Reverse Transcriptase-nested Polymerase Chain Reaction Research Articles

One-Year Investigation of Hepatitis A Virus and Rotavirus in Two Waste Water Treatment Plants in Egypt

Background: Waterborne enteric viruses are evolving source of disease outbreaks and represent a major threat to global public health. The repetitive assessment of water environments is necessary to manage enteric virus-mediated fecal infection and the probable emergence of different variants. Methods: Here, we detected human rotavirus and hepatitis A virus circulating in two wastewater treatment plants, for one year in sewage and sludge samples from two largest wastewater treatment plant (El-Gabal El-Asfar and Zenin) located in Egypt. For this purpose, we examine predefined viruses' prevalence in 72 samples (24 raw sewage samples, 24 treated effluent samples, and 24 sludge) collected from August 2022 to July 2023. Viral RNA was extracted and detected by nested Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) in case of HAV and by multiplex semi nested RT-PCR In case of rotavirus with the Results: Percentage of detection rate 58.3%, 25% 41.6%, 50%, 16.6%58.6%, 41.6%, 8.3%, 41.6%41.6%, 8.3% and 41.6% for inlet, outlet, sludge in WWTP zenin and WWTP El-Gabal El-Asfar for HAV, RV respectively. Using generic primer result indicated G1P8 was the most common although G2P [4], G1, G2, G3P? G9P [8], G9P [11], G1 P [4] were detected with different percentage. Conclusion: These results demonstrated that HAV and RV still remains in the environment after sewage treatment and could play an important role in maintaining the endemicity of HAV and RV infection. Routine and continuous monitoring of waterborne viruses are most useful approach to understand disease occurrence in communities.

Molecular detection and characterization of Rift Valley fever virus in humans and domestic ruminants in Nigeria

Rift Valley fever virus (RVFV) causes a zoonotic mosquito-borne disease of domestic livestock, wildlife and humans majorly in Africa and the Arabian Peninsula. The virus is a Phlebovirus belonging to the Family Phenuiviridae and constitutes a threat to both human and animal health. Although there has been serological evidence of RVFV in humans and animals in Nigeria, reports on its molecular detection and characterization are absent. Blood samples (n = 1101) were collected between January 2019 and March 2021 from humans, cattle and sheep in four States of Nigeria. The harvested plasma samples were tested using a commercial ELISA kit. All the ELISA-positive, ‘-doubtful’ and some ‘-negative’ samples (n = 436) were further analyzed using nested reverse transcriptase-polymerase chain reaction (nRT-PCR). Eight samples (humans, n = 1; cattle, n = 6; sheep, n = 1) that gave the expected band size of approximately 668 base pairs following gel electrophoresis were sequenced by Sanger method. The sequence data were edited using BioEdit software and RVFV was successfully identified in all of them. Nucleotide sequence and phylogenetic analyses revealed that these eight RVFV strains were 98.3–99.9% identical and clustered with the Ugandan Smithburn strain. Genotyping showed that they belonged to Lineage K. This report, which is the first molecular detection and characterization of RVFV in Nigeria, confirms the circulation of the virus in the country. It also highlights the need for large-scale surveillance for RVFV in Nigeria, including among mosquito vectors, to provide better understanding of its spatiotemporal epidemiology, and thus, aid in preventing potential RVF outbreaks in the country.

Higher Risk of Chronic Hepatitis E Virus Infection in Patients with Human Immunodeficiency Virus-1: An Iranian Cross-sectional Study.

Occurrence of Hepatitis E Virus (HEV) infection may be common in Human Immunodeficiency Virus (HIV-1) patients and may lead to chronic infection as well as cirrhosis. We intended to determine the incidence of HEV infection among HIV-1 patients compared to individuals without HIV-1 infection. In our cross-sectional study, 87 HIV-1-positive patients were compared to 93 healthy individuals in Kerman, Iran. Plasma and peripheral blood mononuclear cells (PBMCs) were obtained from all the participants. Plasma samples were evaluated for HEV IgM and IgG using the ELISA kit. Then, reverse transcriptase-nested polymerase chain reaction (RT-nested PCR) was used in RNA extractions from PBMCs to check for the presence of HEV RNA. Among the subjects examined in our study, 61 (70.1%) and 71 (77.4%) out of patients with HIV-1 infection and healthy individuals were male, respectively. The average ages of patients with HIV-1 and the control group were 40.2 years and 39.9 years, respectively. No discernible differences were found between the two groups based on IgM and IgG seropositivity against the HEV. However, HEV-RNA was found in 8% of patients with HIV-1 and 1.1% of HIV-1-negative individuals (P=0.03). There was also an association between the HEV genome and anti-HEV and anti-HCV antibodies in HIV-1-positive patients (P=0.02 and P=0.014, respectively). HEV infection may be more common in HIV-1 patients and may develop a chronic infection in immunocompromised individuals. Molecular-based HEV diagnostic tests, including RT-PCR assays, should be performed in HIV-1 patients with unknown impaired liver function tests.

Development of Nested Polymerase Chain Reaction for Detection of Rabies

Rabies is an acute progressive and zoonotic disease which is caused by the rabies virus. Rabies virus is the prototype virus belonging to the genus lyssavirus in the family Rhabdoviridae of the order Mononegavirales. The aim of this study was to develop a nested reverse-transcriptase polymerase chain reaction (nRTPCR) for the detection of this virus along with a canine internal control to monitor the integrity of the extracted RNA. A pair of outer and inner primer targeting the N gene of the virus was evaluated along with β-actin gene as the internal control. This nRTPCR can detect up to 20 viral copy number.

Utility of Nested Reverse-Transcriptase Polymerase Chain Reaction of Clinical Specimens for Early Diagnosis of Hemorrhagic Fever with Renal Syndrome

Hemorrhagic fever with renal syndrome (HFRS) is confirmed by the isolation of hantavirus from serum, detection of virus-specific IgM, or a four-fold change in IgG titers during the acute and convalescent periods measured using an immunofluorescence assay (IFA). However, these tests are inefficient for early diagnosis. Therefore, this study investigated the usefulness of reverse-transcriptase nested polymerase chain reaction (RT-nPCR) for early diagnosis of HFRS using clinical samples such as urine and serum. Electronic medical records of eight patients with confirmed HFRS using IFA and RT-nPCR between May 2016 and May 2020 at Chosun University Hospital were reviewed. The virus was detected in all patients using RT-nPCR targeting the large (L) segment of hantavirus during the early phase in urine and serum. Importantly, the virus was identified in urine at a time when it was not identified in serum. Additionally, the virus was detected in urine and serum for up to 1 month after initial presentation with illness, but not in saliva, using RT-nPCR. We report eight HFRS cases diagnosed using urine and serum, but not using saliva, with RT-nPCR targeting the L-segment. Hantavirus RNA detection by RT-nPCR in urine and serum may aid the rapid diagnosis of HFRS during the early phase of the disease. In particular, HFRS should not be ruled out based on negative RT-PCR results in serum, and RT-PCR should be performed using urine as well as serum during the early phase of symptoms.

Prevalence of Hepatitis C virus and determination of its genotypes in subjects of Tehsil Daggar District Buner, KP, Pakistan

BackgroundHepatitis C is a noteworthy public health issue around the globe, resulting in liver-related problems. The disease is caused Hepatitis C virus a positive sense RNA virus belongs to Flaviviridae family. Approximately 6% population of Pakistan is infected with HCV. Due to genetic variation, HCV is categorized into 7 genotypes and 67 subtypes. MethodologyIn this study total 1100 ICT positive samples were collected from December 2016 to May 2017 from Tehsil Dagger District Buner. RNA was extracted from ICT positive serum samples of 1100 patients and, were screened for HCV-RNA by using Reverse Transcriptase-Nested polymerase chain reaction (RT-nested PCR) and, then the positive samples were subjected for HCV genotyping. Statistical analysis was carried on SPSS version 10.0. ResultsOut of 1100 ICT positive samples, HCV RNA was detected in 375 individuals, of which 14.9% were males and, 19.2% females. The frequency of HCV genotypes Untypable was 43.5%), followed by 3a (29.6%), 3 b was 9.6%, 2a was 5.9%, 1a was 5.3%, 1 b (4.0%) and, 2 b was 1.9% in these patients. Both genotypes untypable and 3a were the most prevalent genotypes in patients of all age groups and its prevalence was found high among patients with increasing age (>30years). The prevalence rate of HCV in age of 21–30 years (30.1%), of 31–40 years the percentage of positive cases are 24.85% and 11–20 years of age group is (11.4%). Furthermore, genotypes untypable, 3a and 3 b were found to be the most common genotypes in patients with history of barber shops, dental surgery, and receiving multiple injections. ConclusionThe current study reveals HCV untypable variants as the most frequent genotypes in the HCV patients of district Buner KP, Pakistan, with most patients in the age group 21–30 years. HCV various isolates were associated with different routes of transmission. The predominant factors of HCV included shaving in barber shops and intra-venous drugs use. These findings may suggest anti-HCV therapy and monitoring HCV infections.

Clinical Significance of MLL-AF4 Fusion Transcript Expression in the Absence of a Cytogenetically Detectable t(4;11)(q21;q23) Chromosomal Translocation

AbstractLeukemic cells from bone marrow (BM) of 17 infants and 127 children with newly diagnosed ALL, as well as fetal liver and BM and normal infant BM samples, were analyzed for presence of a t(4;11) translocation using standard cytogenetic techniques and expression of an MLL-AF4 fusion transcript using standard reverse transcriptase-polymerase chain reaction (RT-PCR) assays as well as nested RT-PCR that is 100-fold more sensitive than standard RT-PCR. Overall, 9 of 17 infants and 17 of 127 noninfant pediatric ALL patients were positive for expression of MLL-AF4 fusion transcripts, as determined by standard and/or nested RT-PCR assays. None of theMLL-AF4+ cases were positive for E2A-PBX1or BCR-ABL fusion transcript expression. Although 8 of 9MLL-AF4+ infants had cytogenetically detectable t(4;11)(q21;q23), 15 of the 17 MLL-AF4+noninfants were t(4;11)−. Infants withMLL-AF4+ ALL had poor outcomes, whereas non-infant MLL-AF4+/t(4;11)−patients had favorable outcomes similar toMLL-AF4− patients. Notably, MLL-AF4transcripts also were detected by nested RT-PCR in 4 of 16 fetal BMs, 5 of 13 fetal livers, and 1 of 6 normal infant BMs, but not in any of the 44 remission BM specimens from pediatric ALL patients. Our results provide unprecedented evidence that MLL-AF4 fusion transcripts can be present in normal hematopoietic cells, indicating that their expression is insufficient for leukemic transformation of normal lymphocyte precursors.© 1998 by The American Society of Hematology.

A prospective study of residual-disease monitoring of theALL1/AF4 transcript in patients with t(4;11) acute lymphoblastic leukemia

AbstractTwenty-five patients (22 adults and 3 infants) withALL1/AF4-positive acute lymphoblastic leukemia (ALL) were prospectively monitored by reverse transcriptase-polymerase chain reaction (RT-PCR) between January 1992 and July 1999. After high-dose induction and consolidation chemotherapy without bone marrow transplantation, all patients had a complete hematologic remission. Using nested RT-PCR (sensitivity 10−4), we observed conversion to PCR negativity in 11 (44%) of the patients. Thirteen of the 14 patients who did not have a molecular remission had a relapse at a median time of 4 months (range, 1 - 20 months). Of the 11 patients who had a conversion to PCR negativity, 5 reconverted to PCR positivity within 1 to 14 months. These 5 patients all progressed to hematologic relapse after 2, 3, 4, 4, and 7 months, respectively. Of the remaining 6 patients, 4 are in persistent hematologic and molecular remission at 12, 14, 88, and 96 months, whereas 2 are early in their follow-up. Actuarial probabilies of relapse and overall survival were 100% and 0% at 14 and 24 months and 67% and 43% at 96 and 100 months, respectively, in patients who had persistent RT-PCR positivity and in those who had a molecular remission. For both relapse and survival, the differences observed between the two groups were significant (P = .003 andP < .005, respectively). This study, which represents the first prospective analysis of residual-disease monitoring carried out in a substantial series of patients with t(4;11)-positive ALL, emphasizes the clinical relevance of RT-PCR-based methods to monitor minimal residual disease in this leukemia subset. (Blood. 2000;95:96-101)

Occult HCV and occult HBV coinfection in Iranian human immunodeficiency virus-infected individuals.

The presence of hepatitis C virus (HCV) genome in liver biopsy or peripheral blood mononuclear cell (PBMC) specimens in the absence of detectable HCV-RNA in plasma of the people with or without anti-HCV antibodies has defined as occult HCV infection (OCI), whereas occult hepatitis B virus infection (OBI) is detection of hepatitis B virus (HBV) genome in the absence of traceable hepatitis B surface antigen in the plasma samples of patients. The purpose of this study is to determine the presence of OBI and OCI in human immunodeficiency virus (HIV)-infected individuals. In this cross-sectional research, 190 Iranian HIV-infected individuals were enrolled from September 2015 to February 2019. All participants were tested regarding various serological markers for HCV and HBV infections. Viral RNA and DNA were extracted from plasma and PBMC specimens, and the presence of HCV-RNA in plasma and PBMC samples was tested using reverse transcriptase-nested polymerase chain reaction (PCR), HBV viral load was determined in plasma samples using COBAS TaqMan 48 Kit, and also the presence of the HBV-DNA in PBMC samples was tested by real-time PCR. In this study, the prevalence of OBI and OCI in HIV-infected individuals was 3.1% and 11.4%, respectively. The genotypes of HCV in the patients with OCI were as follows: 57.1% were infected with subtype 3a, 35.7% were infected with subtype 1a, and 7.1% was infected with subtype 1b. It is noteworthy that in this study, two patients (1.1%) had OCI/OBI coinfections. The present study revealed that 1.1% of Iranian HIV-infected individuals had OBI and OCI at the same time. Therefore, it seems that designing prospective surveys to determine the presence of this coinfection in HIV-infected individuals is informative.

Significance of Occult Hepatitis C Virus Infection in Liver Transplant Patients With Cryptogenic Cirrhosis.

Investigations into the viral causes of end-stage liver disease in liver transplant patients with cryptogenic underlying disease remain of interest. Hepatitis C virus infection, especially in its silent (occult) form, may play a key role in the introduction and development of cryptogenic cirrhosis. We aimed to determine the prevalence of occult hepatitis C virus infection in liver transplant recipients with cryptogenic cirrhosis. In this cross-sectional study, 127 liver transplant recipients confirmed to have cryptogenic cirrhosis were included. Plasma samples of the patients underwent evaluation for hepatitis C virus antibody using the enzyme-linked immunosorbent assay method. Plasma samples and paraffin-embedded liver tissue samples were tested for hepatitis C virus RNA using nested reverse transcriptase-polymerase chain reaction. Hepatitis C virus RNA was detected in liver tissue sections of 10 patients (7.9%). However, none of the cryptogenic patients had hepatitis C virus RNA or antibody in their plasma samples. None of the patients had hepatitis C or G virus coinfection, but simultaneous detection of hepatitis B and hepatitis C virus was diagnosed in 4 liver tissue samples. A finding of hepatitis C virus RNA in liver tissue samples of transplant recipients presents the historical possibility of occult hepatitis C virus infection as underlying disease in our patients with cryptogenic cirrhosis. Results present an important and determinative role of occult hepatitis C virus infection in the pathogenesis of cryptogenic cirrhosis, which needs further confirmation in additional studies.

Detection of HCV genome in peripheral blood mononuclear cells of Iranian seropositive and HCV RNA negative in plasma of patients with beta-thalassemia major: Occult HCV infection.

Beta (β) thalassemia major is a genetic blood disorder with a deficiency in the hemoglobin beta chain, requiring blood transfusion therapy. Multiple blood transfusions increase the risk of transmitting blood-borne infections. The aim of this study is to determine the frequency of hepatitis C virus (HCV) infection in Iranian individuals with β-thalassemia major. A total of 164 patients with β-thalassemia major were recruited for this study. HCV RNA testing was done on plasma and peripheral blood mononuclear cells (PBMCs) from the HCV seropositive samples (with reverse transcriptase-nested polymerase chain reaction [PCR] method using primers from the 5'-untranslated region [UTR]), and all HCV RNA positive samples were genotyped by the restriction fragment length polymorphism assay. For confirmation of the HCV genotyping in PBMCs of occult HCV infection [OCI]-positive patients, the PCR products of two different regions of HCV (5'-UTR and nonstructural protein 5B [NS5B]) were sequenced. Of 164 patients, 29.3% were positive for anti-HCV antibodies, and HCV RNA was detected in the plasma specimens of 13.4% patients and in the PBMC samples of 15.2% participants. The genomic HCV-RNA was detected in PBMC samples in 3 (6.3%) of the total 48 individuals who were HCV seropositive, and plasma HCV-RNA negative (occult HCV infection). The subtypes of HCV in the plasma and PBMC samples of three participants were not identical. This study shows that among this group of Iranian patients with β-thalassemia major, 13.4% had active HCV infection and 6.3% had occult HCV infection as evidenced by HCV RNA detected in PBMC specimens. Therefore, the design of a prospective study that focuses on the diagnosis of OCI can be very valuable and provide more information.

PCR for equine infectious anemia virus detection in whole blood from naturally infected horses in Colombia

The equine infectious anemia virus is associated with a variety of clinical symptoms that include anorexia, weight loss, pirexia, and anemia. Serological tests and reverse transcriptase nested polymerase chain reaction (RT-nPCR) have been used to detect EIAV in infected horses. In the present study, the use of non-nested PCR for the detection of EIAV gag gene sequences in peripheral blood cells DNA from horses bred in farms around Medellín (Colombia) is reported. EIAV DNA was detected by PCR in blood cells from fifteen horses, which had been found positive in an agar gel immunodiffusion test, despite the absence of clinical symptoms of expression. An amplified product to about 1.300 bp was obtained with a round of PCR without requiring nested PCR or RT-PCR.

Shedding of Rubella Virus among Infants with Congenital Rubella Syndrome Born in Tokyo, Japan, 2013-2014.

Rubella is usually a mild illness, with febrile rash being its main symptom. However, serious consequences of rubella infection can result when the infection occurs during the early stages of pregnancy. After the occurrence of a rubella outbreak in Japan that was observed from 2012 to 2013, 45 infants were reportedly born with congenital rubella syndrome (CRS). We prospectively followed the 15 CRS cases reported in Tokyo to determine the virus shedding periods by using nested reverse transcriptase-polymerase chain reaction to detect rubella virus genes. Throast swabs were used for virus detection. The virus shedding period was measured from birth until the time when the sample last tested positive followed by 2 consecutive negative samples. Kaplan-Meier method was used to estimate the proportion of cases remaining positive for rubella virus genes over time. The proportion of CRS cases shedding virus dropped steadily after birth, dropping to 33.8% at 6 months and 16.9% at 12 months. Our findings also suggested that the earlier the mother's onset of rubella during pregnancy, the longer the infant remained positive. Based on our findings, we believe that infants with CRS should be monitored for rubella virus shedding until 1 year of age.

Occult hepatitis C virus infection in Iranian hemodialysis patients.

Background: Occult hepatitis C virus (HCV) infection is defined as the presence of HCV-RNA in liver or peripheral blood mononuclear cells (PBMCs) in the absence of detectable hepatitis C antibody (anti-HCV) or HCV-RNA in the serum. Low concentrations of HCV-RNA may be detected in PBMCs of hemodialysis (HD) patients and this could have a great impact on the management of HD patients. Objectives: The aim of this study was to detect the occult HCV infection in Iranian HD patients. Patients and Methods: A total of 70 anti-HCV negative HD patients from three dialysis units in Tehran, Iran were included in this study. In these cases, presence of HCV-RNA in plasma samples was tested by reverse transcriptase-nested polymerase chain reaction (RT-nested PCR). In cases with negative anti-HCV and plasma HCV-RNA, genomic HCV-RNA was checked in PBMC specimens by RT-nested PCR. Results: Seventy anti-HCV negative HD patients were enrolled in the study. 32.85% and 1.43% of cases had elevated levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) respectively. 7.14% of patients had elevated levels of both ALT and AST. HCV-RNA was negative in plasma samples of all anti-HCV negative HD subjects. The genomic HCV-RNA was not detected in any PBMC samples of HD cases with negative anti-HCV and plasma HCV-RNA. Conclusions: Occult HCV infection was not detected in our HD patients despite of elevated levels of liver enzymes in some participants. Further studies involving larger number of HD patients are required to elucidate the rate of occult HCV infection in HD cases.

Emergence of dengue in tribal villages of Mandla district, Madhya Pradesh, India.

Background & objectives:Dengue (DEN) is a rapidly spreading arboviral disease transmitted by Aedes mosquitoes. Although it is endemic in India, dengue virus (DENV) infection has not been reported from tribal areas of Madhya Pradesh. Investigations were conducted to establish the aetiology of sudden upsurge of cases with febrile illness in June 2013 from tribal villages of Mandla district of Madhya Pradesh, India.Methods:The rapid response team of the National Institute for Research in Tribal Health, Jabalpur, conducted clinical investigations and field surveys to collect the samples from suspected cases. Samples were tested using molecular and serological tools. Collected mosquitoes were identified and tested for the presence of virus using semi nested reverse transcriptase-polymerase chain reaction (nRT-PCR). The sequences were analysed to identify serotype and genotype of the virus.Results:Of the 648 samples collected from 18 villages of Mandla, 321 (49.53%) were found to be positive for dengue. The nRT-PCR and sequencing confirmed the aetiology as dengue virus type 2. Eighteen per cent of patients needed hospitalization and five deaths were attributed to dengue. The virus was also detected from Aedes aegypti mosquito, which was incriminated as a vector. Phylogenetic analysis revealed that the dengue virus 2 detected belonged to cosmopolitan genotype of the virus.Interpretation & conclusions:Dengue virus serotype 2 was detected as the aetiological agent in the outbreak in tribal villages of Mandla district of Madhya Pradesh. Conducive man-made environment favouring mosquitogenic conditions and seeding of virus could be the probable reasons for this outbreak. Urgent attention is needed to control this new threat to tribal population, which is already overburdened with other vector borne diseases.

Prevalence of hepatitis C virus genotypes in district bannu, khyber pakhtunkhwa, pakistan.

Determination of an individual's hepatitis C virus (HCV) genotypes prior to antiviral therapy has become increasingly important for the clinical management and prognosis of HCV infection. Therefore, this study was conducted to investigate the prevalence of HCV genotypes in HCV infected patients of district Bannu in Khyber Pakhtunkhwa region of Pakistan. Serum samples of 117 seropositive patients were screened for HCV-RNA by using reverse transcriptase-nested polymerase chain reaction (RT-nested PCR) and then PCR positive samples were subjected to HCV genotyping. Out of 117 seropositive samples, 110 samples were found positive by PCR analysis. Genotype 3a was the most prevalent one detected in 38% of patients, followed by genotype 3b in 21% of patients, and then genotype 2a in 12% of patients. However 21% of HCV-PCR positive samples could not be genotyped by method used in this study. Genotype 3a was the most prevalent genotype in patients of all age groups and its prevalence was found high among patients with increasing age (>34 years). Moreover, genotypes 3a and 3b were found to be the most prevalent genotypes in patients with history of shaving by barbers, receiving multiple injections, and dental procedures. In conclusion there is need of further investigation of genotypes of HCV by using more sensitive assays and considering large sample size in district Bannu.

A novel nested reverse-transcriptase polymerase chain reaction method for rapid hepatitis C virus detection and genotyping

Purpose: Rapid and specific detection of viral nucleic acid is increasingly important in the diagnosis of infectious diseases. The objective was to develop a rapid, efficient process of nucleic acid based detection of hepatitis C virus (HCV) infection for its diagnosis and treatment follow-up. Materials and Methods: A two-step nested reverse-transcriptase polymerase chain reaction (RT-PCR) has been standardised on a sample set of 125 individuals from different liver clinics in Kolkata. The method utilises a novel fast nested RT-PCR for HCV detection and genotyping from HCV infected patient plasma with high processivity. Results: The overall time required from ribonucleic acid (RNA) isolation to nested PCR amplified product detection is reduced to 42% when compared with conventional nested RT-PCR amplification. The method is sensitive as conventional PCR and detected all HCV RNA positive samples. Sequencing, phylogenetic analysis of the PCR amplified product by this method showed concordant genotypes with conventional PCR. Conclusion: Though being a two-step process, this method is fast, cost-efficient, reliable and feasible for regular HCV RNA screening and apt even in resource limited settings. This method could be translated to regular nucleic acid screening for other infectious diseases as regular PCR regimen.

An outbreak of West Nile Virus infection in the region of Monastir, Tunisia, 2003

Background:A West Nile (WN) fever epidemic occurred in the region of Monastir, Tunisia, between August and October 2003.Aim of the study:We attempt to describe the epidemiology, clinical presentation, and outcome of patients with confirmed West Nile virus (WNV) infection.Methods:Three groups of specimens were prepared. One was made up of serum only (n = 43), the other of cerebrospinal fluid (CSF) only (n = 30), and the third group was made up of both (n = 40). These specimens were obtained from 113 patients. A serological diagnosis and evidence of WNV genome by nested reverse-transcriptase polymerase chain reaction (nRT-PCR) and TaqMan reverse transcription-polymerase chain reaction (RT-PCR) were carried out.Results:Thirty-eight cases (33.6%) were serologically positive. Results of nRT-PCR showed a total of 10 positive cases of WNV (8.8%) detected in group 1 (n = 1/43), group 2 (n = 5/30), and group 3 (n = 4/40) whereas the PCR TaqMan showed 18 positive samples (15.9%) found in group 1 (n = 3/43), group 2 (n = 9/30), and group 3 (n = 6/40). All TaqMan PCR positive cases were nRT-PCR positive. In addition, four serologically probable cases were confirmed by TaqMan PCR. The attempts to isolate WNV by cell culture were unsuccessful. Considering the results of TaqMan assay and the serological diagnosis, WNV infection was confirmed in a total of 42 patients. The main clinical presentations were meningoencephalitis (40%), febrile disease (95%), and meningitis (36%). Eight patients (19%) died. The highest case-fatality rates occurred among patients aged ≧55 years. The phylogenetic analysis revealed that isolates of WNV were closely related to the Tunisian strain 1997 (PAH001) and the Israeli one (Is-98).Conclusions:West Nile virus is a reemerging global pathogen that remains an important public health challenge in the next decade.

Distribution of Hepatitis C Virus Genotypes Among Patients With Hepatitis C Virus Infection in Hormozgan, Iran

BackgroundMore than 170 million people in the world are infected with Hepatitis C virus (HCV). Determination of HCV genotype before starting the treatment is required, because HCV genotype affects the course of treatment and drug dosageObjectivesWe aimed to evaluate HCV genotypes among patients with positive results for anti-HCV in Bandar Abbas from 2011 to 2012.Patients and MethodsFive hundred and nine consecutive patients with established chronic HCV infection referred to Behavioral Diseases Consultation Center, Blood Transfusion and Center for Special Diseases from March 2011 to March 2012 were enrolled in this cross sectional study. Five mL of peripheral blood was taken from precipitants and viral RNA extracted after plasma separation. Hepatitis C virus RNA was detected by reverse transcriptase-nested polymerase chain reaction (RT-nested PCR) assay and then HCV genotypes analyzed using restriction fragment length polymorphism (RFLP) method.ResultsIn overall, 509 patients enrolled to this study. The mean age of these patients was 38.87 ± 9.55 years ranging from 1 to 90 years. Routs of transmission were: 238 (46.7%) inject of substance, 149 (29.3%) unknown rout, 62 (12.2%) blood transfusion, 50 (9.8%) sexual contact, and 10 (2%) mother to child. Frequency of HCV genotypes were: 316 (62.1%) 1a, 117 (23%) 1b, and 76 (14.9%) 3a. there was no significant association between HCV genotypes and gender, educational degree, risk factor of Hepatitis C, job, monthly income, HIV infection, Hepatitis B virus (HBV) infection, Intravenous drug injection, and underlying disease (P > 0.05).ConclusionsThis results the same as many similar studies demonstrated that common HCV genotypes in Iranian patients were 1a, 3a and 1b, respectively. Patients with 1a and 1b genotypes have lower responses to interferon treatment, and it is reasonable to perform early screening to diagnose and determine HCV genotype for effective treatment and diagnose high-risk cases.

English

This study was carried out to evaluate the diagnostic value of nested reverse transcriptase-polymerase chain reaction (RT-PCR) for HEV (hepatitis E virus) RNA detection relative to anti-HEV immunoglobulin G (IgG) and IgM enzyme-linked immunosorbent assays. One hundred and twenty six (126) patients with sporadic acute hepatitis E were included in this study. We focused on the popular genotype I and IV of HEV-positive patients in China, and selected the conserved region located in ORF2 for designing a set of nested RT-PCR primers. HEV RNA was detected in 53.2% (67/126) of patients, while all control subjects were negative for HEV RNA. The total agreement between IgM and nested RT-PCR detection was 80.9%, showing a fine coincidence. The results further suggested that there was a significant difference between nested RT-PCR detection and IgM ELISA: 3 cases with positive results for HEV RNA showed negative anti-HEV IgM at the early phrase, and presented positive IgM reaction in succession after the trail of detection. HEV RNA was detected in serum samples from sporadic acute hepatitis patient usually by day 1 to 12 after the onset of symptoms, but showed a decreasing sensitivity with the increasing disease course. From these experiments, we can conclude that HEV RNA detection is of great clinical significance, which has an obvious advantage in diagnosis of early infection of HEV. Key words: Hepatitis E virus, nested reverse transcriptase-polymerase chain reaction (RT-PCR), RNA, antibody.