The proportions of uncommon and mixed genotypes were 21.5% and 1.14%, respectively. The large number of genotypes observed, including common, uncommon, and mixed types, indicates a high heterogeneity of RVA strains circulating in Turkey. The current study also exhibited dramatic fluctuations on the prevalences of the common genotypes, with increases in G3 and G1 and decreases in G9 and G2 from 2014-2016.

Prevalence of occult hepatitis B virus infection and Torque teno virus infection and their association with hepatocellular carcinoma in chronic hepatitis C patients

BackgroundThe role of occult hepatitis B virus (HBV) infection and Torque teno virus (TTV) infection in the development of hepatocellular carcinoma (HCC) in chronic hepatitis C patients is still uncertain. AimThe aim of the present study was to investigate the prevalence and significance of OBI and TTV infection, and to examine the genetic diversity of these viruses, in chronic hepatitis C patients with and without HCC. MethodsSera from 151 hepatitis C virus (HCV)-infected patients (49 patients with HCC and 102 without HCC) negative for HBV surface antigen (HBsAg) were tested for the presence of OBI and TTV infection by semi-nested and group-specific multiplex PCR assays, respectively. Nucleotide sequencing of HBV S region was further performed. ResultsOBI and TTV infection were detected in 5 (3.3%) and 68 (45%) patients, respectively. HBV isolates were classified into genotypes A (4/5, 80%) and D (1/5, 20%), and no HBsAg escape mutation was observed. TTV phylogenetic group 3 was the most prevalent among both HCC and non-HCC patients. OBI and TTV infection were significantly more frequent in patients with HCC than patients without HCC (p=0.003, and p=0.009, respectively). Moreover, TTV infection was associated with HCC (OR=2.23, 95%CI=1.04–4.80, p=0.040), independently of liver cirrhosis. ConclusionsA low prevalence of OBI was observed in patients with HCV-related chronic liver disease, and TTV infection was an independent factor associated with the occurrence of HCC. Whether TTV influences the progression of liver disease in chronic hepatitis C patients remains to be elucidated.

FIRST MOLECULAR DETECTION AND VP7 (G) GENOTYPING OF GROUP A ROTAVIRUS BY SEMI-NESTED RT-PCR FROM SEWAGE IN NIGERIA.

SUMMARYRotavirus is the leading cause of viral gastroenteritis worldwide, and sewage is a major source of the virus dissemination in the environment. Our aim was to detect and genotype rotaviruses from sewages in Nigeria. One hundred and ninety sewage samples were collected between June 2014 and January 2015. The two phase concentration method using PEG 6000 and dextran was used to concentrate sewage samples following WHO protocols. Molecular detection was performed by RT-PCR, and VP7 genotyping by semi-nested multiplex PCR. A total of 14.2% (n = 27) samples tested positive. Monthly distribution showed that June to September had a lower rate (3.7% to 7.4%), while October to January recorded 11% to 26%. Genotype G1 predominated followed by G8, G9, G4 and lastly G2, 7.4% (n = 2) of isolates were nontypeable. This is the first report of rotavirus detection in sewages from Nigeria. Genotype G1 remains the most prevalent genotype. This observation calls for an effort by the governmental authorities to implement a molecular surveillance, both clinical and environmental, in order to provide vital information for the control and the vaccine efficacy not only in Nigeria, but globally.

Prevalence of rotavirus genotypes in children younger than 5 years of age before the introduction of a universal rotavirus vaccination program: report of rotavirus surveillance in Turkey.

BackgroundGroup A rotaviruses are the most common causative agent of acute gastroenteritis among children less than 5 years of age throughout the world. This sentinel surveillance study was aimed to obtain baseline data on the rotavirus G and P genotypes across Turkey before the introduction of a universal rotavirus vaccination program.MethodsRotavirus antigen-positive samples were collected from 2102 children less than 5 years of age who attended hospitals participating in the Turkish Rotavirus Surveillance Network. Rotavirus antigen was detected in the laboratories of participating hospitals by commercial serological tests such as latex agglutination, immunochromatographic test or enzyme immunoassay. Rotavirus G and P genotypes were determined by reverse transcription polymerase chain reaction (RT-PCR) using consensus primers detecting the VP7 and VP4 genes, followed by semi-nested type-specific multiplex PCR.ResultsRT-PCR found rotavirus RNA in 1644 (78.2%) of the samples tested. The highest rate of rotavirus positivity (38.7%) was observed among children in the 13 to 24 month age group, followed by children in the age group of 25 to 36 months (28.3%). A total of eight different G types, six different P types, and 42 different G–P combinations were obtained. Four common G types (G1, G2, G3, and G9) and two common P types (P[8] and P[4]) accounted for 95.1% and 98.8% of the strains, respectively. G9P[8] was the most common G/P combination found in 40.5% of the strains followed by G1P[8] (21.6%), G2P[8] (9.3%), G2P[4] (6.5%), G3P[8] (3.5%), and finally, G4P[8] (3.4%). These six common genotypes included 83.7% of the strains tested in this study. The rate of uncommon genotypes was 14%.ConclusionThe majority of the strains analyzed belonged to the G1–G4 and G9 genotypes, suggesting high coverage of current rotavirus vaccines. This study also demonstrates a dramatic increase in G9 genotype across the country.

Evaluation of rotavirus and intestinal parasite infection in a paediatric population in West Cameroon: Clinical characteristics and virus genotyping

The prevalence of Rotavirus (RV)-related gastroenteritis in Cameroon was monitored in this study, identifying the circulating G-P types, and the presence of co-infection with intestinal parasites. The stool samples were examined for rotaviruses using chromatographic and molecular techniques, and parasites were identified by macroscopic and microscopic examination. 38% of samples were RV positive. Data revealed the presence of three different G-genotypes (G1, G2 and G8) and three P-genotypes (P [8], P[6] and P[4]). The main G-P combination was G1+G2P[8]. Entamoeba histolytica was identified in 44.4% of stool samples. The G2P[8] genotype identified is an unusual strain with the G1P[8] or G2P[4] more common. Genotype G8 strains, also associated with animals, have been sporadically recovered from humans and have been considered for inclusion in bovine-human reassortant vaccine. Epidemiological RV strain surveillance should be continued in order to support RV vaccination campaigns. The results also show a high prevalence of intestinal parasite infections. Key words: Gastroenteritis, rotavirus, G/P genotype, semi-nested multiplex PCR, intestinal parasites, Cameroon.