HIV-1 drug resistance mutations in children who failed non-nucleoside reverse transcriptase inhibitor-based antiretroviral therapy.

Non-nucleoside reverse transcriptase inhibitor (NNRTI)-based regimens have recently been used in HIV-1 infected children in resource-limited settings. Treatment failure with this regimen has become more common. A second regimen needs to be prepared for the Thai national program. Genotypic resistance testing was conducted among HIV-1 infected children who experienced virological failure with antiretroviral therapy (ART) using NNRTI-based regimens. Patterns of resistance mutations were studied and options for a second regimen were determined. There were 21 patients with a median (IQR) age of 4.1 (1.9-7.7) years. Sixteen patients were males. The median CD4 cell count and HIV-1 RNA at the time of virological failure were 647 cells/mm3 and 5.3 log copies/ml, respectively. The prevalences of patients with > or =1 major mutation conferring resistance to NRTIs and NNRTIs were 52% and 43%, respectively. Thymidine analoque mutations, M184V/I, and Q151M were observed in 38%, 33%, and 5%. The patterns of resistance mutations suggest that 48% of patients need a protease inhibitor-based regimen for the second regimen and didanosine+lamivudine is the most required nucleoside reverse transcriptase inhibitor backbone.

Multi-drug resistant HIV-1 reverse transcriptase genotype in children treated with dual nucleoside reverse transcriptase inhibitors (NRTIs).

BACKGROUND: Multi-drug resistant HIV mutants have been reported after prolonged dual antiretroviral therapy. OBJECTIVE: To evaluate the prevalence and resistance pattern in HIV-infected children treated with dual NRTIs. MATERIAL AND METHOD: Records of HIV-infected children treated with dual NRTIs at Srinagarind Hospital, Khon Kaen University, Thailand, were reviewed for baseline data and their consensually-stored plasma were checked for the occurrence of HIV mutants by genotyping. RESULTS: Fifty-seven HIV-infected children were treated with dual NRTI regimens (27 males; 30 females). The median age and median CD4+ T-lymphocyte at genotypic testing were 83.5 months and 10.9%, respectively. The median duration of ARV therapy was 22 months. More than half the children (42) were on zidovudine and didanosine. A set of three or more nucleoside analog mutations (NAMs), conferring multi-dideoxynucleoside resistance, was found in 60% of the cases. CONCLUSION: High percentages of NAMs were found in HIV-infected children previously on dual ARV therapy for long periods. Genotypic testing was helpful in designing the second antiretroviral regimen.

Quantitative detection of human immunodeficiency virus type 1 (HIV-1) viral load by real-time RT-PCR assay using self-quenched fluorogenic primers.

HIV-1 viral load is a basic marker to evaluate the severity of HIV-1 related diseases and to monitor the effectiveness of treatment. A method based on real-time RT-PCR technology has been developed to quantify HIV-1 RNA using self-quenched fluorogenic primers known as LUX primers. They were used in this study to recognize a low variable gag region of subtype E and B consensus sequences. Specificity was verified by amplicon melting temperatures. An external standard curve was constructed with 10 fold serial dilutions of synthetic HIV-gag RNA. A broad range linear relationship (10 to 10(6) copies/ml) was observed between the number of PCR cycles needed to detect a fluorescent signal and the number of RNA copies. The intra- and inter-assay coefficients of variation were 0.72 to 2.54% and 3.14 to 8.83%, respectively, thus indicating good reproducibility. Thirty out of fifty HIV-infected individual plasma samples were quantified by this method and compared with the AMPLICOR HIV-1 Monitor assay, which is widely considered the reference technique for HIV-RNA viral load measurement. The results indicate that the AMPLICOR HIV-1 Monitor assay and real-time RT-PCR using LUX primers are in good agreement (mean difference in log10 copies/ml+/-2 standard deviations = 0.21+/-1.34).

Quantification of HIV-1 RNA load by one-tube-one-step RT PCR and real time PCR assay with TaqMan probe.

OBJECTIVES: To develop a less expensive assay to calculate HIV-1 viral load for use in resource-limited countries. MATERIAL AND METHOD: An In-house One-tube-one-step Viral Load Assay (IOVA) was developed by using real-time PCR-based with TaqMan probe. Primers and probe were designed from the conserved region of sequences from all HIV subtypes. A standard curve was generated from reference virus in various dilutions. IOVA was applied on 105 HIV-positive and 25 HIV-negative samples and compared with the results from ROCHE AMPLICLOR. RESULTS: IOVA measured HIV RNA in the samples ranging from 125 to 2 x 10(6) copies/mL. The coefficient of variation of intra- and inter-assay ranged from 0.68% to 7.89%. The sensitivity, specificity, positive and negative predictive values were 92%, 100%, 100% and 79.5% respectively. The parallel quantitative analysis showed high correlation (r=0.95) between IOVA and AMPLICOR. CONCLUSION: A new HIV-1 viral load assay was developed and validated. It was reliable and less expensive.

Establishment of real-time polymerase chain reaction-based assay for quantitation of Epstein-Barr virus DNA in healthy donors and in patients with EBV associated lymphoma.

Epstein-Barr virus (EBV) is associated with several malignancies including nasopharyngeal carcinoma and lymphoma in immunocompromised patients. Quantitative monitoring of EBV DNA in these patients has recently become essential for management of the disease. In the present study the authors developed a rapid and reliable real-time PCR to quantify the EBV DNA in peripheral blood mononuclear cell (PBMC) using hybridization probe technique. The real-time primers and probes in this real-time PCR system were designed based on EBNA-1 sequence. The newly-established real-time PCR demonstrated its high sensitivity (as few as 10 copies of EBV could be detected) and specificity. The intra- and inter-assay variations of the assay were shown to be within a 0.5-log10-difference range. A total of 2 EBV-seronegative, 14 EBV-seropositive healthy donors and 4 patients with PCNSL were enrolled into the study. Our results revealed the median of EBV-DNA in lymphoma patients (7886 copies/10(6) PBMC or 15,150 copies /microg DNA) was higher than that of healthy donors (<10 copies/l0(6) PBMC or <10 copies/microg DNA) with statistic significance (P < 0.01). Assessment of this assay in larger number of donors and patients will provide clinical cut-off values which are essential for monitoring and diagnosis of EBV-associated diseases.

Intratypic variations among Thai herpes simplex virus (HSV) isolates determined by restriction fragment length polymorphism (RFLP) analysis.

Whole genomic polymorphisms for 20 HSV-1 and 20 HSV-2 isolates from Thai patients were analyzed by means of Restriction Fragment Length Polymorphism (RFLP) analysis using 4 restriction endonucleases: BamHI, Kpnl, HindIII, and EcoRI. Variations in cleavage sites among the HSV-1 and HSV-2 isolates were compared to the cleavage patterns of standard HSV-1 strain KOS and HSV-2 strain Baylor 186. Although 70% of HSV-1 isolates with BamHI digestion, 50% with Kpnl, 75% with HindIII and 70% with EcoRI digestion were found to be similar to the standard HSV-1 (KOS) pattern, new BamHI restriction sites were detected in some HSV-1 isolates. For HSV-2 isolates, 85% had the same pattern as the standard HSV-2 (Baylor 186) after digestion with BamHI, HindIII, and EcoRI. No difference was observed with Kpnl digestion. When the patterns from the 4 enzymes were combined, HSV-1 isolates showed more divergence than the HSV-2 isolates. HSV-1 isolates found in both non-genital and genital lesions had more variety than the HSV-2 isolates. This suggests that intratypic variations in HSV-2 are fewer than in HSV-1.

Prevalence of cytomegalovirus, human herpesvirus-6, and Epstein-Barr virus in periodontitis patients and healthy subjects in the Thai population.

Fifty periodontitis patients and 30 healthy patients with oral cavities were selected from the Faculty of Dentistry, Mahidol University, Bangkok, Thailand, from March 2001 to November 2002. Their ages varied between 15 and 70 years. Among the periodontitis patients, specimens were collected from both disease and healthy sites. All samples were evaluated for the presence of CMV, HHV-6, and EBV-1 by nested PCR. Among the periodontitis patients, CMV was found in 34%, of which 8% were at the disease sites, 10% were at the healthy sites, and 16% were from both sites. EBV was not found in this group of the patients, while HHV-6 was found in 4%, at the disease sites only. CMV was found in one (3.3%) healthy control while HHV-6 and EBV-1 were not found. The depth of sample sites, various demographic and baseline characteristics eg sex, age, occupation and root planning were not associated with the presence of these viruses.

Development and comparison of the real-time amplification based methods--NASBA-Beacon, RT-PCR taqman and RT-PCR hybridization probe assays--for the qualitative detection of sars coronavirus.

The aim of this study was to develop a rapid, sensitive and robust procedure for the qualitative detection of SARS coronavirus RNA. Three unique detection formats were developed for real-time RNA amplification assays: a post amplification detection step with a virus-specific internal capture probe based on Taqman (RT-PCR TaqMan assay), hybridization probe (RT-PCR hybridization probe assay) and a real-time assay with virus-specific molecular beacon probes (NASBA-Beacon assay). The analytical sensitivity or reproducibility of the test results among those three assays was compared. All assays yielded results by detecting SARS coronavirus targeting the BNI-1 region in less than 2 hours. RNA detection by all the formats was unaffected by the presence of human sputum. The limits of detection were at least 10 copies of input RNA for both RT-PCR formats (RT-PCR TaqMan and RT-PCR hybridization probe assays), while the NASBA-Beacon assay could detect as little as 1 copy per reaction, with high reproducibility of the coefficient of variation (CV) of <10. These results demonstrate that real-time NASBA provides a rapid and sensitive alternative to RT-PCR for the routine qualitative assay of sputum for SARS corona viral RNA detection.

Prevalence of HIV-1 drug resistance in antiretroviral-naive pregnant Thai women.

HIV-1 drug resistance may limit the use of antiretrovirals when attempting to reduce the vertical transmission rate. Establishing the prevalence of the HIV-1 mutations associated with antiretroviral resistance in pregnant women will enable clinicians to maximize the chances of preventing vertical transmission. In order to determine the prevalence of HIV-1 resistant strains among antiretroviral-naive pregnant Thai women, the nucleotide sequences of the HIV-1 polymerase (pol) gene were evaluated. The plasma samples were collected from the women during the 34th week of pregnancy: numerous secondary mutations could be found in the reverse transcriptase (RT) and protease gene, while no primary mutations in the pol gene were found. The result also showed that by detecting the delta32bp deletion within the CCR 5 locus, it was evident that none of HIV-1 infected individuals had homozygous or heterozygous delta32bp deletions of the CCR5 gene; moreover, no CCR5 gene mutations were found in any individual.

Detection of JC virus infection in progressive multifocal leukoencephalopathy: the first documented case in Thailand.

Progressive Multifocal Leukoencephalopathy (PML) is a demyelinating brain disease caused by human polyoma JC virus (JCV). This disease is an important cause of morbidity and mortality in AIDS patients. Definite diagnosis currently requires a brain biopsy. PCR for JCV of CSF, an emerging diagnostic tool, has a high specificity for the diagnosis of PML in patients with characteristics on clinical and neuroradiological findings. The authors report a 36-year-old woman who presented with prolonged fever, progressive weakness, and slow speech for 2 months. Clinical features and MRI findings were compatible with PML. Qualitative PCR for JCV of CSF showed a positive result. This report emphasizes the yield of PCR, the CSF for JCV in a diagnosis of PML, which may reduce the need for a brain biopsy in such cases.

The effects of antiretroviral dose modification on the re-emergence of HIV-1 wild-type strains.

Four human immunodeficiency virus type 1 (HIV-1) treatment-naïve Thai patients began antiretrovival therapy with a triple drug regimen -zidovudine plus lamivudine plus indinavir; this regimen was modified at week 20 of therapy because of drug toxicity. The virus in all patients was suppressed to lower than 400 copies/ml while they were taking the triple antiretroviral drug regimen. However, suppression was lost after changing the antiretroviral regimen. A comparison of HIV-1 DNA sequences taken from the baseline (day 0) and week 24 showed no significant overgrowth in HIV-1 drug-resistant strains. There was no difference in the protease and reverse transcriptase (RT) mutation profiles. Resistant variants did not emerge, even after sub-therapeutic levels of antiretroviral drugs had been introduced to these patients for 4 weeks. These findings may have clinical implications for long-term treatment strategies.

Prevalence of HIV-1 polymerase gene mutations in pre-treated patients in Thailand.

To determine the prevalence of drug resistance-conferring mutations in human immunodeficiency virus type 1 (HIV-1), 83 HIV-1 infected Thai patients who had been treated with any antiretroviral drug were studied. HIV-1 RNA was reverse transcribed and amplified by RT-PCR. The direct sequencing of HIV-1 reverse transcriptase (RT) and protease was then performed. Changes in nucleotide and amino acid sequences were determined by comparison with a pNL4-3 reference sequence. Data on mutations associated with resistance to antiretroviral drugs were obtained from literature. The mutations associated with lamivudine resistance (M184V/I) were found most often (in 45.7% of individuals). Zidovudine-resistant mutants: T215Y/F (36%), M41L (28%) and K70R (25.3%) were common; but mutations linked to didanosine (L74V) and multinucleoside-resistant genotypes (Q151M) were rarely recognized (2.4% and 3.6%, respectively). The stavudine-resistant mutant (V75T) and T69 insertions were not found. All subjects who had a significant exposure to antiretroviral drugs and current virological failure in the past carried drug-resistant genotypes. Genotypic resistance to zidovudine, lamivudine, zalcitabine, indinavir and ritonavir appeared in more than one third of the samples, which suggested that the prevalence of the HIV-1 resistance-conferring genotype resisting reverse transcriptase inhibitors and/or protease inhibitors was high in treatment experienced patients.