共查询到20条相似文献,搜索用时 31 毫秒
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Wallis CL Mahomed I Morris L Chidarikire T Stevens G Rekhviashvili N Stevens W 《Journal of virological methods》2005,125(2):99-109
The oligonucleotide ligation assay (OLA) has been proposed as an affordable alternative to sequence-based HIV-1 drug resistance testing in resource poor settings. The aim was to evaluate OLA for detecting mutations K103N, Y181C, K65R, Q151M, M184V and T215Y/F in subtype C. Forty-four subtype C and 8 subtype B HIV-1 positive individuals were analysed using the ViroSeqtrade mark HIV-1 genotyping assay (Applied Biosystems, Foster City, CA). A one-step RT-PCR and nested PCR were performed using subtype B specific primers from the OLA kit (NIH AIDS Research and Reference Reagent Program). Seventy-eight subtype C sequences were used to design subtype C specific primers. Ligation and detection steps were followed according to OLA kit protocol. For codons, K103N, Y181C, K65R, Q151M, M184V and T215Y/F, four or more mismatches compared to the probe or mismatches less than four bases from the ligation site were not tolerated. Results revealed accurate identification of mutations in 2/10, 4/9 3/9, 6/7, 2/7 and 6/7 VQA samples and 5/20, 4/17 0/20, 18/24, 5/24 and 13/24 subtype C positive individuals, respectively. It was concluded that the probes and primers in the NIH reference kit would need modification to optimize detection of mutations in subtype C individuals. 相似文献
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Sensitive oligonucleotide ligation assay for low-level detection of nevirapine resistance mutations in human immunodeficiency virus type 1 quasispecies 总被引:1,自引:0,他引:1
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Lalonde MS Troyer RM Syed AR Bulime S Demers K Bajunirwe F Arts EJ 《Journal of clinical microbiology》2007,45(8):2604-2615
This study has adapted the oligonucleotide ligation assay (OLA) to probe for low-level nevirapine (NVP) resistance mutations K103N and Y181C in the human immunodeficiency virus type 1 (HIV-1) population of infected mother-infant pairs from Uganda. When NVP is used to prevent perinatal transmission, NVP-resistant HIV-1 clones may be rapidly selected due to a low barrier for mutation and a relatively high level of fitness (compared to that of other drug-resistant HIV-1 clones). Monitoring for even a low frequency of NVP resistance mutations may help predict the success of subsequent treatment or warrant the use of another regimen to prevent transmission in a subsequent pregnancy. The standard OLA was optimized by using nonstandard bases in oligonucleotides to allow promiscuous base pairing and accommodate significant HIV-1 heterogeneity. Radiolabeled as opposed to fluorescently tagged oligonucleotides increased the sensitivity, whereas alteration of the template, oligonucleotides, salt, and thermostable DNA ligase concentrations increased the specificity for the detection of minority codons. This modified OLA is now capable of detecting mutants with the K103N or the Y181C mutation present in an HIV-1 population at a frequency of approximately 0.4% and is at least 10- to 30-fold more sensitive than the original protocol. A cohort of 19 Ugandan mothers who received NVP treatment perinatally were sampled 6 weeks postdelivery. Ten of 19 HIV-1 DNA samples extracted from peripheral blood mononuclear cells had a detectable K103N (0.5 to 44%) or Y181C (0.8 to 92.5%) mutation, but only one plasma HIV-1 RNA sample had a viral population with the Y181C mutation. These findings suggest that OLA is a robust, sensitive, and specific method for the detection of low-frequency drug resistance mutations in an intrapatient HIV-1 population. 相似文献
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Human immunodeficiency virus type 1 group m protease in cameroon: genetic diversity and protease inhibitor mutational features 总被引:3,自引:0,他引:3
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Fonjungo PN Mpoudi EN Torimiro JN Alemnji GA Eno LT Lyonga EJ Nkengasong JN Lal RB Rayfield M Kalish ML Folks TM Pieniazek D 《Journal of clinical microbiology》2002,40(3):837-845
To establish a baseline for monitoring resistance to protease inhibitors (PIs) and examining the efficacy of their use among persons in Cameroon infected with human immunodeficiency virus type 1 (HIV-1), we analyzed genetic variability and PI resistance-associated substitutions in PCR-amplified protease (PR) sequences in strains isolated from 110 HIV-1-infected, drug-na?ve Cameroonians. Of the 110 strains, 85 were classified into six HIV-1 PR subtypes, A (n = 1), B (n = 1), F (n = 4), G (n = 7), H (n = 1), and J (n = 7), and a circulating recombinant form, CRF02-AG (n = 64). PR genes from the remaining 25 (23%) specimens were unclassifiable, whereas 2% (7 of 301) unclassifiable PR sequences were reported for a global collection. Two major PI resistance-associated mutations, 20M and 24I, were detected in strains from only two specimens, whereas secondary mutations were found in strains from all samples except one strain of subtype B and two strains of CRF02-AG. The secondary mutations showed the typical PI resistance-associated pattern for non-subtype B viruses in both classifiable and unclassifiable PR genes, with 36I being the predominant (99%) mutation, followed by 63P (18%), 20R (15%), 77I (13%), and 10I or 10V (11%). Of these mutations, dual and triple PI resistance-associated substitutions were found in 38% of all the Cameroonian strains. Compared with classifiable PR sequences, unclassifiable sequences had significantly more dual and triple substitutions (64% versus 30%; P = 0.004). Phenotypic and clinical evaluations are needed to estimate whether PI resistance during antiretroviral drug treatment occurs more rapidly in individuals infected with HIV-1 strains harboring multiple PI resistance-associated substitutions. This information may be important for determination of appropriate drug therapies for HIV-1-infected persons in Cameroon, where more than one-third of HIV-1 strains were found to carry dual and triple minor PI resistance-associated mutations. 相似文献
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We recently reported that interleukin-7 (IL-7), a potent stimulator of lymphopoiesis, is an independent predictor of virological response to antiretroviral therapy (ART) in HIV-1 infected adults. To determine if this cytokine also predicts treatment response in HIV-1-infected children, a longitudinal study was performed over 48 weeks in 36 treatment-na?ve children vertically infected with HIV-1. Subjects who received treatment (n = 29) were stratified as complete virological responders (n = 12), partial virological responders (n = 11), or non-responders (n = 6), based on decline in viral load from baseline to week 48. Median plasma IL-7 levels at baseline were higher in complete responders (4.85 pg/mL, interquartile range [IQR] = 3.35-6.5) than in untreated controls (2.10 pg/mL, IQR = 1.50-3.50; p = 0.05). Linear regression analysis showed that baseline IL-7 levels were positively correlated with changes in HIV-1 viral load between baseline and week 24 (r = 0.40; p = 0.03) and between baseline and week 48 (r = 0.34; p = 0.07), but not with corresponding changes in CD4+ T-cell percentages and absolute counts. Collectively, these results indicate that IL-7 levels at baseline are predictive of virological but not immunological response to ART in children infected with HIV-1. 相似文献
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Eshleman SH Guay LA Wang J Mwatha A Brown ER Musoke P Mmiro F Jackson JB 《Journal of acquired immune deficiency syndromes (1999)》2005,40(1):24-29
BACKGROUND: The HIVNET 012 trial in Uganda demonstrated that single-dose nevirapine (NVP) can prevent HIV-1 mother-to-child transmission. NVP resistance (NVPR) mutations were detected in 25% of women 6 to 8 weeks after NVP, with a higher rate of NVPR in women with subtype D than A. This study examined emergence and fading of specific NVPR mutations in women with these subtypes. METHODS: Plasma HIV-1 was analyzed with the ViroSeq genotyping system (Celera Diagnostics, Alameda, CA). Genotypes were obtained from paired samples collected 7 days and 6 to 8 weeks after NVP from 140 women, 83 with subtype A and 57 with subtype D. RESULTS: The rate of NVPR was similar in women with subtype A vs. D at 7 days but was higher in subtype D than A at 6 to 8 weeks. The higher rate of NVPR in subtype D was explained by at least 2 factors: Y181C faded from detection at a greater rate in women with subtype A (odds ratio = 3.06; 95% CI, 1.04, 8.90) and K103N accumulated at a greater rate in women with subtype D (odds ratio = 1.74; 95% CI, 0.62, 4.87). CONCLUSIONS: HIV-1 subtype influences selection and fading of HIV-1 variants with specific drug resistance mutations after antiretroviral drug exposure. 相似文献
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Karin J. Metzner Pia Rauch Patrick Braun Heribert Knechten Robert Ehret Klaus Korn Rolf Kaiser Nadine Sichtig Britta Ranneberg Jan van Lunzen Hauke Walter 《Journal of clinical virology》2011,50(2):156-161
Background and objectivesMinority drug-resistant HIV-1 variants, undetected by conventional genotyping, may impair the outcome of antiretroviral therapy (ART). Thus, we retrospectively analyzed the prevalence of minority drug-resistant HIV-1 variants before ART in chronically HIV-1 infected patients initiating first-line therapy and assessed the impact on clinical outcome in the prospective German Truvada cohort.Study designSamples from 146 antiretroviral treatment-naïve patients were collected between April 2005 and August 2006. K65R, K103N, and M184V variants at low frequencies were detected by allele-specific real-time PCR.ResultsMinority drug-resistant HIV-1 variants were detected in 20/146 patients (13.7%): the M184V mutation in 12/146 patients (8.2%), the K103N mutation in 8/146 patients (5.5%), and the K65R mutation in 4/146 patients (2.7%). Four patients with the M184V mutation also harbored the K65R or the K103N mutation. The 12- and 24 months virological efficacy data revealed that the rate of treatment failure was not increased in the group of patients harboring minority drug-resistant HIV-1 variants prior to ART.ConclusionsMinority drug-resistant HIV-1 variants can be frequently detected in treatment-naïve, chronically HIV-1 infected patients. Despite the presence of those mutations as minority variants before initiating ART, most of the patients were successfully treated. 相似文献
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目的 研究北京市2006年新确认HIV-1感染者毒株的耐药突变本底数据.方法 随机选取北京市2006年新确认HIV-1感染者抗凝全血标本50份,提取血浆病毒RNA,用逆转录聚合酶链反应扩增HIV-1 pol区基因片段,并进行序列测定及耐药基因型分析.结果 成功扩增出34份标本的pol区基因;在1例样本的蛋白酶编码区检测出1个主要耐药突变,7例样本检测出7个次要耐药突变,主要耐药突变为M46L,毒株是CRF01_AE亚型,次要耐药突变有4种,出现的频率分别为A71T(2个)、A71V(3个)、Q58E(1个)、V11IV(1个).在14例样本逆转录酶编码区检测出一种或多种核苷类和(或)非核苷类逆转录酶抑制剂耐药突变,9例标本检出核苷类逆转录酶抑制剂耐药突变,出现频率分别为:V118I(42.9%)、M184V(7.1%)、A62V(7.1%)、K70T(7.1%)、K65R(7.1%)、K219N(7.1%)、T69d(7.1%)、V75LV(7.1%)、K219R(7.1%);10例标本检出核苷类逆转录酶抑制剂耐药突变,出现的频率分别为V1061(35.5%)、Y181C(15.4%)、K103KR(7.7%)、K103R(7.7%)、L100LV(7.7%)、V1081(7.7%)、V179D(7.7%)、V179DV(7.7%).结论 北京市2006年新确认HIV-1感染者毒株中已经存在一定比例耐药突变,有必要定期进行耐药性监测研究. 相似文献
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目的 分离培养体外稳定传代的原代HIV-1耐药毒株,观察失去药物压力下,耐药毒株的体外生长以及主要耐药突变的演化趋势.方法 采集15例服用拉米夫定+司他夫定+萘韦拉平(3TC+D4T+NVP)的HIV-1感染者的外周血单个核细胞(PBMC),用体外共培养的方法从中分离原代HIV-1毒株;RT-PCR扩增耐药毒株历代培养上清的HIV-1 pol区基因并测序,在Stanford HIV Drug Resistance Database数据库进行耐药性分析.结果 15例患者中病毒载量>1000拷贝/ml的有8例,均成功分离出稳定传代的原代毒株,其中2株为耐药毒株,所携带的主要耐药突变分别是K103N/K238T和M184V/K103N/Y181C/H221Y,分别对NVP和3TC/NVP高度耐药;无药物压力的体外培养过程中,M184V、K103N、Y181C和H221Y等耐药突变可以稳定传代,但是K238T发生了回复突变.结论 分离出2株稳定传代的HIV-1耐药毒株,无药物压力情况下,携带K103N突变的毒株具有较好的复制适应性,可稳定传代;携带M184V和K103N/Y181C/H221Y的毒株也能够稳定复制;本研究中发现K238T耐药突变在失去药物的条件下稳定性差,提示该位点易发生回复突变. 相似文献