Perfil sociodemográfico e farmacoepidemiológico de crianças infectadas pelo HIV / Sociodemographic and pharmacoepidemiology profile of HIV infected children

RESUMO A terapia antirretroviral interfere na replicação do vírus HIV, impede a progressão da infecção para a Aids e previne a mortalidade precoce das crianças infectadas. Esta pesquisa investigou o perfil sociodemográfico e os parâmetros relacionados com o tratamento antirretroviral das crianças HIV positivas residentes no estado do Paraná. Trata-se de um estudo observacional descritivo e analítico realizado com dados secundários do ano de 2020 referentes às crianças com até 12 anos de idade. Foram investigados: perfil, prevalência, medicamentos em uso, abandono da terapia, resistência e supressão viral. Foram identificadas 148 crianças, com uma prevalência igual a 8,1/100 mil no Paraná. Apesar de todas as crianças diagnosticadas com HIV terem iniciado o tratamento, 17,2% encontravam-se em abandono da terapia antirretroviral. Entre as crianças que permaneciam em tratamento, 9,8% não atingiram a supressão viral e suas cargas virais comumente ultrapassavam mil cópias virais/mL. Houve um predomínio de esquemas medicamentosos provavelmente prescritos após falhas terapêuticas. Os resultados indicam que o Paraná apresenta bons resultados quanto ao início rápido da terapia e à supressão viral das crianças. Entretanto, existe um número considerável de abandonos da terapia e de falhas terapêuticas, indicando a necessidade de reforçar a vinculação desta população aos serviços de saúde.

ABSTRACT Antiretroviral therapy interferes with the replication of the HIV virus, stops the progression of infection, and prevents early mortality in infected children. This research investigated the sociodemographic profile and parameters related to the antiretroviral treatment of HIV positive children living in the state of Paraná. This is a descriptive observational and analytical study, carried out with secondary data from the year 2020, referring to children up to 12 years of age. The profile, prevalence, medicines in use, treatment abandonment, viral resistance, and viral suppression were investigated. A total of 148 children were identified, with a prevalence equal to 8.1/100,000 in Paraná. All infants had begun their treatment, but 17,2% abandoned it. Among children who remained on treatment, 9.8% did not achieve viral suppression and their viral loads commonly exceeded 1000 viral copies/mL. There was a predominance of drug regimens probably prescribed after treatment failures. The results indicate that Paraná presents good results in terms of rapid initiation of therapy and viral suppression in children. However, there is a considerable number of abandonments of therapy and therapeutic failures, indicating the need to strengthen the link between this population and health services.

Treatment of Hepatitis C with Direct-Acting Antivirals does not Induce Significant Arrhythmias

Abstract Background Chronic Hepatitis C (CHC) therapy with direct-acting antivirals (DAAs) has high efficacy and safety, but some cases of bradyarrhythmias have been described. Objective To evaluate heart rhythm disorders during DAA treatments. Methods Forty-eight patients with CHC (mean 61 years of age; 56% males; 73% HCV genotype 1) were evaluated before and during treatment with DAAs, analyzed by a resting 12-lead ECG [PR, QRS, and QT corrected (QTc) intervals measured] and a 24-h-Holter system, to evaluate the heart rate (HR) and the occurrence of arrhythmias. The Student's t-test or the Wilcoxon-Mann-Whitney test for continuous, independent variables were performed with a statistically significant p-value < 0.05. Results The electrocardiographic parameters before and during treatment were: PR interval (147.2 ± 15.6 vs 144.9 ± 15.6 ms; p = 0.21), QTc interval (427 ± 22.3 vs 421.7 ± 25.3 ms; p = 0.24), minimum HR (52.7 ± 8.4 vs 53.2 ± 8.5 bpm; p = 0.49), median HR (74.2 ± 10.4 vs 75.2 ± 9 bpm; p = 0.83), and maximum HR (117.4 ± 16.8 vs 117.9 ± 16.3 bpm; p = 0.25). These parameters proved to be similar among 11 beta-blockers or 22 ribavirin users. During treatment, the 21 cirrhotic patients presented significantly lower median HRs (72.1 ± 9.0 vs 77.9 ± 8.2 bpm; p = 0.02) and maximum HRs (108.9 ± 15.2 vs. 125.1 ± 13.2 bpm, p < 0.0001) through a 24-h-Holter monitoring than the patients without cirrhosis. No clinically relevant arrhythmias were detected. Conclusion DAAs do not significantly influence heart rate or induce significant cardiac arrhythmias in patients with CHC.

Selection dynamics of HCV genotype 3 resistance-associated substitutions under direct-acting antiviral therapy pressure

ABSTRACT The chronic hepatitis C (CHC) treatment is currently based on the use of direct-acting antivirals (DAAs), and patients infected with hepatitis C virus genotype 3 (GT3) have emerged as a more difficult-to-cure population. The NS5A inhibitor daclatasvir (DCV) and sofosbuvir (SOF), an NS5B viral polymerase inhibitor, are among the drugs that compose more effective and safer treatment regimens. The virus genetic variability is related to resistance-associated substitutions (RASs) that adversely impact DAAs effectiveness. The aims of this study were to analyze the association of NS5A and NS5B RASs and other clinical factors with DAAs regimens effectiveness in patients with GT3 CHC infection. This was a prospective cohort study performed in a Brazilian university hospital. Individuals older than 18 years with GT3 CHC treated with SOF + DCV ± ribavirin (RBV) or SOF + peginterferon (PEG) + RBV were included. Blood samples were collected at baseline and post-treatment. A total of 121 patients were included. Sustained virological response rates were 87.6% for the SOF + DCV ± RBV group and 80.0% for the SOF + PEG + RBV arm. Cirrhosis, prior treatment with interferon/PEG + RBV, and baseline NS5A RAS were associated with higher risk of treatment failure. The NS5A analysis suggested that A30K, Y93H, and RAS at site 62 were related to failure. Interestingly, a likely compensatory effect was shown between A30K and A62T. Emergence of Y93H was always associated with RAS at position 62. The RASs dynamics comprehension is an important tool to indicate more effective treatment for GT3 patients.

Research advances in tenofovir in treatment of chronic hepatitis B patients with drug resistance / 临床肝胆病杂志

Currently, tenofovir is the first-line drug for the treatment of chronic hepatitis B. This article reviews the research advances in its therapeutic effects in patients who are resistant to lamivudine and adefovir dipivoxil, respond poorly to entecavir, or have multidrug resistance and introduces the results of the comparative study on the therapeutic effects of tenofovir monotherapy and combined treatment. It is pointed out that tenofovir has good safety and a good therapeutic effect in patients with drug resistance, including pregnant women; however, there are no significant differences in study results between tenofovir monotherapy and combined treatment.

Advances in research of HBV quasispecies / 中华检验医学杂志

Hepatitis B virus exists in the form of quasispecies.Quasispecies are an important basis for the pathogenesis and drug resistance of hepatitis B virus.Researches for quasispecies could contribute to evaluating the progress of disease, predicting drug resistance, adjusting the therapeutic regimen and performing individual treatment.

Advances in detection technology for HBV phenotypic resistance / 中华检验医学杂志

Long-term use of nucleoside ( nucleotide ) analogues for anti-HBV treatment care easily lead to drug resistance. As compared to genotypic resistance detection, the detection technology of phenotypic resistance is a method used to analysis the sensitivity of antiviral drugs in vitro cell model, the result can directly reflect the relationship between drug-resistance mutations and drug susceptibility, thus perform a better prediction of clinical resistance.Among those technologies, some analysis strategies like transient transfection assay, stable cell lines and enzymatic assay, have been developed for early diagnoses as well as dynamic monitoring of drug resistance, and contribute greatly to proper selection of therapeutic regimen, reduction of resistance rate and implementation of individual treatment.

Primary drug resistance of human immunodeficiency virus (HIV) among the treat-ment-naive individuals with HIV in China:a meta-analysis / 北京大学学报(医学版)

Objective:To estimate the prevalence of antiretroviral drug resistance in treatment-naive in-dividuals with human immunodeficiency virus ( HIV ) in China. Methods: Five electronic databases [ Chinese BioMedical Literature Database ( CBM) , Chinese Journal Full-text Database ( CNKI) , Chinese Science-Technology Journal Database ( VIP) , Wanfang Data, and PubMed] were searched for studies of HIV drug resistance in untreated individuals. Drug resistance data were abstracted then pooled using the random effect model. Subgroup analysis was done across sampling time, location, study population ( mean age and infection status) , and sample size. Results: Seventy-six studies were included for our meta-analysis (46 in Chinese, 30 in English). The pooled rates of drug resistance to total, to non-nucleoside reverse transcriptase inhibitor ( NNRTI ) , to nucleoside reverse transcriptase inhibitor (NRTI), and to protease inhibitor ( PI) were 4. 7% (95%CI:4. 0% -5. 4%), 2. 3% (95%CI:1. 8% -2. 8%), 1. 8% (95%CI:1. 3% -2. 3%), and 1. 4% (95%CI:1. 1% -1. 8%), respective-ly. All the rates before 2007 were higher than those for 2008 or later. Meanwhile, significant differences were found in the sample areas (P <0. 05), in which, the rates in South-central and Southwest were both higher than 5%. The difference was complex between mean age and infection status subgroup, and we found the total prevalence in the group under 25 years and the newly infected, and confirmed group was lower than in the others. For sample size, all the rates in the group under 100 samples were higher than in the others, and the difference was significant (P<0. 05). Conclusion: The prevalence of HIV primary drug resistance in China was 4. 7%, which stayed low, but was also close to the line set by WHO. Enhanced surveillance for drug resistance is necessary in high epidemic areas including the South-central and Southwest China whose prevalence has crossed the line.

An innovative HBV genotyping and drug-resistant mutation detection platform / 中华检验医学杂志

Despite decades of the prevention and control.China is still one of the countries with high prevalence of hepatitis B virus (HBV).It is imperative for us to innovate.Lab diagnostic platforms for drug-resistant mutation and HBV genotyping to enhance the management level in patients with CHB.Specifically,the following three strategies should be included:firstly,choose an appropriate platform for drug-resistant mutation detection according to the actual situation in laboratory.Secondly,establish a practical and simple genotyping platform for real-time monitoring of HBV genotypes.Thirdly,combined with clinical,explore the correlation among genotypic resistance,phenotypic resistance and clinical resistance when different medications are adopted,confirm the percentage or threshold of resistant strains causing clinical resistance,and ascertain the influence of HBV genome polymorphism (genotype) on clinical prognosis.These strategies above mentioned will lay a foundation for CHB personalized treatment and prognosis.

Design and validation of a clinical DNA sequencing assay for drug-resistance genes in hepatitis B virus / 中华检验医学杂志

Objective Under the ISO15189 and America Association of Pathologists ( CAP ) laboratory accreditation system, to establish the performance verification standards for detecting hepatitis B virus resistance gene by sanger sequencing.Methods 25 cases of HBV drug resistance outpatients and inpatients were collected from August 2012 to December in Hepatitis Clinic of Huashan Hospital Affiliated to Fudan University.Analytical performance parameters including analytical sensitivity, precision, accuracy, analytical specificity, reference range/reportable range, etc were evaluated.Sequencing quality evaluation parameters included fluorescence signal intensity overall sequencing chromatogram, signal to noise ratio, trace scores and QV value.Results 10%-20% mutation could be detected under wild-type background. The methool had good precision and accuracy.No obvious interference and cross contamination were observed.Conclusions Performance validation of the sequencing should combine with the practical application.Especially in view of the different detection subjects, and appropriately adjusted to meet the clinical needs.Detection of hepatitis B virus resistance gene by the in the test method in this study can be used in clinical detection.

Molecular inversion probe-based single base mutations of hepatitis B virus resistance gene analysis / 中华检验医学杂志

Objective To establish a molecular inversion probe ( MIP) method for detection of single base drug-resistance mutation in Hepatitis B virus ( HBV) gene.Methods The HBV wild type and YVDD mutant strain were isolated by Daping Hospital of the Third Military Medical University.The MIP was designed and applied to detect the HBV drug-resistance YVDD mutation in one case of wild type and one case of YVDD mutant HBV strain isolated previously.The results of MIP method were compared with that of sequencing to evaluate the detection accuracy.Results Thermal cycling single-base extension and connection reaction performed by Taq DNA Ligase and Ampligase DNA Ligase could ensure the specificity of the detection.The optimum probe concentration of MIP was 1 nmol/L.Through detection of the target gene with different DNA concentrations , the detection sensitivity of MIP was determined as 1 nmol/L.The results of MIP were consistent with that of sequencing method in detection of the clinical samples.Conclusion MIP is successfully used to detect single-base drug-resistance mutation in HBV gene.

Pyrosequencing for detection of drug resistant relevant mutation in the polymerase gene of Hepatitis B virus and its clinical application / 中华检验医学杂志

Objective To explore the accuracy and clinical application of Pyrosequencing for detection of drug resistant relevant mutation in the polymerase gene of Hepatitis B virus (HBV).Methods Compared with Sanger sequencing,the accuracy and sensitivity of Pyrosequencing were assessed.Pyrosequencing was used to determine the serum of 1164 patients with chronic Hepatitis B and its results were analyzed.Results The sensitivity of Pyrosequencing was 1 × 103 KIU/L,the same as Sanger sequencing.But Pyrosequencing was more stable and specific than Sanger sequencing to detect low rate of mutation with over 10％ mutation.The mutations couldn't be detected in 248 patients with chronic Hepatitis B,but positive results occurred in 919 patients,among them,61.5 ％ without mutation,38.5 ％ with mutation (including 47.5％ of the single locus mutation and 52.5％ of the combined mutation).Other sites had different degree of mutations except the sites of rtI169T and rtA194T; the main mutation sites in patients with chronic Hepatitis B were rtM204V/I (32.1％),rtL180M (19.8％),rtA181V/T (6.7％) and rtN236T (5.3％),in which fractional mutation had high ratio in the sites of rtA181V/T (6.7％) and rtN236T (5.3％).Conclusions Pyrosequencing has good sensitivity,specificity and accuracy and can early detect the drug resistant relevant mutation in the polymerase gene of HBV,which is suitable for monitoring patients with chronic Hepatitis B for the treatment of nucleoside analogues (acid).The different sites and frequencies of mutations may be associated with different habits of doctors for using different anti-HBV drugs.

Analysis of genotype resistance mutations sites among 123 antiretroviral-therapy failure in HIV/AIDS patients in Yunnan province, China / 中华检验医学杂志

Objective To analyze the prevalence and diversity of genotype resistant mutations in 123 HIV/AIDS patients experiencing failure of high antiretroviral therapy (HAART) in Yunnan province.Methods A cross-sectional survey was conducted among 151 HIV/AIDS patients experiencing failure of antiretroviral therapy form January 2011 to January 2012 in Yunnan province.The HIV-1 pol gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR),the sequences were then submitted to the Stanford University HIV drug resistance database to analyze the prevalence of resistant mutations.The resistant mutations were statistically analyzed by gender,ethnic groups,transmission route and subtype,respectively.The chi-square or fisher's exact test was used for statistical test.Results Of the 151 cases,123 plasma samples were amplified successfully for protease PR (codon 1-99) and reverse transcriptase RT (codon 1-272) fragments.The most common mutation was M184 (72.4％),followed by the mutation at position K103 (47.2％),T215 (26.0％),D67 (15.4％),G190 (34.1％),Y181 (29.3％),K101 (17.1％).The frequency of mutations at position V75,A62 and M230 was higher in male population than that in female population (x2 =7.001,6.975,5.446,P ＜ 0.05).The frequency of variants at position Tl215,K70 and T69 was higher in the Han population than in the other ethnic population(x2 =5.290,4.060,3.860,P＜ 0.05).It was interesting that the variant M41L was rare in the other ethnic groups.The significant difference existed at various transmission routes.Frequencies of variants at position T215 and T69 were significantly higher among people infected HIV-I through sexual contact than the intravenous drug users (x2 =10.431,7.952,P ＜ 0.05).Frequencies of variants at position G190 were significantly higher among the intravenous drug users than the population infected HIV-1 through sexual contact(x2 =6.669,P ＜ 0.05),but the variant M230L never occurred in intravenous drug users.The RT mutations V75,T69,M230 were more frequently occurred in patients infected with CRF01_AE than in patients with subtype B (P＜ 0.05).The mutation L74 was never seen in patients infected with CRFOI_AE (P ＜ 0.05).Conclusions The HIV/AIDS patients with failure of high antiretroviral therapy (HAART) were attributed to HIV-1 genotype resistance mutations.The mutation sites among the HAART failure patients from the regions of Dehong,Gejiu,Wenshan and Yuxi were significant difference accordance among the gender,ethnicith,transmission route and subtype,respectively.

Drug resistance mutation of HIV-1 in HIV/AIDS patients infected by blood transfusion / 解放军医学杂志

Objective To study the characteristic of HIV-1 gene mutation in HIV/AIDS patients infected by blood transfusion, and analyze the resistance to anti-HIV drugs. Methods Plasma samples were collected from 37 HIV/AIDS patients infected by blood transfusion for extraction of HIV-1 RNA. The gene fragments of HIV pol domain were amplified by RT-PCR and nested-PCR, and the electrophoresis positive products were sequenced. The sequencing result was landed to the website http:// HIV-ldb.stanford.edu to analyze the drug resistance mutations. Results Drug resistance mutations were found in 20 patients, including 19 cases of virological or immunological failure. Mutation of gene locus V32AV of protease inhibitors (Pis) occurred in 3 patients during the treatment, but it did not cause the drug resistance of Pis. Mutation of the coding regions of reverse transcriptase was found in 23 patients, including M184V, TAMs, Q151M complexus, K103N, Y181C and so on. Of the 23 patients mentioned above, the HIV-1 gene mutation induced the resistance to reverse transcriptase inhibitors (RTIs) in 20 patients, and the mutation rate of RTIs was 54.05% (20/37). Conclusion The drug resistance rate of HIV-1 in patients infected by blood transfusion may be high for antiviral therapy, so the drug resistance of HIV-1 should be monitored and treatment plan should be adjusted timely.

Detection of human cytomegalovirus UL97 gene mutations conferring ganciclovir resistance / 中华检验医学杂志

Objective To explore human cytomegalovirus UL97 mutations related to ganciclovir resistance in hematopoietic stem cell transplant (HSCT) recipients.Methods A total of 43 patients,including 24 males and 19 females,with an average age of 21 years old,who had HCMV DNAemia for more than two weeks after HSCT between 2008 and 2010 in Peking University People's Hospital,were included in this prospective study.UL97 GCV resistance mutations were investigated in 49 plasma specimens collected from those patients.GCV resistance mutations such as UL97 M460V/I,H520Q,A591V,A594V,L595S/F,and C603W,were analyzed by modified PCR-RFLP methods.UL97 mutations related to GCV resistance were assayed by the method of PCR-direct sequencing (PCR-DS).An amplified refractory mutation system real-time PCR (ARMS RT-PCR) was developed for the detection of UL97 A594V mutation.Results Eight known UL97 ganciclovir resistance mutations were not detected by PCR-RFLP and PCR-DS.Four new UL97 mutations like UL97 R494P,T502A,N558D,and G561S,were detected by PCR-DS.The ARMS RT-PCR for detecting of UL97 A594V was established successfully.The lower limit of detection of the method was at least 7.5 × 102 copies/ml combined with the use of nucleic acid extraction reagent.UL97 A594V resistance mutation was identified by the method of ARMS RT-PCR in two HSCT recipients.The rate of UL97 A594V mutation was 4.7％ (2/43) in HSCT recipients.Conclusion The ARMS RT-PCR assay represented a sensitive method for the identification of UL97 A594V mutation.

Genotyping and resistant mutants analysis of P gene in HBV fragment sequencing / 中华检验医学杂志

Objective To establish a method for efficient,accurate genotyping and nucleoside drug-resistant mutation analysis for hepatitis B virus ( HBV ).Methods The 48 HBV serum samples were collected from the Third People's Hospital of Taiyuan from July to August 2011,and HBV DNA were extracted using the commercial kit.The HBV whole genome and P gene were amplified and sequenced.Each HBV sample was genotyped by both constructing phylogenetic trees and genotyping software analysis.The results from two strategies were compared for every sample.Results A total of 48 HBV full genome sequences were identified into 12 B and 36 C genotype's by both constructing phylogenetic trees and genotyping software analysis,which was exactly the same as the analysis using P gene fragment sequencing.Seven forms of nucleoside drug-resistant mutation were found in the P gene for all the samples,with the ratio of 27.1％ ( 13/48 ),in which all the mutation forms were associated with lamivudine or adefovir,and no other nucleotide drugs-related resistance mutations existed.In addition,there were 11 B and 35 C genotype and 2 B/C hybrid type with the analysis using Real-time PCR genotyping for the 48 samples.Conclusion P gene sequencing can be used as a new clinical method for efficient,accurate HBV genotyping and resistant mutation analysis,which provides guidance for hepatitis B treatment.

Efficacy and drug resistance profiles of nucleosides retreatment in nucleoside experienced chronic hepatitis B patients / 中华传染病杂志

Objective To evaluate the efficacy and drug resistance profiles of nucleosides (NA) retreatment in NA experienced chronic hepatitis B (CHB) patients. Methods Totally 104 NA experienced CHB subjects were enrolled in this study.All these subjects had received at least 3 months NA monotherapy and stopped the treatment,and then received NA retreatment for at least one year.The subjects were divided into three groups according to the following criteria:reached the therapy endpoint of China guideline when they stopped NA-naive treatment (group A,n =39); did not reach the therapy endpoint when they stopped NA-naive treatment but hepatitis B virus (HBV) DNA＜1.0× 103 copy/mL (group B,n=33); and with HBV DNA＞1.0× 103 copy/mL (group C,n=32).The efficacy and drug resistance profiles of retreatment were compared among three groups. The effects of baseline alanine aminotransferase (ALT) levels,HBV DNA levels and HBeAg titers on the retreatment efficacies were analyzed. The mutations of HBV P gene were detected by nested polymerase chain reaction (PCR) and direct sequencing.The data were analyzd by Wilcoxon test and x2 test.Results The time to ALT normalization in patients with baseline ALT＜ 1.3 × upper limit normal (ULN) was shorter than that in patients with ALT≥1.3×ULN (2 months vs 4 months; Z=2.281,P=0.023).The time to virological response in patients with baseline HBV DNA＜5 lg copy/mL was shorter than that in patients with HBV DNA≥5 lg copy/mL (1 month vs 2 months; Z=2.054,P =0.040). The time to virological response and ALT normalization in baseline HBeAg negative were both shorter than those in patients with baseline HBeAg positive patents ( 1 month vs 3 months and 2 months vs 4.5 months,respectively; Z=2.580 and 2.304,respectively; both P＜0.05). The subjects in group A achieved virological response and HBeAg seroconversion after retreatment earlier compared to previous NA-naive therapy ([1.61 ± 1.76] months vs [3.48±4.066]months and [3.38 ± 3.34] months vs [9.92-11.22] months,respectively; Z=-2.854 and-1.094,respectively; both P＜0.05).The cumulative HBeAg seroconversion rate in group A was higher compared to those in group B and group C (80.0％ vs 36.8％ and 37.5％,respectively; x2 =4.368 and 5.100,respectively; both P＜0.05).Thirteen patients developed clinical resistance and four of them developed genotypic resistance proved by PCR direct sequencing.Among the patients retreated with the same regimen as previous in the C group,the cumulative resistance rate was highest compared to group A and B (44％ vs 9％ and 0,respectively; x2 =5.019 and 6.588,respectively;both P＜0.05).No resistance was detected in the 14 patients retreated with combined NA treatment without cross resistance.Conclusions For NA experienced CHB patients who fulfill the indication of antiviral therapy,the retreatment should be started as earlier as possible. Retreatment with NA combination without cross resistance can prevent (reduce) the risk of developing drug resistance.

Comparison of drug resistance mutations associated with hepatitis B virus covalently closed circular DNA and relaxed circular DNA / 中华传染病杂志

Objective To detect nucleos(t)ide-resistance mutations in hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) isolated from hepatocytes of patients with chronic HBV infection and to analyze the correlation between the mutations found in cccDNA and relaxed circular DNA (rcDNA). MethodsForty patients with chronic HBV infection were investigated. Preoperation serum samples and non-tumor liver tissues were collected.Intrahepatic HBV cccDNA and rcDNA were selectively extracted by co-precipitation of sodium dodecyl sulphate-protein and QIAamp DNA Mini Kit, and further purification with plasmid-safe ATP-dependent DNase (PSAD).Thereafter,cccDNA were amplified by selective polymerase chain reaction (PCR) or nested PCR using the primers spanning both the two gaps in HBV genome and covering the common mutations associated with nucleoside analogues resistance (rt169- rt250).Intrahepatic HBV rcDNA and pre-operation serum HBV rcDNA were also extracted and then amplified by PCR.The PCR products were then purified and sequenced.Results Among the 40 patients,intrahepatic HBV cccDNA were detected in 31 patients,and HBV rcDNA were detected in liver samples of 35 patients and pre-operation serum samples of 21 patients. The PCR products amplified from these samples were all successfully sequenced.rtM204Ⅰ mutation was detected in intracellular HBV cccDNA,rcDNA and serum HBV rcDNA in 2 patients.Both rtM204Ⅰ and rtQ215H were detected in intrahepatic HBV cccDNA and rcDNA in 2 patients,while no corresponding mutation was observed in serum HBV rcDNA of these two patients.Three variants including rtM204V,rtM204V and rtV173L-rtL180M-rtM204V were detected in serum HBV rcDNA in 3 patients,while corresponding mutants were not detected in intracellular HBV cccDNA or rcDNA of these patients.Condsions The results suggest that antiviral nucleos(t) ide resistance mutations can be found in HBV cccDNA in chronic hepatitis B patients. The dominant resistant mutation found in intrahepatic HBV cccDNA/rcDNA may be different from that in serum HBV rcDNA.

Virological surveillance and antiviral resistance of human influenza virus in Argentina, 2005–2008 / Vigilancia virológica y resistencia a los antivíricos del virus de la gripe humana en la Argentina, 2005–2008

Objective. To describe the virological characteristics of the influenza strains circulating in Argentina in 2005–2008 and to assess the prevalence of antiviral resistance. Methods. On the basis of their geographical spread and prevalence, influenza A and B isolates grown in Madin–Darby canine kidney cells were selected after antigenic and genomic characterization to be analyzed for antiviral resistance by enzymatic assay and pyrosequencing. Amantadine susceptibility was evaluated by pyrosequencing for known resistance markers on 45 strains of influenza A. Susceptibility to oseltamivir and zanamivir was evaluated by enzymatic assay of 67 influenza A and 46 influenza B strains, some of which were further analyzed by sequencing the neuraminidase gene. Results. Resistance to amantadine was observed only on A(H3N2) strains (29/33); all of them carried the mutation S31N in their M2 sequence. Oseltamivir resistance was observed in 12 (34.3%) of the 35 A(H1N1) strains from 2008; all of them carried the mutation H275Y in their neuraminidase sequence. All these viruses remained sensitive to zanamivir. Conclusions. This study describes a high incidence of amantadine-resistant influenza A(H3N2) viruses since 2006 and an unprecedented increase in oseltamivir resistance detected only in influenza A(H1N1) viruses isolated in 2008. Influenza A and B viruses were more sensitive to oseltamivir than to zanamivir, and influenza A viruses were more sensitive to both neuraminidase inhibitors than the influenza B viruses. The national data generated and analyzed in this study may help increase knowledge about influenza antiviral drug resistance, which is a problem of global concern.

Objetivo. Describir las características virológicas de las cepas de virus de la gripe que circulaban en la Argentina entre el 2005 y el 2008, y evaluar la prevalencia de la resistencia a los antivíricos. Métodos. Según su diseminación geográfica y su prevalencia, se seleccionaron aislados de gripe A y B cultivados en células renales caninas de Madin-Darby después de su caracterización antigénica y genómica, y se analizó su resistencia a los antivíricos mediante análisis enzimático y pirosecuenciación. La sensibilidad a la amantadina se evaluó por pirosecuenciación para los marcadores conocidos de resistencia en 45 cepas de gripe A. La sensibilidad al oseltamivir y al zanamivir se evaluó mediante análisis enzimático de 67 cepas de gripe A y 46 cepas de gripe B, algunas de las cuales se analizaron en mayor profundidad mediante la secuenciación del gen de la neuraminidasa. Resultados. Se observó resistencia a la amantadina solo en las cepas de gripe A (H3N2) (29/33); todas ellas tenían la mutación S31N en su secuencia de M2. Se observó resistencia al oseltamivir en 12 (34,3%) de las 35 cepas de gripe A (H1N1) aisladas en el 2008; todas ellas tenían la mutación H275Y en su secuencia de neuraminidasa. Todos estos virus conservaron su sensibilidad al zanamivir. Conclusiones. En este estudio se describe una incidencia elevada del virus de la gripe A (H3N2) resistente a la amantadina desde el 2006 y un aumento sin precedentes de la resistencia al oseltamivir detectada solo en los virus de la gripe A (H1N1) aislados en el 2008. Los virus de la gripe A y B fueron más sensibles al oseltamivir que al zanamivir y los virus de la gripe A fueron más sensibles a ambos inhibidores de la neuraminidasa que los virus de la gripe B. Los datos nacionales generados y analizados en este estudio pueden ayudar a aumentar los conocimientos acerca de la resistencia a los fármacos antivíricos dirigidos contra el virus de la gripe, lo que es un motivo de preocupación mundial.

Prevalence and patterns of HIV transmitted drug resistance in Guatemala / Prevalencia y patrones de farmacorresistencia transmitida del VIH en Guatemala

Objective. To assess human immunodeficiency virus (HIV) diversity and the prevalence of transmitted drug resistance (TDR) in Guatemala. Methods. One hundred forty-five antiretroviral treatment-naïve patients referred to the Roosevelt Hospital in Guatemala City were enrolled from October 2010 to March 2011. Plasma HIV pol sequences were obtained and TDR was assessed with the Stanford algorithm and the World Health Organization (WHO) TDR surveillance mutation list. Results. HIV subtype B was highly prevalent in Guatemala (96.6%, 140/145), and a 2.8% (4/145) prevalence of BF1 recombinants and 0.7% (1/145) prevalence of subtype C viruses were found. TDR prevalence for the study period was 8.3% (12/145) with the Stanford database algorithm (score > 15) and the WHO TDR surveillance mutation list. Most TDR cases were associated with non-nucleoside reverse transcriptase inhibitors (NNRTIs) (83.3%, 10/12); a low prevalence of nucleoside reverse transcriptase inhibitors and protease inhibitors was observed in the cohort (< 1% for both families). Low selection of antiretroviral drug resistance mutations was found, except for NNRTI-associated mutations. Major NNRTI mutations such as K101E, K103N, and E138K showed higher frequencies than expected in ART-naïve populations. Higher literacy was associated with a greater risk of TDR (odds ratio 4.14, P = 0.0264). Conclusions. This study represents one of the first efforts to describe HIV diversity and TDR prevalence and trends in Guatemala. TDR prevalence in Guatemala was at the intermediate level. Most TDR cases were associated with NNRTIs. Further and continuous TDR surveillance is necessary to gain more in-depth knowledge about TDR spread and trends in Guatemala and to optimize treatment outcomes in the country.

Objetivo. Evaluar la diversidad del virus de la inmunodeficiencia humana (VIH) y la prevalencia de la farmacorresistencia transmitida en Guatemala. Métodos. Entre octubre del 2010 y marzo del 2011 se incluyeron en el estudio 145 pacientes no tratados anteriormente con antirretrovirales, derivados al Hospital Roosevelt en la Ciudad de Guatemala. Se obtuvieron las secuencias pol a partir del VIH plasmático y se evaluó la farmacorresistencia transmitida con el algoritmo de Stanford y la lista de mutaciones para la vigilancia de la farmacorresistencia transmitida de la Organización Mundial de la Salud (OMS). Resultados. El subtipo B del VIH fue sumamente prevalente en Guatemala (96,6%, 140/145), y se encontró una prevalencia de formas recombinantes BF1 de 2,8% (4/145) y una prevalencia del subtipo C del virus de 0,7% (1/145). La prevalencia de la farmacorresistencia transmitida durante el período de estudio fue de 8,3% (12/145) según el algoritmo de la base de datos de Stanford (puntuación > 15) y la lista de mutaciones para la vigilancia de la farmacorresistencia transmitida de la OMS. En la mayoría de los casos, la farmacorresistencia transmitida se asoció con los inhibidores de la transcriptasa inversa no análogos de nucleósidos (ITINN) (83,3%, 10/12); en la cohorte se observó una baja prevalencia asociada con los inhibidores de la transcriptasa inversa análogos de nucleósidos y con los inhibidores de la proteasa (< 1% para ambas familias de fármacos). Se encontró una baja selección de mutaciones causantes de farmacorresistencia debidas a los antirretrovirales, excepto en las mutaciones asociadas a los ITINN. Las mutaciones importantes relacionadas con los ITINN, como K101E, K103N y E138K, mostraron frecuencias más elevadas que las esperadas en las poblaciones vírgenes de tratamiento antirretroviral. En las personas con un nivel de escolaridad más elevado se encontró un mayor riesgo de farmacorresistencia transmitida (razón de posibilidades 4,14; P = 0,0264). Conclusiones. Este estudio representa uno de los primeros intentos de describir la diversidad del VIH, y la prevalencia de la farmacorresistencia transmitida y sus tendencias en Guatemala. La prevalencia de la farmacorresistencia transmitida en Guatemala presentó un nivel intermedio y en la mayoría de los casos se asoció con los ITINN. Se necesita una vigilancia más intensa y sostenida de la farmacorresistencia transmitida para conocer más exhaustivamente su grado de diseminación y sus tendencias en Guatemala, al igual que para optimizar los resultados del tratamiento antirretroviral en el país.

Surveillance for Oseltamivir-resistant influenza pandemic A H1N1 viruses in Guangdong province / 中华检验医学杂志

Objective To investigate the susceptibility to oseltamivir of influenza pandemic A (H1N1) 2009 viruses in Guangdong province during 2009,provide valuable information of prescription for clinics,and elucidate the variant trend of the epidemic strain based on phylogenetic analysis.Methods During April to December 2009,clinical specimens were collected from sentinel hospitals covering the whole Guangdong province.Virus isolation was performed by in MDCK cells or embryonated chicken eggs.A fluorescence-based neuraminidase (NA) enzyme inhibition assay was conducted to measure influenza susceptibility.The NAI susceptibility of influenza virus was expressed as the concentration of NAI needed to inhibit the NA enzyme activity by 50％.A subset of 68 viruses were performed NA sequencing for detecting resistant mutations and studying variant trends.Results During surveillance,221 influenza pandemic A ( H1N1 ) viruses were isolated.All strains were sensitive to oseltamivir inhibition assay,with a median IC50 of 0.24 nmol/L (range 0.02 -1.66 nmol/L).No mutation related to resistance was found.Phylogenic analysis illustrated that these NA genes were homology high to 99.5％ - 100.0％ with those from other countries.Conclusions influenza pandemic A (H1N1) 2009 viruses were sensitive to oseltamivir in Guangdong,and useful for prophylaxis and treatment of influenza infection.Little selective pressure was found by phylogenic analysis.Our laboratory will continue to observe antiviral-resistance among circulating influenza viruses.