Jutarat Praparattanapan

Comparison of Immunogenicity and Safety of Four Doses and Four Double Doses vs. Standard Doses of Hepatitis B Vaccination in HIV-Infected Adults: A Randomized, Controlled Trial

Background HBV vaccination is recommended in HIV-infected adults with CD4+ cell count >200/mm3 although the efficacy is only 33.3% -65%. We conducted a randomized, controlled trial to evaluate the efficacy and safety of three regimens of HBV vaccination at Chiang Mai University Hospital, Thailand. Methods From February 4, 2011 to May 4, 2012, 132 HIV-infected adults with CD4+ cell counts >200 cells/mm3, undetectable plasma HIV-1 RNA, and negative for all HBV markers were randomly assigned to receive one of three recombinant vaccine (Hepavax-Gene® Berna, Korea) regimens: 20 μg IM at months 0, 1, and 6 (Standard doses group, n=44), 20 μg IM at months 0, 1, 2, 6 (four doses group, n=44), or 40 μg IM at months 0, 1, 2, and 6 (four double doses group, n=44). The primary outcomes were to compare the immunogenicity and safety between the four-doses groups with the Standard doses group. Results At months 7 and 12, the percentages of responders (anti-HBs ≥10 mIU/mL) were 88.6% and 70.4% in the Standard doses group, 93.2% and 86.4% in the four doses group, (P=0.713 and 0.119), and 95.4% and 88.6% in the four double doses group, (P=0.434 and 0.062), respectively. Factors associated with a high titer level (anti-HBs ≥100 mIU/mL) were vaccination schedule and younger age. The most common adverse event was pain at the injection site (42.4%); this was significantly more frequent in the four double doses group compared to the Standard doses group. No serious adverse events were observed. Conclusions In Northern Thailand, the standard three-doses HBV vaccination in HIV-infected adults with CD4+ cell counts >200 cells/mm3 and undetectable plasma HIV-1 RNA is highly effective. Although regimens of four injections of either standard or double doses could not significantly increase the response rate, these regimens may induce higher levels of antibody to the virus. Trial registration information: ClinicalTrials.gov; NCT1289106; http://clinicaltrials.gov/ct2/show/NCT01289106

Development of TaqMan real-time polymerase chain reaction for the detection and identification of Penicillium marneffei.

Penicillium marneffei is a dimorphic fungus, which is endemic in Southeast Asia and responsible for emerging opportunistic infections. Diagnosis of penicilliosis may be difficult when few yeast cells are present, while a gold standard diagnosis technique requires long‐term culture. In order to provide a more rapid and accurate diagnosis, we developed a TaqMan real‐time PCR to detect and identify P. marneffei DNA coding for 5.8S rRNA in purified yeast DNA and clinical samples. All P. marneffei DNA preparations could be detected using specific primers and TaqMan probe. The assay has a sensitivity to detect at least 10 yeast cells in seeded blood. Moreover, it can detect P. marneffei DNA in peripheral blood samples and blood‐culture bottles. Therefore, the real‐time PCR assay may represent a potential tool for early diagnosis of penicilliosis marneffei.

HIV multi-drug resistance at first-line antiretroviral failure and subsequent virological response in Asia

First‐line antiretroviral therapy (ART) failure often results from the development of resistance‐associated mutations (RAMs). Three patterns, including thymidine analogue mutations (TAMs), 69 Insertion (69Ins) and the Q151M complex, are associated with resistance to multiple‐nucleoside reverse transcriptase inhibitors (NRTIs) and may compromise treatment options for second‐line ART.

Isolation and expression of heat shock protein 30 gene from Penicillium marneffei

Penicillium marneffei is a dimorphic fungus that can cause disseminated mycosis, especially in AIDS patients. The role of heat shock proteins and stress response-related proteins in P. marneffei remains unknown. In this study, we isolated a cDNA encoding for heat shock protein 30 (Hsp30) of P. marneffei using an antibody screening method. The DNA sequence and deduced amino acid sequence analysis showed high homology to other fungal hsp30 genes. Expression of P. marneffei hsp30 in response to temperature increase was determined by Northern blot analysis. A high level of hsp30 transcript was detected in yeast cells grown at 37 degrees C, whereas a very low or undetectable transcript level was observed in mycelial cells at 25 degrees C. A recombinant Hsp30 protein was produced and tested preliminarily for its immunoreactivity with sera from P. marneffei-infected AIDS patients using Western blot analysis. The positive immunoblot result, with some serum samples, confirmed the antigenic property of the Hsp30. Collectively, the high response of hsp30 to temperature increase could indicate it may play a role in heat stress response and cell adaptation. This is the first report showing that this small heat shock protein could elicit the human immune response.

Resistance-associated mutations after initial antiretroviral treatment failure in a large cohort of patients infected with HIV-1 subtype CRF01_AE.

The nucleoside reverse transcriptase inhibitors (NRTIs), zidovudine (AZT) and stavudine (d4T) are thymidine analog drugs recommended as first-line antiretroviral therapy in HIV-1-naive patients. Two thymidine analog mutation (TAM) pathways, TAM-1 and TAM-2, confer high levels of resistance with mutations in the viral RT. The relative prevalence of TAM pathways and their associations with other NRTI resistance mutations acquired under the pressure of drug treatment in a large cohort of 1,876 patients infected with HIV-1 CRF01_AE attending the Infectious Disease Clinic, Chiang Mai University Hospital, Chiang Mai, Thailand, were studied. From 117 patients infected with HIV-1 CRF01_AE who had plasma HIV-1 RNA of ≥500 copies/mL, 69 patients had at least one TAM. The most common mutation associated with NRTI resistance was M184V/I (89.9%). The TAM-2 (89.9%) pathway occurred approximately two times more frequently than the TAM-1 (43.5%) pathway. The presence of TAM and the TAM-1 pathway was significantly more frequent in the AZT- than the d4T-receiving group ((OR, 2.89; 95% CI, 1.12-7.46; P< 0.05) and (OR, 3.33; 95% CI, 1.19-9.37; P< 0.05), respectively). In conclusion, the TAM-2 pathway was selected more frequently than the TAM-1 pathway by thymidine analog drugs in HIV-1 CRF01_AE-infected patients, while the TAM-1 pathway occurred more frequently than the TAM-2 pathway in such patients with AZT-based treatment. Routine monitoring of plasma HIV-1 RNA may result in less exposure to failing regimens and reduce the opportunity for TAMs to accumulate. However, the low frequency of the TAM-1 pathway in our cohort data suggests that these patients should respond well to second-line regimens containing a ritonavir-boosted protease inhibitor.

Impact of the Frequency of Plasma HIV-1 RNA Monitoring on the Outcome of Antiretroviral Therapy

BACKGROUND Current guidelines for HIV management recommend monitoring plasma HIV-1 RNA level every 3-6 months in patients on a stable antiretroviral regimen. However, cost is the major obstacle to follow the guidelines in resource-limited settings. OBJECTIVE This study aimed to compare the outcome of antiretroviral therapy among HIV-infected patients on a stable regimen who had plasma HIV-1 RNA monitoring once vs. twice yearly. METHODS A retrospective cohort study was conducted among HIV-infected patients receiving antiretroviral therapy since 2002 at Chiang Mai University Hospital, Thailand. We evaluated the incidence of virological failure and number of reverse transcriptase (RT) mutations between groups. RESULTS Of 551 patients on a stable antiretroviral regimen, 405 (73.5%) and 146 (26.5%) patients had plasma HIV-1 RNA measurement once and twice yearly, respectively. Forty-seven of 405 patients (11.6%) in once-yearly group and 15 of 146 patients (10.3%) in twice-yearly group developed virological failure, giving the incidence rate of 2.03/100 and 1.95/100 person-years, respectively. The probability of virological failure did not differ between groups (p=0.897, log-rank test). The number of RT mutations was not statistically different between groups (all p-values>0.05). The predicting factors for virological failure from a multivariate analysis were adherence rate <95% and baseline CD4 cell count <50 cells/mm3 but not the frequency of HIV-1 RNA monitoring. CONCLUSIONS The incidence of virological failure and the number of RT mutations were not different between groups. Therefore, in resource-limited settings, the recommendation to perform plasma HIV-1 RNA measurement once yearly in patients on a stable antiretroviral regimen is justified.

Itraconazole vs Fluconazole as a Primary Prophylaxis for Fungal Infections in HIV-Infected Patients in Thailand

BACKGROUND Disseminated fungal infections are common presenting opportunistic infections among AIDS patients in developing countries. Primary prophylaxis with itraconazole has been shown to be effective in northern Thailand. This study aimed to compare the efficacy of fluconazole vs itraconazole as primary prophylaxis for fungal infections in HIV-infected patients. METHODS A retrospective cohort study was conducted among HIV-infected patients who received primary prophylaxis with fluconazole 400 mg once weekly or itraconazole 200 mg once daily at Chiang Mai University Hospital. We compared the incidence of systemic fungal infections and the probability of disease-free survival between groups. RESULTS From January 2000 to June 2010, 308 HIV-infected patients who received primary fungal prophylaxis were enrolled; 148 were male (48.1%) and the mean age was 38.2 ± 8.0 years. 276 patients received fluconazole and 32 received itraconazole. Baseline CD4+ cell count was 35 (IQR 15, 70) and 50 (IQR 21,75) cells/mm(3) in fluconazole and itraconazole groups, respectively (p=0.159). The median follow-up time was 12 months (IQR 7, 19) in fluconazole group and 15.5 months (IQR 9, 21.5) in itraconazole group. Seven patients (2.5%) who received fluconazole and 2 patients (6.3%) who received itraconazole developed systemic fungal infections, giving the incidence of 17.0 and 34.8/10000 person-months, respectively (p=0.261). The probability of developing any systemic fungal infections or death did not differ between groups. CONCLUSIONS Although P. marneffei has a reduced susceptibility in in vitro to fluconazole, our study has demonstrated that once-weekly fluconazole is at least as effective as once-daily itraconazole as primary prophylaxis for systemic fungal infections in AIDS patients in northern Thailand.

Rilpivirine resistance-associated mutations among antiretroviral-naive patients infected with HIV-1 in Asia.

To the Editors: HIV-1 infection in Asia accounts for a major proportion of the global HIV-1 epidemic. After rapid scale-up of combination antiretroviral therapy (ART), HIV-associated mortality and morbidity in this region have been significantly reduced. Nonnucleoside reverse transcriptase inhibitor (NNRTI)–based regimens are used in the majority of patients on first-line ART in Asia, particularly in developing countries. Rilpivirine (RPV), a new NNRTI of the diarylpyrimidine family, was recently approved by the US Food and Drug Administration to be used in ART-naive patients based on its safety and sustained efficacy. RPV-based regimens would thus be a valid alternative to firstgeneration NNRTI-based regimens in Asia, particularly in patients who cannot tolerate both nevirapine (NVP) and efavirenz (EFV). A previous study demonstrated the significant (6.5%) prevalence of resistance-associated mutations (RAMs) to NVP, EFV, or etravirine among treatment-naive patients, raising the concern of primary drug resistance to NNRTIs in Asia. However, primary drug resistance to RPV in this region has never been evaluated. It has been well established that primary drug resistance can threaten the effectiveness of ART among HIV-1–infected patients who are initiating ART. Fifteen reverse transcriptase mutations (K101E/P, E138A/G/K/Q/R, V179L, Y181C/I/V, H221Y, F227C, and M230I/ L) associated with decreased susceptibility to RPV have been described in the International Antiviral Society–USA (IAS-USA) drug resistance mutations list. TREAT Asia (Therapeutics, Research, Education and AIDS Training in Asia) is a network of clinics, hospitals, and research institutions working to ensure safe and effective delivery of HIV/AIDS treatment throughout the Asia Pacific. This network has developed the TREAT Asia Studies to Evaluate Resistance (TASER) to monitor HIV-1 drug resistance in Asia. The majority of patients enrolled in TASER studies are infected with HIV-1 non–B subtypes, mostly CRF01_AE. It is therefore relevant to study RPV RAMs among ART-naive patients infected with HIV-1 non–B subtypes in this cohort. The primary objective of this study was to determine the prevalence of RPV RAMs among this group of ART-naive HIV-1–infected patients, to estimate the potential use of RPV as a first-line NNRTI in Asia. Secondary objectives were to compare the prevalence of RPV RAMs between patients infected with HIV-1 non–B subtypes and B subtypes and to determine other clinical factors that are associated with the presence of RPV RAMs. ART-naive patients with available pretreatment reverse transcriptase genotypes from the TASER-Monitoring Study (TASER-M) cohort were selected for inclusion in the study. TASER-M recruitment began in 2007, with a total of 11 participating sites from Thailand, Hong Kong, Malaysia, the Philippines, and Indonesia. Ethics approvals were obtained from the institutional review boards at the participating clinical sites and coordinating and data management centers. Informed consent was obtained from participants before enrollment. FASTA files were interpreted using the Stanford University HIV Drug Resistance Database tools (Stanford HIVdb, version 6.1.1) for genotyping and REGA HIV-1 Subtyping Tool— version 2.0 for subtyping. Mutations were analyzed using the IAS-USA 2011 mutation list. Fifteen reverse transcriptase mutations (K101E/P, E138A/ G/K/Q/R, V179L, Y181C/I/V, H221Y, F227C, and M230I/L) associated with decreased susceptibility to RPV. Comparison of categorical data was performed using x2 or Fisher exact test, whereas the Wilcoxon rank sum test was used to compare continuous data. Baseline factors associated with the presence of at least 1 or more RPV RAMs were analyzed using a logistic regression model. We defined the baseline as the period up to 6 months before starting ART. If multiple FASTA files, CD4 and HIV RNA measurements were available during the baseline period, only those closest to start of ART were chosen for analysis. In the logistic model, factors with significant univariate P values at the 10% level were chosen for inclusion in the multivariate model, using a forward stepwise method. Covariates were considered significant in the multivariate model if P values were ,0.05. Site’s income levels were grouped according to World Bank classifications (June 22, 2012). The statistical analyses were performed using SAS (Version 9.2, SAS Institute Inc, Cary, NC) and STATA (Version 12.1, StataCorp, College Station, TX). A total of 1627 HIV treatmentnaive patients were analyzed. The median age was 36 (IQR: 30–44) years, with 68% of the patients being male. The predominant mode of exposure was heterosexual contact (68%), and the median pretreatment CD4 and HIV RNA were 107 cells per microliter and 100,000 copies per milliliter, respectively. The main subtypes were of non–B types (83%), with CRF01_AE representing 74% of the overall subtypes. The TREAT Asia Studies to Evaluate Resistance (TASER) is an initiative of TREAT Asia, a program of amfAR, The Foundation for AIDS Research, with major support provided by the Dutch Ministry of Foreign Affairs through a partnership with Stichting Aids Fonds, and with additional support from amfAR and the National Institute of Allergy and Infectious Diseases (NIAID) of the U.S. National Institutes of Health (NIH) and the National Cancer Institute (NCI) as part of the International Epidemiologic Databases to Evaluate AIDS (IeDEA) (grant no. U01AI069907). Queen Elizabeth Hospital and the Integrated Treatment Centre are supported by the Hong Kong Council for AIDS Trust Fund. The Kirby Institute is funded by the Australian Government Department of Health and Ageing, and is affiliated with the Faculty of Medicine, The University of New South Wales. The content of this publication is solely the responsibility of the authors and does not necessarily represent the official views of any of the institutions mentioned above. The authors have no conflicts of interest to disclose. The Members of The TASER Study are listed in Appendix I.

Transmitted drug resistance in recently infected HIV-positive Individuals from four urban locations across Asia (2007–2010) – TASER-S

BackgroundThe availability of HIV antiretroviral therapy (ART) has been associated with the development of transmitted drug resistance-associated mutations (TDRM). TDRM can compromise treatment effectiveness in patients initiating ART and the prevalence can vary in different clinical settings. In this study, we investigated the proportion of TDRM in treatment-naïve, recently infected HIV-positive individuals sampled from four urban locations across Asia between 2007–2010.MethodsPatients enrolled in the TREAT Asia Studies to Evaluate Resistance – Surveillance Study (TASER-S) were genotyped prior to ART initiation, with resulting resistance mutations analysed according to the WHO 2009 list.ResultsProportions of TDRM from recently infected individuals from TASER-S ranged from 0% to 8.7% - Hong Kong: 3/88 (3.4%, 95% CI (0.71%-9.64%)); Thailand: Bangkok: 13/277 (4.7%, 95% CI (2.5%-7.9%)), Chiang Mai: 0/17 (0%, 97.5% CI (0%-19.5%)); and the Philippines: 6/69 (8.7%, 95% CI (3.3%-18.0%)). There was no significant increase in TDRM over time across all four clinical settings.ConclusionsThe observed proportion of TDRM in TASER-S patients from Hong Kong, Thailand and the Philippines was low to moderate during the study period. Regular monitoring of TDRM should be encouraged, especially with the scale-up of ART at higher CD4 levels.

Thymic Function during 12 Months of Highly Active Antiretroviral Therapy in Thai HIV-Infected Patients with Normal and Slow Immune Recovery.

The aim of this study was to determine and compare thymic output during 12 months of highly active antiretroviral therapy (HAART) in HIV-infected patients with different types of immune recovery. In total, 18 Thai HIV-infected patients with normal immune recovery (NR) and 13 Thai HIV-infected patients with slow immune recovery (SR) were enrolled. T-cell receptor rearrangement excision circle (TREC) levels in peripheral blood mononuclear cells (PBMCs) and CD4(+) T cells were quantified at baseline, and after 6 and 12 months of HAART. CD4(+) T-cell counts in NR patients were significantly higher than those in SR patients after 6 and 12 months of HAART. However, the median TREC levels in PBMCs and CD4(+) T cells in both groups were comparable. Moreover, TREC levels showed similar trends in PBMCs and CD4(+) T cells in both groups during 12 months of HAART. Only patients with SR had significant increases in median TREC levels in PBMCs and CD4(+) T-cells during the first 6 months of HAART. No correlations were found between CD4(+) T-cell count and TREC levels in PBMCs and CD4(+) T cells. These results imply that the increase in CD4(+) T-cell count in SR patients after 12 months of HAART is likely attributable to thymic output and other sources.