Pretreatment HIV drug resistance in adults initiating antiretroviral therapy in China, 2017.

BACKGROUND: After the scale-up of antiretroviral therapy (ART) for HIV infected people, increasing numbers of patients have pretreatment drug resistance (PDR). In this study, the prevalence of PDR was evaluated in adults initiating antiretroviral therapy in China. METHODS: Blood samples were obtained from 1943 patients who initiated antiretroviral therapy (ART) in 2017 from 13 provinces or cities in China. Pol sequences were used to analyze drug resistance and construct transmission networks. Logistic regression model was used to estimate the potential factors associated with PDR. RESULTS: In total, 1711 eligible patients (76.0% male; 87.8% aged ≥ 25 years) were included, of which 117 (6.8%) had PDR. The highest rates of PDR were 12.2% in Liangshan Prefecture of Sichuan and 9.3 and 8.9% in Dehong and Lincang Prefecture of Yunnan. A multivariate logistic regression analysis revealed that PDR was significantly higher among intravenous drug users (adjusted Odds Ratio (aOR) = 2.64, 95% CI: 1.57-4.44) and individuals from Liangshan, Dehong, and Lincang (aOR = 2.04, 95% CI: 1.26-3.30). In total, 754 sequences were used to generate 164 transmission networks. Five transmission networks had two or three sequences containing the same mutations, two networks contained subjects from Liangshan, and one network contained subjects from Dehong. CONCLUSIONS: Overall, the PDR prevalence was moderate, with a particularly high prevalence in areas with severe HIV epidemics. These results indicate the importance of continuous PDR monitoring in patients initiating antiretroviral therapy.

Genetic Characteristics of CRF01_AE Among Newly Diagnosed HIV-1-Infected 16- to 25-Year Olds in 3 Geographic Regions of Guangxi, China.

The aim of this study was to analyze the distribution of clusters and drug resistance of CRF01_AE among newly diagnosed, treatment-naïve HIV-infected teenagers and young adults in 3 major HIV-affected geographic regions of Guangxi Province, including the cities of Hezhou, Liuzhou, and Nanning. Samples were sequentially collected from newly diagnosed HIV-infected 16- to 25-year olds in these 3 regions from 2009 to 2013. The viral genome was extracted, and the partial pol gene was amplified and sequenced. Phylogenetic analyses were used to determine HIV-1 subtypes and CRF01_AE clusters. Transmitted drug resistance (TDR) mutations were identified using the 2009 WHO list of TDR mutations. A total of 216 sequences were obtained from CRF01_AE strains, which accounted for 83.1% of the 260 genotyped samples, of which 36 were from Hezhou, 147 from Liuzhou, and 33 in Nanning. Most (83.3%, 180/216) were from heterosexuals, followed by injection drug users (5.6%), homosexuals (4.2%), and unknown risk group (6.9%). Based on phylogenetic analyses by the maximum likelihood method, 5 distinct clusters (cluster 1-5) were identified with 213 (98.6%) sequences, whereas 3 (1.4%) sequences were ungrouped. In Hezhou, 88.9% (32/36) of CRF01_AE infections were caused by cluster 2, and 11.1% (4/36) were caused by cluster 1. In Liuzhou, 83.0% (122/147) of the CRF01_AE strains were found in cluster 1, 11.6% (17/147) from cluster 2, 1.4% (2/147) from cluster 3, 2.7% (4/147) from cluster 4, and 0.7% (1/147) from cluster 5. The distribution of CRF01_AE clusters was more even in Nanning than it was in the other 2 regions, with 18.2% (6/33) from cluster 1, 36.3% (12/33) from cluster 2, 9.1% (3/33) from cluster 3, 18.2% (6/33) from cluster 4, and 12.1% (4/33) from cluster 5. The most frequent TDR mutations were M46I (2) in the protease region and Y181C (2) from the reverse transcriptase fragment. Clusters 1 and 2 of CRF01_AE strains were prevalent in Liuzhou and Hezhou, respectively. However, multiple CRF01_AE clusters existed in Nanning. This can be partially explained by the high mobility of laborers in Nanning, the capital city of Guangxi. The prevalence of TDR was low.

[The study of an in-house method for drug resistance genotyping testing on HIV-1 strains prevailing in China].

OBJECTIVE: To evaluate the amplification rate and the lowestlower detection limit of an in-house HIV-1 Drug resistant (HIVDR) genotyping test. METHODS: A total of 30 plasma samples were selected, which covered all major HIV-1 subtypes predominating prevailing in China (B', CRF07_BC, CRF01 _AE). The viral loads of the 30 selected samples were detected in triplicate by Easy Q method and the average values were taken as the viral loads of the samples. Each sample was diluted to the concentration of > 1000 copies/ml, 401-1000 copies/ml, 101-400 copies/ml, 50-100 copies/ml and < 50 copies/ml with HIV-negative plasma. After extraction of nucleic acids, RT-PCR and nested PCR amplification were performed, the efficiency of amplification of each subtype and the minimum detection limit were determined statistically based on the PCR results. RESULTS: The viral loads of the selected samples ranged from 2.03 x 10(2)-5.92 x 10(4) copies/ml. The sample of 50-1000 copies/ml have a high amplification rate (86%). CONCLUSION: The In-house method for HIV-1 drug resistance genotyping has a high sensitivity with a high successful amplification rate, especially in the samples with low viral load. This method can be used to the detection of drug-resistant virus and to provide scientific data to treatment options for patients.

[Impacts of HIV-1 resistance mutations associated with nucleoside reverse transcriptase inhibitors on viral fitness].

Nucleoside reverse transcriptase inhibitors which act as a major component of highly active antiretroviral therapy regimens are widely used in treatment of Acquired Immune Deficiency Syndrome. However, the emergence of drug-resistant variants of HIV-1 severely limits the effectiveness of these drugs. Many drug resistance mutations confer a fitness cost, which can be partially overcome by compensatory mutations or other molecular mechanisms. This review focuses on the impacts of resistance mutations emerging during treatment with nucleoside reverse transcriptase inhibitors on viral fitness, and inter actions between these mutations.

[Drug resistance and associated factors on HIV in Liangshan prefecture, Sichuan province].

OBJECTIVE: To investigate the HIV drug resistance among HIV/AIDS patients who had received highly active antiretroviral treatment (HAATR) in Liangshan prefecture and related factors. METHODS: This investigation was conducted from August to October 2010. Data on epidemiology, treatment, CD4(+) T cell, viral load and drug resistance tests were collected. RESULTS: 233 (73.50%) had a viral load of < 1000 copy/ml, with the median CD4(+) T cell count as 329 cell/µl. 26 samples appeared to be drug resistant, with the rate as 8.20%. Among 84 patients with antiviral therapy failure, the overall drug resistance rate was 30.95% (26/84). While 24 (28.57%) were resistant to non-nucleoside reverse transcriptase inhibitor (NNRTI) drugs. Among nucleoside reverse transcriptase inhibitors (NRTI), 7 (8.33%) were resistant. 1 (1.19%) had protease inhibitor (PI) resistance mutations identified. Factors that significantly associated with drug resistance would include: being injecting drug users (AOR = 3.37, 95%CI: 1.06 - 10.66, P = 0.0390), having had chronic diarrhea > 1 month (AOR = 8.38, 95%CI: 1.87 - 37.69, P = 0.0055), having had CD4(+) T cell < 200 (AOR = 3.48, 95%CI: 1.29 - 9.39, P = 0.0139), being residents from Butuo area (AOR = 17.68, 95%CI: 4.97 - 62.86, P < 0.0001). When comparing with other areas, data from Butuo showed that people who carried Yi ethnicity (AOR = 17.35, 95%CI: 2.01 - 149.73, P = 0.0095) and were literate (having had primary or higher levels of education) (AOR = 0.18, 95%CI: 0.08 - 0.42, P < 0.0001), being married or having cohabited relations (AOR = 8.17, 95%CI: 2.35 - 28.39, P = 0.001) were found to be less adherent (AOR = 0.05, 95%CI: 0.02 - 0.13, P < 0.0001) to the treatment. CONCLUSION: Successful antiviral outcomes were seen among those AIDS patients under treatment, in Liangshan prefecture. Resistance rates were significantly different in regions. For IDUs, enforcement on subjects including prevention on drug resistance, adherence to HAART and treatment for drug addiction should be strengthened and programs being integrated.

[Feasibility of using dried blood spots to detect HIV drug resistance genotyping].

OBJECTIVE: This study aimed at exploring the feasibility of using dried blood spots (DBS) to detect HIV drug resistance genotyping in China by comparing the results of drug resistance from DBS, plasma and whole blood samples. METHODS: Blood samples were collected from 39 AIDS patients from Anhui (10), Yunnan (13), Hunan (6) and Xinjiang (10) provinces and autonomous regions. The HIV strains that infected these patients covered all the major HIV-1 subtypes prevailing in China (B, CRF01_AE, CRF07_BC). HIV drug resistance genotyping assay was performed on DBS as well as on the whole blood and plasma samples from the same patients simultaneously by using an in-house nest RT-PCR method. Drug resistance levels were determined based on Stanford University HIV drug resistance database, and the results from these three types of samples were compared. RESULTS: The percentages of successful amplification of protease and reverse transcriptase regions in the pol gene were 95% (37/39) from DBS, 92% (36/39) from whole blood and 100% (39/39) from plasma samples. The sequences from the three types of samples showed more than 99% identity.86% (31/36) of the DBS samples had the same set of drug resistance mutations as those which were detected from plasma samples. The differences probably resulted from mixed bases. CONCLUSIONS: There was no major difference in detecting HIV drug resistance genotyping among DBS, plasma and whole blood samples. Therefore, DBS is useful for detection of HIV drug resistance genotyping and is particularly valuable in developing countries like China, especially in remote rural regions.

[Genotypic drug-resistance of HIV-1 CRF01_AE in Zhejiang province, 2004-2007].

OBJECTIVE: To study the genotypic drug-resistant mutation among treat-naïve or treated patients infected with HIV-1 CRF01_AE in Zhejiang province during 2004-2007. METHODS: HIV-1 pol amplicons (PR + RT) from 13 treated and 43 treat-naïve patients were obtained by reverse transcription-polymerase chain reaction (RT-PCR). The sequences were analyzed for genotypic antiretroviral resistance through online tools (http://hivdb.stanford.edu). RESULTS: The median count of CD4+ T lymphocytes in 43 treat-naïve patients was 229 cells /mm3 and the median log10 viral load was 3.41. Some drug-resistant mutations were seen in these samples including amino acid 10, 46, 71, in the genes ofprotease (PR) and 103, 118, in the genes of reverse transcriptase (RT) whereas twenty-nine resistance mutations in the genes of PR and RT were obtained in the 13 treated patients (8/13, 61.5%). The high prevalence of drug-resistant mutations was observed in patients who had been receiving HAART (hight active antiretroviral therapy). Among them, cross drug resistance was dominant. Correspondingly, the median counts of CD4+ T lymphocytes and the log10 viral load were 186 cells/mm3 and 3.91. CONCLUSION: There was a low prevalence of genotypic drug-resistant mutations in treat-naïve patients, but higher drug-resistant mutation in treated patients. More attention should be paid to the transmission of drug-resistant HIV strains and the antiretroviral therapy recipe should be adjusted correspondingly for the development of ART drugs, intervention as well as clinical therapy programs.

[Apoptosis in neonatal rat lung exposed to hyperoxia].

OBJECTIVE: Exposure to high concentrations of oxygen in the neonatal period may impair lung growth and is a major contributing factor to the development of bronchopulmonary dysplasia (BPD). Cell death from hyperoxic injury may occur through either an apoptotic or nonapoptotic pathway. The aim of the present study was to investigate the effect of hyperoxia on caspase-3 and p53 gene expression and apoptosis in the lungs of neonatal rats, so as to determine the type of cell death that occurs in the lungs of neonatal rats exposed to hyperoxia. METHODS: Hyperoxic lung injury model was established by exposing to 95% O(2) in the neonatal period of Spraque-Dawley rats. The levels of caspase-3 mRNA and p53 mRNA expression in the lungs of neonatal rats exposed to 95% hyperoxia or room air were detected by RT-PCR. To quantify PCR products, PCR products were electrophoretically separated with 1.5% agarose gels. The optical density (A) values of the DNA bands were quantified by complete gel documentation and analysis system. The A ratios of p53/beta-actin denoted the relative content of p53 mRNA, results were showed as mean +/- standard deviation. The specific positive or negative bands of caspase-3 in electrophoresis gels were counted, Fisher's exact test of propabilities was used to determined statistically significant differences between two groups. We determined the extent of apoptosis taking place in the lungs of neonatal rats exposed to 95% hyperoxia using terminal deoxyribonucleotide transferase-mediated deoxyuridine triphosphate-fluorescence nick-end labeling (TUNEL) in 7-d-old neonatal lung. Under light microscope, five areas of lung parenchyma were systematically and randomly photographed from each animal and positive cells among 500 lung cells were calculated. Results were showed as mean +/- standard deviation. RESULTS: We found increased levels of p53 messenger RNA, a gene associated with apoptosis, in the lungs of neonatal rat born and raised in 95% hyperoxia. Moderate increase in the level of p53 mRNA was found in the hyperoxic-treated group at 24 h (q = 3.2305, P > 0.05). Significant increase in the level of p53 mRNA was found in the hyperoxic-treated group at 48 h (q = 7.2941, P < 0.01). The levels of p53 mRNA expression in neonatal rat lungs exposed to 95% O(2) for 72 h or 96 h returned to normal level. The levels of caspase-3 mRNA expression were very low or absent in the hyperoxic-treated groups at 12 h, 24 h, 48 h, 72 h and 96 h or in the air-breathing groups at 12 h, 24 h, 48 h, 72 h and 96 h. An increase in the number of cells undergoing apoptosis was found in the hyperoxic-treated group at 7 d (F = 56.5010, P < 0.001) which was significantly greater than the number of apoptotic cells found in the lungs of rats of the same age exposed to room air. CONCLUSION: Our results suggested that 95% hyperoxia could temporarily up-regulate the gene expression of p53, which induced the transcription of p21(WAF/CIP1) mRNA. Furthermore, p21(WAF/CIP1) could lead to cell cycle arrest and inhibit proliferation of lung cells. Meanwhile, p53 could also promote apoptosis of lung cells. Therefore, exposure to high concentrations of oxygen in the neonatal period may impair lung growth and is a major contributing factor to the development of bronchopulmonary dysplasia (BPD), and hyperoxia may affect the future lung growth and lead to barrier of lung development. The treatments of anti-apoptosis and promoting alveoli growth hold a promising perspective in hyperoxic lung injury. The level and ratio of caspase-3 gene expression were very low or absent in the lungs of neonatal rats exposed to 95% O(2) or room air. We speculated that caspase-3 gene expression was not essential in the hyperoxia induced lung cell apoptosis in neonatal rats.

[Effects of U74389G on pulmonary macrophage influx and lung development in 95% O2 exposed newborn rats].

OBJECTIVE: Oxygen toxicity is believed to play a critical role in the pathogenesis of bronchopulmonary dysplasia (BPD). U74389G, a potent 21-aminosteroid antioxidant, was applied to the 95% O(2) induced acute lung injury in newborn rat model. The present study aimed to investigate the mechanism of hyperoxic lung injury and the interaction of possible mediators, and to explore the effect of antioxidant intervention. METHODS: Newborn Sprague-Dawley rats were randomly divided into four groups: air-exposed control, air-exposed treated with U74389G, hyperoxia-exposed control, hyperoxia-exposed treated with U74389G. Hydroxyl radical formation (2,3-DHBA and 2,5-DHBA) was assessed by an aromatic hydroxylation assay using GC/MS with salicylate as the probe. The 8-isoprostane, a specific marker for in vivo lipid peroxidation, was quantitated by enzyme immunoassay. Pulmonary macrophage influx and nitrotyrosine formation were measured by means of immunohistochemistry. (3)H-TdR (autoradiography) incorporation was assessed as an index of active lung cell growth. RESULTS: Exposure to 95% O(2) for 7 days induced significant lung injury and mortality. The contents of hydroxyl radical in the hyperoxia-exposed lungs were dramatically increased [(2,3-DHBA 49.2 +/- 3.5 pmol/mg), (2,5-DHBA 55.8 +/- 2.3 pmol/mg), P < 0.05) and were decreased by treatment with U74389G [(2,3-DHBA 37.9 +/- 2.4 pmol/mg), (2,5-DHBA 31.3 +/- 1.9 pmol/mg), P < 0.05). The level of 8-isoprostane in the lungs of 95% O(2)-exposed newborn rats was significantly raised (546.6 +/- 32.2 pg/mg, P < 0.05) and lowered down by U74389G (358.5 +/- 24.1 pg/mg, P < 0.05). This phenomenon was also observed in the air-exposed animals. Remarkable pulmonary macrophage infiltration was evident in hyperoxia-exposed newborn rats and was attenuated by U74389G treatment. Nitrotyrosine distributed in the lung parenchyma and epithelial cells of large airway of hyperoxia-exposed newborn rats. The extent of protein nitration was reduced by U74389G, but the oxygen induced morphological change was not significantly improved by U74389G treatment. Exposure to 95% O(2) induced lung growth arrest as shown by (3)H-TdR incorporation. U74389G partially preserved active lung cell growth in hyperoxia-exposed rats, but showed an inhibitory effect on normal lung cell growth. CONCLUSION: Through scavenging hydroxyl radical and lipid peroxides, U74389G could block pulmonary macrophage influx and partly avert alveolar development arrest in hyperoxia-exposed newborn rats. Antioxidant intervention holds promising in hyperoxic lung injury though cautions should be taken as possible interference on normal cell development.