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Objective:To investigate the prevalence of pretreatment drug resistance and the genetic polymorphism of CRF08_BC strains among HIV-1 patients in China.Methods:This cross-sectional survey involved the plasma samples of HIV patients in a national pretreatment HIV drug resistance survey conducted in 2018. RNA was extracted from the samples. The fragments containing protease and partial reverse transcriptase (PR/RT) regions were obtained and sequenced. Drug resistance was analyzed using Stanford HIVdb Program. Differences in polymorphic mutations between drug-resistant and non-drug-resistant HIV-1 strains were analyzed by Chi-square test or Fisher′s exact test. The association between drug-resistant and polymorphic mutations was evaluated using CorMut R package. Molecular transmission networks were constructed using HIV-TRACE software. Results:Totally 465 partial pol sequences were obtained from individuals with CRF08_BC infection in 25 provinces and cities. The total pretreatment drug resistance rate was 17.8% (83/465). The pretreatment drug resistance rates to non-nucleoside reverse transcriptase inhibitors (NNRTIs), nucleoside reverse transcriptase inhibitors (NRTIs) and protease inhibitors (PIs) were 16.6% (77/465), 1.1% (5/465) and 0.9% (4/465), respectively. The resistance rate to rilpivirine (RPV) was the highest (15.7%, 73/465). The most common mutation was E138A (11.6%, 54/465). There were six polymorphic mutations (S162C, K102Q, T200A, V179E, I202V, T200M) that co-variated with E138A. The molecular transmission network showed that patients infected with CRF08_BC strains carrying the resistant mutations at position E138 mainly gathered in clusters in Yunnan and Sichuan, and the highest degree of connection was in Lincang, Yunnan. Conclusions:In China, HIV-1 CRF08_BC-infected patients showed a high rate of pretreatment resistance to one of the second-generation NNRTIs, namely RPV. Further researches were warranted to evaluate the impacts of co-mutations of the E138A mutation and polymorphic sites on HIV resistance and replicative capacity.
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Objective @#To learn the characteristics of second-line drug resistance and related gene mutations of multidrug-resistant Mycobacterium tuberculosis ( MDR-TB ) Beijing genotype strains. @*Methods@#The MDR-TB isolates in Hwa Mei Hospital from 2017 to 2019 were enrolled and detected using RD105 deletion-targeted multiplex polymerase chain reaction (PCR). The proportion method for drug susceptibility test was used to detect the drug-resistant profiles against kanamycin, amikacin, capreomycin, ofloxacin and levofloxacin. The gene sequencing of rrs, tlyA, eis, gidB, gyrA and gyrB was conducted by PCR compared with H37RV strain. The differences in the rates of drug resistance and mutation between Beijing and non-Beijing genotype strains were examined to understand the characteristics of Beijing genotype strains. @*Results@#There were 106 Beijing genotype and 27 non-Beijing genotype strains in 133 MDR-TB isolates. The drug resistance rates of kanamycin, amikacin, capreomycin, ofloxacin and levofloxacin in Beijing genotype strains were 9.43%, 7.55%, 3.77%, 32.08% and 32.08%, respectively. The rates of quasi-extensive and extensive drug resistance in Beijing genotype strains were 30.19% and 7.55%. The gene mutation rates of rrs, tlyA, eis, gidB, gyrA and gyrB in Beijing genotype strains were 7.55%, 7.55%, 1.89%, 2.83%, 36.79% and 2.83%, respectively. There were no significantly differences between Beijing and Non-Beijing genotype strains in the factors above ( P>0.05 ). The gene rrs, tlyA, eis, gidB, gyrA and gyrB had 2, 1, 2, 2, 5 and 3 mutation types, respectively, with single base substitution as the main type. @*Conclusion@#Beijing genotype strains are dominant in MDR-TB, with high resistance to fluoroquinolones and mainly gyrA gene mutation.
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Objective To analyze the clinical occurrence and phenotypic characteristics of rtA181S-related novel multidrug-resistance mutation in reverse transcription region of hepatitis B virus (HBV). Methods The clinical data and sequence information of 16 443 patients with chronic HBV infection who received nucleos(t)ide analogues (NAs)-resistance testing at the original PLA 302 Hospital from 2012 to 2017 were retrospectively analyzed. rtA181S-related mutation patterns were analyzed and cloning-sequencing (≥20 clones/sample) was performed on the mutation samples of rtA181S+T184I+M204I with the highest detection rate. Phenotypic analysis was performed to evaluate the viral replication capacity and drug susceptibility. Results The rtA181S mutation was detected in 0.75% (124/16 443) of the patients. Among them, 21 patients were detected with coexistence of lamivudine (LAM)-resistance mutation and 74 patients were detected with coexistence of entecavir-resistance (ETVr) mutation. The rtA181S+T184I+M204I novel mutation accounted for 77.0% (57/74) of the rtA181S+ETVr mutation. Dynamic clinical data analysis showed that the rtA181S+T184I+M204I mutation emerged after adefovir dipivoxil (ADV), ETV, and LAM/telbivudine (LdT) treatment, accompanied by virological breakthrough or inadequate virological response. Compared to wild-type strain, rtA181S+T184I+M204I mutant had 53.7% decreased replication capacity, over 1000-, 3.9-, and 383.3-fold increased LAM, ADV, and ETV resistance, respectively, and remained sensitive to tenofovir (TDF). Conclusions rtA181S+T184I+M204I mutation is a novel multidrug-resistance mutation, which is related to the ADV, ETV or LAM/LdT sequential or combined treatment. TDF-based rescue therapy should be considered for patients harboring this mutation in clinical practice.
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Objective To analyze HIV-1 drug resistance gene mutations in AIDS patients who failed first-line antiviral therapy in Hubei Province from 2017 to 2018, and to provide references for clinical treatment. Methods Plasma samples of HIV patients who had received first-line antiviral treatment for more than 12 months and had a viral load greater than 103 copies / ml were collected in Hubei Province, and drug resistance genotypes were detected. The prevalence and characteristics of drug resistance were analyzed. Results A total of 198 patients were selected, and 182 target gene sequences were successfully detected. The gene subtypes were mainly CRF01_AE, with a total drug resistance rate of 69.23%. The proportion of NRTIs, NNRTIs and PIs resistance mutations was 46.15%, 65.38% and 0.55%, respectively. The occurrence of cross resistance mutations of NRTIs and NNRTIs reached 40.66%. The mutation sites related to NRTIs were mainly M184V and K65R, while the mutation sites related to NNRTIs were mainly V179D, K103N and Y181C. There was only one case of PIs related mutation at the site of M46I. Conclusion HIV-1 genotyping demonstrated a high proportion of drug resistance in the HAART failure population in Hubei Province, and multi-drug resistance occurred frequently. It is necessary to strengthen the monitoring of drug resistance, implement timely adjustments to antiviral treatment programs, and reduce the occurrence and spread of drug-resistant strains.
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Objective To investigate the relationship between HBV -DNA load and serum markers in chronic hepatitis B( CHB) patients in Hohhot,Inner Mongolia,and to explore the mutation of HBV genotype and nucleoside analogue.Methods From January 2015 to December 2017,one hundred and ninety-three CHB patients hospitalized in the People＇s Hospital of Inner Mongolia were selected randomly.The clinical diagnostic criteria for all admitted patients were based on the " Guidelines for the Prevention and Treatment of Chronic Hepatitis B" jointly formulated by the Infectious Diseases Society of 2010. The HBV -DNA load of HBV was detected by real -time quantitative PCR,and the correlation between HBV -DNA load and serum markers was analyzed. Seventy -nine patients were selected from 193 hospitalized patients,PCR-reverse dot blot hybridization was used to analyze HBV genotyping and the drug resistance mutations of different genotypes.Results The differences of HBeAb level and HBV-DNA load between HBeAg positive patients and negative patients were statistically significant(all P<0.001). Of 79 serum specimens of HBV infected people,9 cases(11.4% ) were B genotypes,and 70 cases of C genotype (88.6% ).Of them,25 cases had different loci variation,the rate of variation was 31.6% (25/79),with the unit point rtS213T mutation dominated,accounting for about 24.0% (6/25).Conclusion In Hohhot Inner Mongolia patients with CHB,HBV-DNA load with HBeAg and HBe Ab level are correlated;genotype in patients including B type and C type,which is mainly genotype C;patients with CHB mainly had drug resistance to lamivudine and adefovir dipivoxil, mutations including rtS213T,and hybrid mutation.
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ObjectiveThe genotypes and drug-resistance mutation of hepatitis B virus (HBV) are closely related to the progression of chronic hepatitis B (CHB) and effects of medication. This study was to identify the genotypes and drug-resistance mutation genes of HBV in West Guangxi and provide some laboratory evidence for anti-HBV treatment.MethodsWe collected serum samples from 869 CHB patients with HBV DNA >1.0×103 IU/mL, all from West Guangxi, between January 2016 and March 2018. We detected the genotypes and drug-resistance mutation genes in the patients by PCR and reverse dot blot (PCR-RDB).ResultsA total of 7 genotypes were identified in the patients, including genotypes B in 304 cases (34.98%), C in 268 (30.84%), D in 147 (16.92%), B+C in 28 (3.22%), B+D in 22 (2.53%), C+D in 1 (0.12%) and B+C+D in 1 (0.12%). Drug-resistance mutation was observed in 63 cases (7.25%), including 31 cases with genotype C (49.21%) and 19 with genotype B (30.16%). Fourteen gene mutation patterns were found in all, including rt204I, rt181V, rt236T, rt180M+rt204V, and rt180M+rt204V+rt204I, among which, rt180M+rt204V exhibited the highest mutation frequency (17.46%). There was a statistically significant difference between the mutation rates of genotypes B and C (19% vs 31%, P < 0.05).ConclusionCHB patients in West Guangxi have a wide variety of genotypes of HBV, mainly including genotypes B and C, with a high rate and complicated patterns of drug-resistance mutation, which has provided some reference for anti-HBV treatment in West Guangxi.
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Objective To analyze the heterogeneities of hepatitis B virus ( HBV ) reverse transcriptase domain (RT) gene mutations related to nucleos (t)ide analogues (NAs) resistance.Methods Blood samples from 2765 chronic hepatitis B patients with virological breakthrough or poor drug response treated in Ningbo No .2 Hospital and Ningbo Fourth Hospital from April 2011 to March 2018 were collected . According to the medication status , it was divided into LAM monotherapy group ( n =603 ) , LdT monotherapy group (n=147), ADV monotherapy group (n=68), ETV monotherapy group (n=10) and the sequential or combined drug resistance of NAs group (n=365).The resistance mutation sites and drug resistance patterns (pathways) of each group were analyzed .The SPSS 19.0 software was used to analyze the data.Results Among 2765 serum samples, the NAs-related HBV-RT resistance mutations were detected in 1193 cases with an overall mutation rate of 43.15％.The mutation rate of LAM monoclonal resistance group was 62.62％ (603/963) with 19 mutation types, the most common single point mutation was rtM204I/V (40.30％, 243/603).The mutation rate of LdT monoclonal resistance group was 45.51％(147/323), and there were 3 mutation types, with the single point mutation rtM204I/V being the most common (59.86％, 88/147).The mutation rate of the ADV monoclonal resistance group was 17.80％(68/382), mainly rtA181T single point mutation (64.71％, 44/68).The mutation rate of the ETV monoclonal resistance group was 4.06％(10/246), and the single point mutation of rtT184A/G/S/I/L/F was the most common one (80.00％, 8/10).The mutation rate of the sequential or combination therapy group was 41.91％ (365/871), among which the mutation rate of the LAM/LdT poor response or the resistance with the sequential ADV group was 63.39％(142/224), and the most single mutation point was rtA181V/T ( 35.21％, 50/142 );the mutation rate of LAM/LdT poor response or drug-resistant with combined ADV group was 42.19％ (54/128), and the most common mutation point was rtA181V/T (46.30％, 25/54);the mutation rate of LAM/LdT with poor response or resistance after sequential ETV 1.0 mg was 44.66％(117/262), and the most common mutation point was rtL180M+M204I/V+S202G/I (31.62％, 37/117);the LAM/LdT poor response or the drug-resistant ETV combined with ADV group had a mutation rate of 7.14％(5/70), all of which were multi-site mutations;the mutation rate of poor response to ADV or resistant with sequential ETV 0.5 mg group was 28.14％(47/167), all of which were multi-site mutations.Secondary ( compensation ) sites such as rtV173L, rtL180M, and rtV214A, and single-point mutations such as rtV207I/L/G, rtS213Tand rtN238T, which were not fully defined , were detected.The resistance patterns ( pathways ) of NAs monotherapy were relatively simple , and the resistance patterns ( pathways ) of NAs experienced patients ( sequential or combined treatment group ) were complex and diverse, and multiple resistance patterns (pathways) existed, along with NAs increasing in species.Non-first-line NAs-related resistance patterns ( pathways ) showed an overall downward trend sand ETV-related drug-resistant mutation showed an overall upward trend .Conclusion The NAs-related HBV resistance mutation sites ( patterns ) are complex and diverse , especially multi-site mutations , refractory drug resistance mutations, multidrug resistance mutations and cross-resistance mutations.Therefore, the optimization of antiviral treatment strategies and drug resistance management concepts need to be continuously updated .
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Objective@#To understand the incidence and related factors of HIV-1 drug resistance among HIV/AIDS patients experiencing treatment failure in Jiangsu province, China.@*Methods@#The HIV/AIDS integrated prevention and control data information management system of China were used to collect the basic data of patients, blood specimens were collected from patients who had antiretroviral therapy (ART) failure with ≥12 months and older than 18 years in 2016 in Jiangsu, excluding cases with missing information, 713 cases were enrolled in this study. HIV-1 RNA was extracted, and then pol gene region was amplified and sequenced. The obtain sequences were submitted to Stanford University HIV Drug Resistance Database to interpret and analyse HIV-1 drug resistance and sub-types. Multivariate logistic regression model was used to explore the related factors of drug resistance.@*Results@#A total of 579 subjects were amplified successfully, male accounted for 85.66% (496 cases), and the median age was 39 years old. The main route of infection was sexual transmission (553 cases, 95.51%). A total of 331 patients with drug resistance gene mutation were detected, drug resistance mutation rate was 57.18%. Compared with patients with baseline CD4+T cell count >500 cells/μl, patients with CD4+T cell count in 201-500 cells/mm3 and ≤200 cells/μl had a higher incidence of genetic drug resistance, the odds ratio was 3.33 and 6.87, respectively. Compared with patients with treatment less than 24 months, patients treated for 25-48 months had a higher incidence of drug resistance, the odds ratio was 1.88. Compared with patients infected by CRF07_BC strains, patients infected by CRF01_AE strains were associated with higher incidence of drug resistance, the odds ratio was 2.22 and 3.32, respectively. Protease inhibitor (PI) resistance mutations, nucleoside reverse transcriptase inhibitor (NRTI) and non-nucleoside reverse transcriptase inhibitor (NNRTI) were found in 3.80%, 33.16% and 53.37% of patients, respectively. 31.95% of patients harbored NRTI and NNRTI resistance mutations simultaneously. M184V/I and K103N/Q were the highest frequency of NRTI and NNRTI resistance mutation, the prevalence of M184V/I and K103N/Q were 28.15% and 22.28%, respectively.@*Conclusion@#The status of HIV-1 drug resistance mutations are complex and diverse among patients experiencing failure of ART in Jiangsu. Patients with lower baseline CD4+T cell count, longer treatment time and HIV-1 CRF01_AE and B strains infection were associated with higher incidence of drug resistance mutation.
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Objective To investigate the mutation characteristics and genotype of hepatitis B virus resistance gene in Nantong area,and provide scientific basis for clinical rational drug.Methods A total of 158 cases of chronic hepatitis B (CHB)pa-tients who were received with nucleos (T)ide analogues therapy for at least 2 years as the research object,and 30 cases of CHB patients who were not received nucleos (T)ide analogues for the treatment as the control group.PCR-sequencing method was used to detect the HBV P resistant gene and genotype,meanwhile,observe the relationship between three main mutation model and the levels of ALT and HBV DNA was also investigated.Results B genotype was detected in 42 (26.58%)out of 158 CHB patients,and 116 cases (73.42%)were C genotype.A total of 131 patients with different site mutations in P region,the mutation rate were 82.91%.There were totally 11 HBV mutation sites,including the main muta-tion site:M204I,L180M,M204V,A181V and A181T,the frequency of drug resistance were 41.14%,37.34%,22.15%, 11.39% and 10.13%,respectively.Moreover,11 mutation sites had 21 mutation patterns.In lamivudine (LAM)resistance associated mutations,the L180M and M204V sites were mainly co-occurrence,followed by M204I alone.In adefovir dipivoxil (ADV)resistance associated mutations,A181V was the main mutation site.Whereas,the drug resistance rate of entecavir (ETV)was low.Conclusion The main genotypes of HBV were type B and C in Nantong area,and C type was the dominant genotype.The resistance mutations mainly concentrated in LAM and ADV resistance associated mutations,while the resist-ance rate of ETV was low.Multi-locus drug-resistant mutation detection may help to detect viral resistance and guide clinical treatment better.
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Objective To explore correlations between hepatitis B virus (HBV)genotyping,other clinical information and drug resistance mutations.Methods 358 cases of patients with chronic hepatitis B (CHB)were selected as subjects,and the resistance loci and genotypes were detected by using the polymerase chain reaction(PCR)-reverse dot blot technique.Clinical data,such as ser-um HBV DNA loads,serum levels of alanine aminotransferase(ALT)and hepatitis B virus e antigen(HBeAg),gender,age and length of nucleoside analogues use were collected.Results All samples were successfully amplified positive band.Type B(267 ca-ses)was the main HBV genotype,followed by type C(81 cases)and type D(10 cases).In the 31 1 cases of patients taking nucleoside analogues,269 cases were completely wild type.No drug resistance mutation was found in 47 cases of patients not taking medicine. The drug resistance mutations mainly occured in 204 and 180/204 site.There was no significant correlation between resistance mu-tations and gender,age,serum HBV DNA loads,genotype,serum levels of ALT and HBeAg(P >0.05).While the medication time was longer,the incidence of resistant mutants was greater(P <0.05).The 180 mutation had a certain correlation with 204 site mu-tation(P <0.05).Conclusion PCR-reverse dot blot technology can effectively detect the HBV genotype and mutations,which could effectively guide the clinical medication.
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HIV-1 gp41 is an envelope protein that plays an essential role in virus entry. The mutation of gp41 affects HIV-1 entry and susceptibility to the fusion inhibitor T-20. Therefore, we analyzed the natural polymorphism of gp41 of 163 HIV-1 isolates from T-20-naive Koreans infected with HIV-1. This study of gp41 polymorphisms showed that insertions in the fourth threonine (74.8%) and L7M substitutions (85.3%) were more frequent in the fusion peptide motif in Korean HIV-1 isolates compared with those from other countries. Minor T-20 resistance mutations such as L45M (1.2%), N126K (1.2%), and E137K (6.7%) were detected, but the critical T-20 resistance mutations were not detected in the gp41 HR1 and HR2 regions. In addition, the N42S mutation (12.9%) associated with T-20 hypersusceptibility was detected at a high frequency. These results may serve as useful data for studies considering T-20 for use in the development of a more effective anti-retroviral treatment in Korea.
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Humans , Anti-HIV Agents/pharmacology , Drug Resistance, Viral/genetics , HIV Envelope Protein gp41/genetics , HIV Infections/virology , HIV-1/genetics , Peptide Fragments/pharmacology , Polymorphism, Genetic , Protein Structure, Tertiary/genetics , Republic of Korea , Virus InternalizationABSTRACT
Objective To understand the hepatitis B virus(HBV)genotyping and pre-existing antiviral resistance mutation in the patients with clinical chronic HBV infection in Shenzhen area.Methods Serum samples in 244 cases of chronic HBV infection were detected for HBV genotyping and pre-existing antiviral resistance mutation by using PCR combined with reverse dot hybridization. Results In 244 cases of chronic HBV infection,3 kinds of genotype were found,including 143 cases(58.6%)of genotype B,100 ca-ses(41%)of genotype C and 1 case(0.4%)of genotype D;38 cases(15.57%)of pre-existing antiviral resistance mutation were de-tected out.The total detection rate of pre-existing antiviral resistance mutation was 15.57%,in which 22 cases(9.01%)were related with lamivudine resistance and 16 cases(6.56%)were related with adefovir dipivoxil resistance.Conclusion The genotype B and C are the main genotypes in Shenzhen area,and the incidence rate of pre-existing antiviral resistance mutation in the patients with chronic HBV infection is relatively high.The detection of genotyping and pre-existing antiviral resistance mutation of HBV has the important significance to predict the disease progression and guide the individulized treatment.
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Objective To investigate the distribution characteristics of hepatitis B virus (HBV) genotypes and the variation situ-ation of drug resistance gene in Shenyang area by the gene chip technology .Methods Serum samples were randomly collected from 90 inpatients of hepatitis B in 202 Hospital of PLA from August 2012 to July 2013 .The HBV genotypes and drug resistance associ-ated 5 sites in common 3 types of antiviral drug were detected by the gene chip technology of nested PCR (NPCR) combined with reverse dot blot(RDB) .Results Among 90 samples ,HBV genotypes included type B 6 .7% (6/90) ,type C 86 .6% (78/90) ,mixed type B and C 6 .7% (6/90) .The serum HBV DNA level in the patients with HBV-C genotype were significantly higher than that with genotype HBV-B and mixed genotype B and C .There were 4 cases of drug resistance gene mutation in the detected samples ,3 cases of mutation were placed in 180M+204V ,1 case of mutation was placed in 180M+204I .All the drug resistance mutations ap-peared in the patients with HBV-C genotype .Conclusion The main HBV genotype is the genotype C in Shenyang area .Genotype B and genotype mixed B and C are unusual .The HBV DNA level in the patients with the genotype C is relatively high ,which is more likely to happen resistance to lamivudine .Moreover the mixed mutation in 180M is more likely to happen .
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Estima-se que a prevalência global da população mundial com hepatite C é de 3%. Pouco se sabe sobre a resposta ao tratamento com respeito à resistência viral. Algumas mutações no fragmento de 109 aminoácidos da NS5B são associadas com resistência ao interferon (IFN) e ribavirina (RBV). Estudos moleculares e clínicos identificaram fatores associados com o hospedeiro e vírus relacionados associada com a resposta ao tratamento, tal como o gene que codifica a IL-28B. Este estudo foi dividido em duas fases, cujos objetivos foram caracterizar a frequência de mutações que conferem resistência ao HCV e avaliar a relevância das mutações em pacientes Respondedores (R) ou Não Respondedores (NR) ao tratamento e caracterizar geneticamente as populações sobre polimorfismos genéticos nos SNPs da IL-28B em relação ao prognóstico da resposta ao tratamento. As amostras dos pacientes foram submetidas a testes de genotipagem e carga viral. As sequências geradas foram comparadas no BLAST e no banco de dados Los Alamos HCV. Realizamos o alinhamento das sequências homólogas e as mutações identificadas. Com base no genótipo e carga viral determinamos a classificação dos pacientes de acordo com a resposta à terapia. O DNA genômico foi isolado a partir de sangue periférico para a realização da tipagem de SNPs de IL-28B. A metodologia utilizada foi de PCR em tempo real utilizando sondas TaqMan SNP específico. A análise dos dados foi realizada utilizando GraphPad Prism com qui-quadrado, risco relativo (RR), Odds Ratio (OR) e intervalo de confiança de 95%, com um nível de significância de P <0,05. Foi encontrado na primeira fase deste estudo uma taxa significativa mutações associadas ao tratamento nas amostras estudadas. A prevalência de mutações associadas à resistência ao IFN e RBV bem como a novos medicamentos antivirais localizados no fragmento de 109 aminoácidos da NS5B foi examinado em 69 indivíduos infectados naïve no Rio de Janeiro, Brasil. Na segunda fase, as mutações foram ...
It is estimated that the overall prevalence of the average world population with hepatitis C is 3%. Little is known about the treatment response with respect to viral resistance. Some mutations in the 109-aminoacid fragment of NS5B are associated to Interferon (IFN) and Ribavirin (RBV) resistance. Molecular and clinical studies have identified factors associated with the host and related viruses associated with response to treatment, as the gene encoding IL-28B. This study was divided into two phases whose objectives were to characterize the frequency of mutations conferring resistance to HCV viral evaluating the relevance of these in Responders (R) or Non-Responders (NR) patients to treatment and to characterize genetically the populations regarding genetic polymorphisms SNPs IL-28B in relation to prognosis of response to treatment for HCV. Patient samples were subjected to tests for genotyping and viral load. The sequences generated were compared in the BLAST and the Los Alamos database HCV. We conducted the alignment of homologous sequences and mutations identified. Based on virological parameters genotype and viral load determined the classification of patients according to response to therapy. Genomic DNA was isolated from peripheral blood for carrying out the typing of SNPs of IL-28B. The methodology used was real-time PCR using TaqMan probes specific SNPs. Data analysis was performed using GraphPad Prism with chi-square, relative risk (RR), Odds Ratio (OR) and confidence interval of 95% with a significance level of P <0.05. To study these biological parameters we associated the responsive patients, non-responders, the viral load, genotype, and IL-28B polymorphism to treatment outcome. We found in the first phase of this study a significant rate of treatment-associated mutations in the samples studied. The prevalence of mutations associated to resistance to interferon and ribavirin (IFN/RBV) as well new antiviral drugs located in the 109 aminoacid ...
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Humans , Drug Resistance, Viral/genetics , Hepatitis C/virology , Mutation , Antiviral Agents/administration & dosage , Antiviral Agents/pharmacology , Genotyping Techniques , Hepacivirus/pathogenicity , Interferons/pharmacology , Polymorphism, Genetic , Real-Time Polymerase Chain Reaction/methods , Ribavirin/pharmacology , Treatment Outcome , Viral LoadABSTRACT
BACKGROUND/AIMS: Molecular diagnostic methods have enabled the rapid diagnosis of drug-resistant mutations in hepatitis B virus (HBV) and have reduced both unnecessary therapeutic interventions and medical costs. In this study we evaluated the analytical and clinical performances of the HepB Typer-Entecavir kit (GeneMatrix, Korea) in detecting entecavir-resistance-associated mutations. METHODS: The HepB Typer-Entecavir kit was evaluated for its limit of detection, interference, cross-reactivity, and precision using HBV reference standards made by diluting high-titer viral stocks in HBV-negative human serum. The performance of the HepB Typer-Entecavir kit for detecting mutations related to entecavir resistance was compared with direct sequencing for 396 clinical samples from 108 patients. RESULTS: Using the reference standards, the detection limit of the HepB Typer-Entecavir kit was found to be as low as 500 copies/mL. No cross-reactivity was observed, and elevated levels of various interfering substances did not adversely affect its analytical performance. The precision test conducted by repetitive analysis of 2,400 replicates with reference standards at various concentrations showed 99.9% agreement (2398/2400). The overall concordance rate between the HepB Typer-Entecavir kit and direct sequencing assays in 396 clinical samples was 99.5%. CONCLUSIONS: The HepB Typer-Entecavir kit showed high reliability and precision, and comparable sensitivity and specificity for detecting mutant virus populations in reference and clinical samples in comparison with direct sequencing. Therefore, this assay would be clinically useful in the diagnosis of entecavir-resistance-associated mutations in chronic hepatitis B.
Subject(s)
Adult , Humans , Antiviral Agents/therapeutic use , Cross Reactions , DNA, Viral/blood , Drug Resistance, Viral/genetics , Genotype , Guanine/analogs & derivatives , Hepatitis B virus/genetics , Hepatitis B, Chronic/drug therapy , Mutation , Polymerase Chain Reaction/standards , Reagent Kits, Diagnostic/standards , Reference Standards , Sequence Analysis, DNA , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/standardsABSTRACT
Mutations located in the 109-amino acid fragment of NS5B are typically associated with resistance to interferon (IFN) and ribavirin (RIB) and to new antiviral drugs. The prevalence of these mutations was examined in 69 drug-naïve individuals with hepatitis C virus (HCV) infections in Rio de Janeiro, Brazil. Mutations related to non-response to IFN/RIB were observed in all subtypes studied (1a, 1b, 2b, 3a and 4). The most common mutation was Q309R, present in all subtypes, except subtype 2b with frequency above 20 percent. D244N was detected only in subtype 3a and A333E was detected only in subtype 2b. We did not detect the S282T, S326G or T329I mutations in any of the samples analysed. Of note, the C316N mutation, previously related to a new non-nucleoside compound (HCV796 and AG-021541), was observed in only eight of 33 (24 percent) samples from subtype 1b. Site 316 was under positive selection in this HCV variant. Our data highlight the presence of previously described resistance mutations in HCV genotypes from drug-naïve patients.