Screening for and verification of novel mutations associated with drug resistance in the HIV type 1 subtype B(') in China.

OBJECTIVE: Mutations associated with HIV drug resistance have been extensively characterized at the HIV-1 polymerase domain, but more studies have verified that mutations outside of the polymerase domain also results in resistance to antiviral drugs. In this study, mutations were identified in 354 patients experiencing antiretroviral therapy (ART) failure and in 97 naïve-therapy patients. Mutations whose impact on antiviral drugs was unknown were verified by phenotypic testing. METHODS: Pol sequences of HIV subtype B(') obtained from patients experiencing ART failure and from naïve-therapy patients were analyzed for mutations distinct between two groups. Mutations that occurred at a significantly higher frequency in the ART failure than the naïve-therapy group were submitted to the Stanford HIV Drug Resistance Database (SHDB) to analyze the correlation between HIV mutations and drug resistance. For mutations whose impact on the antiviral drug response is unknown, the site-directed mutagenesis approach was applied to construct plasmids containing the screened mutations. 50% inhibitory concentration (IC(50)) to AZT, EFV and NVP was measured to determine the response of the genetically constructed viruses to antiviral drugs. RESULTS: 7 mutations at 6 positions of the RT region, D123E, V292I, K366R, T369A, T369V, A371V and I375V, occurred more frequently in the ART failure group than the naïve-therapy group. Phenotypic characterization of these HIV mutants revealed that constructed viruses with mutations A371V and T369V exhibited dual resistance to AZT and EFV respectively, whereas the other 5 mutations showed weak resistance. Although the impact of the other six mutations on response to NVP was minimal, mutation T369V could enhance resistance to NVP. CONCLUSIONS: This study demonstrated that mutations at the RT C-terminal in subtype B' could result in resistance to RT inhibitors if the mutations occurred alone, but that some mutations could promote susceptibility to antiviral drugs

Immobilization of Cadmium by Molecular Sieve and Wollastonite Is Soil pH and Organic Matter Dependent

The excessive cadmium (Cd) concentration in agricultural products has become a major public concern in China in recent years. In this study, two amendments, 4A molecular sieve (MS) and wollastonite (WS), were evaluated for their potential passivation in reducing Cd uptake by amaranth (Amaranthus tricolor L.) in six soils with different properties. Results showed that the responses of amaranth biomass to these amendments were soil-property-dependent. The effects of MS and WS on soil available Cd were in turn dependent on soil and amendment properties. The application of WS and MS at a dose of 660 mg·kg−1 Si produced the optimum effect on inhibiting Cd accumulation in amaranth shoots (36% and 34%, respectively) and did not affect crop yield. This was predominantly attributed to the marked increase in pH and exogenous Ca or Na, which facilitated the adsorption, precipitation, and complexation of Cd in soils. The immobilization effects of WS and MS were dependent on soil properties, where soil organic matter may have played an important role. In conclusion, MS and WS possess great potential for the remediation of Cd-contaminated acidic soils

Comparison of susceptibility of HIV-1 variants to antiretroviral drugs by genotypic and recombinant virus phenotypic analyses

Objective: This study utilized genotypic and in-house recombinant virus phenotypic assays to examine HIV-1 variant susceptibility to antiretroviral (ARV) drugs; comparisons were made between the analyses. Methods: A nested PCR was employed to amplify the HIV-1 gag-pol gene, which comprised the entire PR gene (codons 1–99) and the former RT gene (codons 1–312). Genetic resistance was determined by submitting the sequences to the Stanford University Network HIV-1 Database. Phenotypic susceptibilities to six ARV drugs were measured using a high-throughput, multi-cycle, recombinant virus phenotypic assay. Results were expressed in terms of the IC50 (half maximal inhibitory concentration) and fold-change values. The relationship between phenotypic drug resistance and genetic polymorphisms was determined. Results: Nineteen fragment sequences for which recombinant viruses were successfully constructed were translated and compared with the consensus B sequences in the Stanford University Network HIV-1 Database. No recognizable genotypic resistance-associated mutations were noted, except in one sample. Each homologous replication-competent recombinant viral fold-change in the presence of six ARV drugs used widely in China was measured. According to the clinical and statistical criteria, 16 of the 19 samples were susceptible to the six drugs tested. The majority of phenotypic and genotypic results obtained were in agreement, with a concordance rate of 97.4%. Both phenotypic and genotypic assays suggested that sample HN2009001 was resistant to all drugs tested. All phenotypic and genotypic results obtained regarding the susceptibility of the 19 recombinant viruses to nucleoside reverse transcriptase inhibitors (NRTIs) were in agreement. With regard to the genotypic results for the non-nucleoside reverse transcriptase inhibitors (NNRTIs), 7.9% (3/38) were inconsistent with the phenotypic results. Conclusions: The in-house recombinant virus phenotypic assay was able to provide a straightforward quantitative assessment of resistance. In most cases, the genotypic and novel phenotypic assays yielded similar results. The disparity in HIV-1 susceptibility indicates a need to further investigate the clinical outcomes of antiretroviral therapy in certain individuals

The Antiviral Activity of Approved and Novel Drugs against HIV-1 Mutations Evaluated under the Consideration of Dose-Response Curve Slope.

OBJECTIVES:This study was designed to identify common HIV-1 mutation complexes affecting the slope of inhibition curve, and to propose a new parameter incorporating both the IC50 and the slope to evaluate phenotypic resistance. METHODS:Utilizing site-directed mutagenesis, we constructed 22 HIV-1 common mutation complexes. IC50 and slope of 10 representative approved drugs and a novel agent against these mutations were measured to determine the resistance phenotypes. The values of new parameter incorporating both the IC50 and the slope of the inhibition curve were calculated, and the correlations between parameters were assessed. RESULTS:Depending on the class of drug, there were intrinsic differences in how the resistance mutations affected the drug parameters. All of the mutations resulted in large increases in the IC50s of nucleoside reverse transcriptase inhibitors. The effects of the mutations on the slope were the most apparent when examining their effects on the inhibition of non-nucleoside reverse transcriptase inhibitors and protease inhibitors. For example, some mutations, such as V82A, had no effect on IC50, but reduced the slope. We proposed a new concept, termed IIPatoxic, on the basis of IC50, slope and the maximum limiting concentrations of the drug. The IIPatoxic values of 10 approved drugs and 1 novel agent were calculated, and were closely related to the IIPmax values (r > 0.95, p < 0.001). CONCLUSIONS:This study confirms that resistance mutations cannot be accurately assessed by IC50 alone, because it tends to underestimate the degree of resistance. The slope parameter is of very importance in the measurement of drug resistance and the effect can be applied to more complex patterns of resistance. This is the most apparent when testing the effects of the mutations on protease inhibitors activity. We also propose a new index, IIPatoxic, which incorporates both the IC50 and the slope. This new index could complement current IIP indices, thereby enabling predict the efficacy of pre-clinical drugs for which human pharmacokinetic is not available

The prevalence of drug resistance among treatment-naïve HIV-1-infected individuals in China during pre- and post- 2004

Abstract Background The widespread use of antiretroviral therapies has led to considerable concerns about the prevalence of drug-resistant, as transmission of drug-resistant (TDR) strains poses a challenge for the control of the HIV-1 epidemic. Methods We conducted an epidemiological study enrolling treatment-naïve HIV-1-positive subjects at the Peking Union Medical College Hospital since 1991. Drug resistance was determined by submitting the sequences to the Stanford University Network HIV-1 database. Results Of 521 participants, 478 samples were amplified and sequenced successfully. HIV Transmitted drug resistance prevalence in China was determined to be 6.7 %. We did not find significant differences in the TDR rate by demographic characteristics. No significant time trend in the prevalence of overall TDR was observed (p > 0.05). Conclusions We identified an intermediate prevalence of transmitted drug resistance (TDR), exhibiting a stable time trend. These findings enhance our understanding of HIV-1 drug resistance prevalence and time trend, and provide some guidelines for the comprehensive public health strategy of TDR prevention