A complete molecular biology assay for hepatitis C virus detection, quantification and genotyping

Casanova, Yara Silva; Boeira, Thaís da Rocha; Sisti, Elisa; Celmer, Álvaro José; Fonseca, André Salvador Kazantzi; Ikuta, Nilo; Simon, Daniel; Lunge, Vagner Ricardo

doi:10.1590/0037-8682-0040-2014

Cited by 10 publications

(4 citation statements)

References 29 publications

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“…6,22 We found more number (63.4%) of HCV-positive pediatric cases in the age group of >10 years that is similar to a work performed in Brazil. 23 Age has been argued to be a major factor in HCV studies, with infection more predominant in older people. 24 Our study reports 79.3% prevalence of genotype 3a, 11.1% 3b and 9.6% type 1a.…”

Section: Discussionmentioning

confidence: 99%

Prevalence, Genotypic Distribution and the associated Risk Factors of Hepatitis C Infection in Pakistan Pediatric Patients

Numan¹,

Jabbar²,

Zafar³

et al. 2022

J. Pure Appl. Microbiol.

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Hepatitis C virus (HCV) is an important contributor to chronic morbidity and mortality in developing countries. The study’s objective was to determine the genotype distribution and risk factors associated with the transmission of HCV infections in pediatric patients. Rapid screening and confirmation by the enzyme-linked immunosorbent assay (ELISA) were used to analyze 585 pediatric blood specimens hospitalized and visited the outpatient department of the largest tertiary care hospital in Pakistan. Detection and genotyping of HCV RNA were performed using a real-time polymerase chain reaction (RT-PCR). Demographic data and a history of risk factors were gathered through a survey questionnaire. HCV RNA was detected in 323 (72.4%) cases which showed viral load ranging from Log10 IU/mL < 3 to > 6 IU/mL. HCV genotype 3a was detected in 256 (79.3%) cases while type 3b and 1a was observed in 36 (11.1%) and 31 (9.6%) patients, respectively. HCV positivity was significantly associated with the cases from rural areas [p = 0.005; odds ratio (OR) 1.65; 95% CI 1.16-2.23] and also significantly associated with low-income group [p < 0.001; OR 5.75; 95% CI 3.90-8.40]. The primary risk factors associated with HCV transmission in children were family history (p = 0.002), blood transfusion (p = 0.03), surgical procedures (p = 0.02), and history of injections (p = 0.05). HCV genotype 3a is the most common genotype in children. The main risk factors for HCV transmission in children are blood transfusion, surgical procedures, and injection practices by informal health care providers.

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Section: Discussionmentioning

confidence: 99%

Prevalence, Genotypic Distribution and the associated Risk Factors of Hepatitis C Infection in Pakistan Pediatric Patients

Numan¹,

Jabbar²,

Zafar³

et al. 2022

J. Pure Appl. Microbiol.

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“…Then, they were reversely transcribed into cDNA with an RNA reverse transcription kit (PrimeScript™ RT reagent Kit with gDNA Eraser, Takara Bio Inc., Dalian, China). HCV RNA was detected through nested real-time PCR ( Casanova et al, 2014 ).…”

Section: Methodsmentioning

confidence: 99%

CpG Island Methylation of Suppressor of Cytokine Signaling-1 Gene Induced by HCV Is Associated With HCV-Related Hepatocellular Carcinoma

Liu

Xing

et al. 2022

Front. Microbiol.

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Suppressor of cytokine signaling 1 (SOCS-1) is implicated in both virus infection and carcinogenesis. This study investigated the role of HCV infection on SOCS-1 in normal and HCV-infected tissues and revealed a possible mechanism underlying HCV-induced hepatocellular carcinoma (HCC) genesis. In total, 10 HCV-HCC tissues, seven adjacent tissues, seven distal tissues, and 16 normal liver tissues were collected. SOCS-1 expression in tissue sections was detected by immunohistochemistry. After viral load was quantified, the correlation between SOCS-1 expression and viral load was analyzed in different tissues. Then, HCV replicon model was used to detect a relationship between HCV and SOCS-1. Subsequently, methylation-specific PCR (MSP) was applied to show the methylation status of SOCS-1 genes in normal tissues and HCV-replicating cell lines. A correlation between gene methylation, SOCS-1 expression, and HCV was analyzed. The lowest expression of SOCS-1 was observed in HCV-HCC tissues. Tissues with a higher HCV viral load showed lower SOCS-1 expression (p = 0.0282). Consistently, SOCS-1 mRNA and protein were lower in HCV-replicating cell lines than in uninfected ones. Furthermore, gene methylation was found in all examined tissues but higher in HCC tissues, and it is positively correlated with HCV viral load (r2 = 0.7309, p < 0.0001). HCV infection would upregulate methylation of the SOCS-1 gene in HCV-replicating cell lines. The downregulation of SOCS-1 in normal and HCV-replicating cell lines may result from HCV infection through epigenetic regulation, in which gene methylation in the CpG island of SOCS-1 promoters upon HCV infection suppresses its expression.

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“…This high-grade conservation makes the 5ʹ UTR the region of choice for performing (RT)-PCR detection tests, such as the HCV amplicor test 12 . A number of genotyping schemes have been established and utilized in this region to obtain phylogenetic genotype information 13 , 14 . Sequencing data of the 5ʹ UTR and other regions, such as the NS-3, NS-4, core and NS-5, have been used in phylogenetic studies and genotyping of HCV 15 – 17 .…”

Section: Introductionmentioning

confidence: 99%

Phylogenetic analysis of the 5ʹ untranslated region of HCV from cirrhotic patients in Khyber Pakhtunkhwa, Pakistan

Ullah

Rehman

Ahmad

et al. 2021

Sci Rep

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Hepatitis C virus (HCV), a small, single-stranded RNA virus with a 9.6 kb genome, is one of the most common causes of liver diseases. Sequencing of the 5ʹ untranslated region (UTR) is usually used for HCV genotyping, but it is less important in numerous subtypes due to its scarce sequence variations. This study aimed to identify genotypes using the 5ʹ UTR of HCV from cirrhotic patients of Khyber Pakhtunkhwa (KP). Serum RNA samples (44) were screened by real time PCR to determine the HCV viral load. Nested PCR was performed to identify cDNA and the 5ʹ UTR. The HCV 5′ UTR was sequenced using the Sanger method. MEGA-7 software was used to analyze evolutionary relatedness. After 5ʹ UTR sequencing, 26 samples (59%) were identified as genotype 3, and 2 samples (6%) were identified as genotypes 1, 2 and 4. The most predominant genotype was 3a, and genotype 4 was rarely reported in the phylogenetic tree. Analysis of the HCV 5ʹ UTR is an efficient alternative method for confirmation of various genotypes. Phylogenetic analysis showed that genotype 3 was dominant in the area of KP, Pakistan.

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A complete molecular biology assay for hepatitis C virus detection, quantification and genotyping

Cited by 10 publications

References 29 publications

Prevalence, Genotypic Distribution and the associated Risk Factors of Hepatitis C Infection in Pakistan Pediatric Patients

Prevalence, Genotypic Distribution and the associated Risk Factors of Hepatitis C Infection in Pakistan Pediatric Patients

CpG Island Methylation of Suppressor of Cytokine Signaling-1 Gene Induced by HCV Is Associated With HCV-Related Hepatocellular Carcinoma

Phylogenetic analysis of the 5ʹ untranslated region of HCV from cirrhotic patients in Khyber Pakhtunkhwa, Pakistan

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