Ekawat Pasomsub

Emergence of HIV-1 drug resistance mutations among antiretroviral-naïve HIV-1-infected patients after rapid scaling up of antiretroviral therapy in Thailand

BackgroundAfter rapid scaling up of antiretroviral therapy in HIV-1-infected patients, the data of primary HIV-1 drug resistance in Thailand is still limited. This study aims to determine the prevalence and associated factors of primary HIV-1 drug resistance in Thailand.MethodsA prospective observational study was conducted among antiretroviral-naïve HIV-1-infected Thai patients from 2007 to 2010. HIV-1 subtypes and mutations were assayed by sequencing a region of HIV-1 pol gene. Surveillance drug resistance mutations recommended by the World Health Organization for surveillance of transmitted HIV-1 drug resistance in 2009 were used in all analyses. Primary HIV-1 drug resistance was defined as the presence of one or more surveillance drug resistance mutations.ResultsOf 466 patients with a mean age of 38.8 years, 58.6% were males. Risks of HIV-1 infection included heterosexual (77.7%), homosexual (16.7%), and intravenous drug use (5.6%). Median (IQR) CD4 cell count and HIV-1 RNA were 176 (42-317) cells/mm3 and 68,600 (19,515-220,330) copies/mL, respectively. HIV-1 subtypes were CRF01_AE (86.9%), B (8.6) and other recombinants (4.5%). The prevalence of primary HIV-1 drug resistance was 4.9%; most of these (73.9%) had surveillance drug resistance mutations to only one class of antiretroviral drugs. The prevalence of patients with NRTI, NNRTI, and PI surveillance drug resistance mutations was 1.9%, 2.8% and 1.7%, respectively. From logistic regression analysis, there was no factor significantly associated with primary HIV-1 drug resistance. There was a trend toward higher prevalence in females [odds ratio 2.18; 95% confidence interval 0.896-5.304; p = 0.086].ConclusionsThere is a significant emergence of primary HIV-1 drug resistance in Thailand after rapid scaling up of antiretroviral therapy. Although HIV-1 genotyping prior to antiretroviral therapy initiation is not routinely recommended in Thailand, our results raise concerns about the risk of early treatment failure in patients with primary HIV-1 drug resistance. Interventions to prevent the transmission of HIV-1 drug resistance and continuation of surveillance for primary HIV-1 drug resistance in Thailand are indicated.

Pharmacogenetic markers of CYP2B6 associated with efavirenz plasma concentrations in HIV‐1 infected Thai adults

AIMS To investigate the frequency of CYP2B6 polymorphisms and the influence of haplotype structure on plasma efavirenz concentrations in Thai adults with HIV-1 infection. METHODS Genotyping of nine single nucleotide polymorphisms (SNPs, c.64C>T, c.499C>G, c.516G>T, c.785A>G, c.1375A>G, c.1459C>T, g.3003T>C, g.18492C>T and g.21563C>T) of CYP2B6 were performed using real-time PCR-based allelic discrimination on blood samples from 52 HIV-infected adults who had received an efavirenz-based regimen. Plasma efavirenz concentrations were measured by high performance liquid chromatography. RESULTS The minor allele frequencies for c.64C>T, c.516G>T, c.785A>G, g.3003C>T, g.18492T>C and g.21563C>T were 0.087, 0.365, 0.413, 0.308 and 0.356, respectively. However, no variant alleles were identified for three SNPs (c.499 C>G, c.1375 A>G and c.1459 C>T). Efavirenz plasma concentrations were significantly associated with c.516G>T (P= 0.0095), c.785A>G (P= 0.0017), g.21563C>T (P= 0.0036) and g.18492C>T (P= 0.0011). The composite CYP2B6 of three SNPs (c.516G ≥ T, c.785A ≥ G and g.21563C ≥ T) genotypes were significantly associated with higher efavirenz concentrations. CONCLUSIONS Our data indicate that the GAC-CYP2B6 haplotype is associated with higher plasma efavirenz concentrations in HIV-infected Thai adults.

Impaired glucose tolerance and insulin resistance in survivors of childhood acute lymphoblastic leukemia: prevalence and risk factors.

Aim/Purpose Survivors of acute lymphoblastic leukemia (ALL) are at increased risks of impaired glucose metabolism, insulin resistance, and metabolic syndrome. The aim of our study was to determine the prevalence of alterations in glucose metabolism and the predisposing factors of these disturbances in survivors of childhood ALL. Patients and methods In 131 ALL survivors, an oral glucose tolerance test was conducted to determine β-cell function/insulin sensitivity. The particular risk factors were analyzed and 6 single nucleotide polymorphisms of diabetic predisposing genes: PAX4 and TCF7L2 were genotyped to evaluate the association between these factors and &bgr;-cell function/insulin sensitivity. Results Ten out of 131 survivors (7.6%) had impaired glucose tolerance (IGT) whereas 40 out of 131 (30.5%) had insulin resistance (IR) and showed characteristics of the metabolic syndrome (hyperinsulinemia, hypertriglyceridemia, and low HDL-C). In the logistic regression analysis, the most important factor predicting IGT and IR was older age of survivors (P=0.014 and P<0.001, respectively) whereas the PAX4 R192H mutation (rs2233580) was significantly associated with IGT after adjustment for age (P=0.043) (adjusted OR 5.28, 95% CI 1.06-26.40). Conclusions Existing evidence suggests that older age is an independent risk factor for developing IGT and IR in childhood ALL survivors, emphasizing the need for life-long metabolic screening. The PAX4 variant might impact individual susceptibility against IGT and diabetes. However, an identification of underlying risk(s) is the rational for future studies.

A study of seven rule-based algorithms for the interpretation of HIV-1 genotypic resistance data in Thailand.

Since the free therapy program was started by the Thai government, the number of patients infected by HIV-1 with access to antiretroviral drugs has increased. The selection of effective interpretation algorithms for antiretroviral drug resistance has become even more important for clinical management. In this retrospective study, the level of agreement was evaluated in 721 antiretroviral-therapy failing HIV-1 subjects. Regarding genetic diversity, about 89% was recognized as non-B variants (CRF01_AE). The level of complete concordant interpretation score in all seven algorithms was recognized in non-nucleoside reverse transcriptase inhibitors (NNRTIs) and protease inhibitors (PIs) (67%), but not in nucleoside reverse transcriptase inhibitors (NRTIs) (52%). Over 10% of the major discordance score with TRUGENE was revealed in didanosine (Agence Nationale de Recherches sur le SIDA[ANRS]; Detroit Medical Centre [DMC]), abacavir (ANRS; Centre Hospitalier de Luxembourg [CHL]), and also with delavirdine, indinavir and amprenavir (Grupo de Aconselhamento Virológico [GAV]). A good to excellent agreement range of kappa scores was detected for most antiretroviral drugs. However, poor agreement with the TRUGENE system (k<0.40) was seen in the ANRS system with didanosine, abacavir and lopinavir; GAV system in indinavir and amprenavir; and DMC system in ritonavir. These might be an option for resource limited countries when selecting the use of a low cost or free algorithm interpretation, which has excellent agreement as the U.S. Food and Drug Administration (FDA)-approved TRUGENE commercial system.

Association of CYP2D6 and CYP2C19 polymorphisms and disease-free survival of Thai post-menopausal breast cancer patients who received adjuvant tamoxifen

Purpose To investigate the impact of CYP2D6 and CYP2C19 polymorphisms in predicting tamoxifen efficacy and clinical outcomes in Thai breast cancer patients. Methods Polymorphisms of CYP2D6 and CYP2C19 were genotyped by the AmpliChip™ CYP450 Test (Roche Molecular Diagnostics, Branchburg, NJ, USA) for 57 patients, who were matched as recurrent versus non-recurrent breast cancers (n = 33 versus n = 24, respectively, with a 5-year follow-up). Results Based on the genotype data, five CYP2D6 predicted phenotype groups were identified in this study including homozygous extensive metabolizer (13 of 57, 22.80%), extensive/intermediate metabolizer (23 of 57, 40.40%), extensive/poor metabolizer (3 of 57, 5.30%), homozygous intermediate metabolizer (14 of 57, 24.50%), and intermediate/poor metabolizer (4 of 57, 7.00%), and three CYP2C19 genotype groups including homozygous extensive metabolizer (27 of 57, 47.40%), extensive/intermediate metabolizer (27 of 57, 47.40%), and homozygous poor metabolizer (3 of 57, 5.30%). The CYP2D6 variant alleles were *10 (52 of 114, 45.60%), *5 (5 of 114, 4.40%), *41 (2 of 114, 1.80%), *4 (1 of 114, 0.90%), and *36 (1 of 114, 0.90%); the CYP2C19 variant alleles were *2 (27 of 114, 23.70%) and *3 (6 of 114, 5.30%). Kaplan–Meier estimates showed significantly shorter disease-free survival in patients with homozygous TT when compared to those with heterozygous CT or homozygous CC at nucleotides 100C>T and 1039C>T (CYP2D6*10) post-menopausal (log-rank test; P = 0.046). They also had increased risk of recurrence, but no statistically significant association was observed (hazard ratio 3.48; 95% confidence interval 0.86–14.07; P = 0.080). Conclusion The CYP2D6 and CYP2C19 polymorphisms were not involved in tamoxifen efficacy. However, in the subgroup of post-menopausal women, the polymorphisms in CYP2D6 and CYP2C19 might be useful in predicting tamoxifen efficacy and clinical outcomes in breast cancer patients receiving adjuvant tamoxifen treatment. As the number of breast cancer patients was relatively small in this study, results should be confirmed in a larger group of prospective patients.

Association of CYP3A4/5 , ABCB1 and ABCC2 polymorphisms and clinical outcomes of Thai breast cancer patients treated with tamoxifen

Background Pharmacogenetic study of cytochrome P450 (CYP) gene CYP2D6 and tamoxifen outcomes remain controversial. Apart from CYP2D6, other drug-metabolizing enzymes and transporters also play a role in tamoxifen metabolic pathways. The aim of this study is to investigate the impact of CYP3A4/5, ABCB1, and ABCC2 polymorphisms on the risk of recurrence in Thai patients who received tamoxifen adjuvant therapy. Methods Patients with early-stage breast cancer who received tamoxifen adjuvant therapy were recruited in this study. All six single-nucleotide polymorphisms (SNPs), including CYP3A4*1B (−392 A>G)/*18(878 T>C), CYP3A5*3(6986 G>A), ABCB1 3435 C>T, ABCC2*1C(−24 C>T), and ABCC2 68231 A>G, were genotyped using real-time polymerase chain reaction assays. The impacts of genetic variants on disease-free survival (DFS) were analyzed using the Kaplan–Meier method and Cox regression analysis. Results The ABCB1 3435 C>T was found to have the highest allele frequency among other variants; however, CYP3A4*1B/*18 could not be found in this study. Patients with heterozygous ABCB1 3435 CT genotype showed significantly shorter DFS than those with homozygous 3435 CC genotype (P = 0.041). In contrast, patients who carried homozygous 3435 TT genotype showed no difference in DFS from wild-type 3435 CC patients. Cox regression analysis showed that the relative risk of recurrence was increased by five times (P = 0.043; hazard ratio = 5.11; 95% confidence interval: 1.05–24.74) in those patients carrying ABCB1 3435 CT genotype compared to those with ABCB1 3435 CC. Conclusion ABCB1 3435 C>T is likely to have a clinically significant impact on recurrence risk in Thai patients with breast cancer who receive tamoxifen adjuvant therapy.

Pathogen lineage-based genome-wide association study identified CD53 as susceptible locus in tuberculosis.

Tuberculosis (TB) is known to be affected by host genetic factors. We reported a specific genetic risk factor through a genome-wide association study (GWAS) that focused on young age onset TB. In this study, we further focused on the heterogeneity of Mycobacterium tuberculosis (M. tb) lineages and assessed its possible interaction with age at onset on host genetic factors. We identified the pathogen lineage in 686 Thai TB cases and GWAS stratified by both infected pathogen lineage information and age at onset revealed a genome-wide significant association of one single-nucleotide polymorphism (SNP) on chromosome 1p13, which was specifically associated with non-Beijing lineage-infected old age onset cases (P=2.54E-08, OR=1.74 (95% CI=1.43–2.12)), when we compared them to the population-matched healthy controls. This SNP locates near the CD53 gene, which encodes a leukocyte surface glycoprotein. Interestingly, the expression of CD53 was also correlated with the patients’ active TB status. This is the first report of a pathogen lineage-based genome-wide association study. The results suggested that host genetic risk in TB is depended upon the pathogen genetic background and demonstrate the importance of analyzing the interaction between host and pathogen genomes in TB.

6 HCV genotyping 9G test and its comparison with VERSANT HCV genotype 2.0 assay (LiPA) for the hepatitis C virus genotyping

In this article, we describe the 6 HCV Genotyping 9G test and its evaluation by using clinical samples and plasmid DNA standards. In tests with 981 plasmid DNA standards, the 6 HCV Genotyping 9G test showed higher than 92.5% sensitivity and 99.4% specificity. The 6 HCV Genotyping 9G test was compared with the VERSANT HCV Genotype 2.0 assay (LiPA 2.0) for detection and discrimination of HCV genotypes in clinical samples. The results of both tests were verified by genomic sequencing. The 6 HCV Genotyping 9G test demonstrated a 100% agreement with the sequencing results, which was higher than LiPA 2.0. These results indicate that the 6 HCV Genotyping 9G test can be a reliable, sensitive, and accurate diagnostic tool for the correct identification of HCV genotypes in clinical specimens. 6 HCV Genotyping 9G test can genotype six HCV types in 1 PCR in 30min after PCR amplification. The 6 HCV Genotyping 9G test, thus provide critical information to physicians and assist them to apply accurate drug regimen for the effective hepatitis C treatment.In this article, we describe the 6 HCV Genotyping 9G test and its evaluation by using clinical samples and plasmid DNA standards. In tests with 981 plasmid DNA standards, the 6 HCV Genotyping 9G test showed higher than 92.5% sensitivity and 99.4% specificity. The 6 HCV Genotyping 9G test was compared with the VERSANT HCV Genotype 2.0 assay (LiPA 2.0) for detection and discrimination of HCV genotypes in clinical samples. The results of both tests were verified by genomic sequencing. The 6 HCV Genotyping 9G test demonstrated a 100% agreement with the sequencing results, which was higher than LiPA 2.0. These results indicate that the 6 HCV Genotyping 9G test can be a reliable, sensitive, and accurate diagnostic tool for the correct identification of HCV genotypes in clinical specimens. 6 HCV Genotyping 9G test can genotype six HCV types in 1 PCR in 30min after PCR amplification. The 6 HCV Genotyping 9G test, thus provide critical information to physicians and assist them to apply accurate drug regimen for the effective hepatitis C treatment.

Coreceptor tropism determined by genotypic assay in HIV-1 circulating in Thailand, where CRF01_AE predominates†

Chemokine (C‐C motif) receptor 5 (CCR5) inhibitors are a novel class of antiretroviral agents that are promising for treatment of patients who harbour the HIV‐1 R5 strain. Data on coreceptor tropism in non‐B HIV‐1 subtypes are limited. We studied coreceptor tropism in HIV‐1 circulating in Thailand, where CRF01_AE predominates, using a genotypic assay.

Surveillance of Transmitted HIV Drug Resistance in Antiretroviral-Naive Patients Aged less than 25 Years, in Bangkok, Thailand

Emergence of transmitted HIV drug resistance (TDR) is a concern after global scale-up of antiretroviral therapy (ART). World Health Organization had developed threshold survey method for surveillance of TDR in resource-limited countries. ART in Thailand has been scaling up for >10 years. To evaluate the current TDR in Thailand, a cross-sectional study was conducted among antiretroviral-naive HIV-infected patients aged <25 years who newly visited infectious disease clinic in a university hospital, in 2011. HIV genotypic-resistance test was performed. World Health Organization 2009 surveillance drug-resistance mutations were used to define TDR. Of 50 patients, the prevalence of TDR was 4%. Of 2 patients with TDR, 1 had K103N and the other had Y181C mutations. Transmitted HIV drug resistance is emerging in Thailand after a decade of rapid scale-up of ART. Interventions to prevent TDR at the population level are essentially needed in Thailand. Surveillance for TDR in Thailand has to be regularly performed.