Serum levels of MIP-1beta and RANTES in HIV-1 subtype CRF01_AE infected patients with different rates of disease progression.

The beta-chemokines have been shown to inhibit HIV replication in vitro. To evaluate the role of serum beta-chemokines in disease progression and their anti-viral role in vivo, we determined serum levels of macrophage inflammatory protein-1beta (MIP-1beta) and regulated upon activation normal T-cell expressed and secreted (RANTES) of twenty HIV-1 subtype CRF01_AE infected patients: nine progressors (PRs, follow-up CD4+ cell count < 200/mm3 and progression to AIDS or death) and eleven slower progressors (SPs, asymptomatic and/or follow-up CD4+ cell counts > 350/mm3 at the end of follow-up) and determined their plasma viral loads. The subjects were followed for at least 36 months. All had initial CD4 values > 350 cells/mm3. In this longitudinal study, serum levels of MIP-1beta and RANTES in specimens obtained either early or later in the course of HIV infection did not differ significantly between progressors and slower progressors (p > 0.05). There were no significant changes in serum MIP-1beta and RANTES levels over time in either patient group (p > 0.05). No significant associations were observed between plasma viral loads and the measured beta-chemokines (r = -0.205, p = 0.21 for MIP-1beta and r = -0.12, p = 0.492 for RANTES). The results suggest these chemokines do not play a major systemic role in control of viremia or protection against the progression of HIV disease.

Evaluation of HIV-1 viral load detection by modified a commercial real-time PCR reagent used for Light Cycler 1.2 instrument.

OBJECTIVE: Commercial TaqMan real-time PCR reagent was modified and applied on Light Cylcer 1.2 for quantifying HIV-1 RNA in plasma and compared with the reference method; COBAS AmpliPrep/COBAS Amplicor HIV-1 monitor test version 1.5. MATERIAL AND METHOD: Three hundred and eight frozen and fresh plasma samples were used for evaluation. Sequential specimens were also tested for follow-up cases. RESULTS: The correlation between HIV-1 RNA values obtained by reference and modified method with automated and manual sample preparation were significant with r = 0.916 and 0.908 (p < 0.001, p < 0.001) respectively with similar agreement log of mean bias (0.5 versus 0.48). High degree of correlation and agreement were observed between the assays in blind fresh plasma, r = 0.953 (p < 0.001) with 0.15 log difference in HIV-1 RNA level. Among follow-up samples, both methods gave 100% concordant results. CONCLUSION: This modified protocol provided evidence for using modified commercial real-time PCR reagent for HIV-1 RNA quantitative detection as a monitoring tool for HIV/AIDS patients in Thailand.

Surveillance of subtype and genetic variation of the circulating strains of HIV-1 in Thailand.

Two HIV-1 strains, CRF01_AE and subtype B', were reported in Thailand during the early years of the epidemic. Recently, an intersubtype recombination of HIV-1 strain was found in Thailand. Eight-hundred and twenty-eight samples collected during years 1995-2004 from high-risk groups in Bangkok, northern, northeastern, and southern region of Thailand were studied. HIV-1 env nucleotide sequences were used for phylogenetic analysis of the circulating HIV-1 strain. By single HIV-1 region (env) genotyping, CRFO1_AE was found in 97.3% and HIV-1 subtype B was found in 2.7%. A predominance of CRF01_AE was found in all geographic regions. Parallel analysis of the HIV-1 gag and env genes demonstrated that 2.1% and 4.0% of recombinant HIV-1 strains were found using p17 and p24 region sequences, respectively. The recombinant gag gene was also found in one southern isolate. Phylogenetic analysis of HIV-1 isolated from 20 provinces in 2002 suggested the northern and northeastern isolates were more related than the southern isolates which had the lowest genetic diversity of 0.13. The GPGQ V3 loop tip was also present in isolates from all regions. The molecular epidemiological data from this study may be useful for surveillance design as well as targeting prevention efforts. It also provides information regarding new antigenic regions of circulating strains responsible for the HIV-1 epidemic in Thailand.

HIV-1 subtyping using gag/env heteroduplex mobility assay and peptide enzyme-linked immunosorbent assay.

Two HIV-1 subtypes have accounted for virtually all infections in Thailand: subtype B', found mainly in injection drug users (IDUs), and CRF01_AE (initially subtype E), found in over 90% of sexually infected persons and increasingly in IDUs in recent years. During 1997-1998, 227 blood samples were collected from HIV-1 infected individuals consisting of 92 mothers, 35 children and 100 IDUs. The blood samples were subtyped by heteroduplex mobility assay (HMA) and peptide enzyme-linked immunosorbent assay (PEIA). Using gag and env HMA, CRF01_AE and subtype B' accounted for 96-97% and 3-4% of both the mothers and the children, respectively. In the IDU group, 10% of the plasma samples could only be performed by gag HMA and gave the result as CRF01_AE. CRF01_AE and subtype B' using PEIA accounted for 67% and 33% of the IDUs. There was 100% concordance of the results between gag HMA and env HMA. Ninety-five percentages of concordant results were observed between HMA and PEIA. Of the 6/134 (5%) subjects with discordant results, nucleotide sequencing, used as a gold standard, confirmed the HMA result. In this study, HIV-1 was successfully genotyped by HMA and PEIA. However, a comparison of the subtyping results between HMA and PEIA revealed that HMA was slightly more accurate than PEIA.

Initiation of antiretroviral treatment with dual nucleoside reverse transcriptase inhibitors in human immunodeficiency virus-infected infants with less advanced disease in a resource-limited setting: a multi-center study in Thailand 1998-2000.

OBJECTIVES: To evaluate the feasibility, duration of efficacy, and outcome of therapy with dual nucleoside reverse transcriptase inhibitors (NTRI) initiated in HIV-infected infants with mild to moderate disease. MATERIAL AND METHOD: During 1998-2000, a multi-center prospective open-labeled operational study was conducted. Antiretroviral naôve HIV-infected infants were enrolled in seven hospitals to receive either zidovudine (AZT) plus lamivudine (3TC) or AZT plus didanosine (ddI). Infants who were in CDC stage "C3" were excluded from the study. RESULTS: Of the 88 infants, the mean age of treatment initiation was 6.8 months, and the mean initial CD4 was 1538 cells/mm3 (21.4%). The z-scores for weight and height increased after 4-8 months of treatment, and by the 24th month, were +0.89 and +0.69 higher than at enrollment. The CD4% peak increased at 8 months of treatment, by a mean increment of 4.19%, but decreased to the level of 1.08% above baseline by the 24th month of treatment. Three (3.4%) infants died, 11 (12%) had disease progression, 7 (8%) was prematurely discontinued from the study protocol due to poor compliance, and 37 (42%) were lost to follow-up. At the end of 24 months, all remaining 30 children were in stable condition with a chance of clinical and immunological stability of 34% and 68% by intention-to-treat and on-treatment analysis, respectively. CONCLUSION: Clinical and immunological benefit from dual NRTI was limited. Treatment of HIV-infected infant with mild to moderate disease in a resource-limited setting may have limited feasibility due to the high drop-out rate.

Specific immune response and pathological findings in BALB/c mice inoculated with recombinant BCG expressing HIV-1 antigen.

Recombinant BCGs (rBCGs) containing extrachromosomal plasmids with different HIV-1 insert sequences: nef, env (V3J1 and E9Q), gag p17 or whole gag p55 were evaluated for their immunogenicity, safety and persistent infection in BALB/c mice. Animal injected with, rBCG-plJKV3J1, rBCG-pSO gag p17 or rBCG-pSO gag p55 could elicit lymphocyte proliferation as tested by specific HIV-1 peptides or protein antigen. Inoculation with various concentration of rBCG-pSO gag p55 generated satisfactory specific lymphocyte proliferation in dose escalation trials. The rBCG-pSO gag p55 recovered from spleen tissues at different time interval post-inoculation could express the HIV protein as determined by ELISA p24 antigen detection kit. This result indicated that the extrachromosomal plasmid was stable and capable to express Gag protein. It was also demonstrated that rBCGs did not cause serious pathological change in the inoculated animals. The present study suggested the role of BCG as a potential vehicle for using in HIV vaccine development.

Characteristic of HIV-1 in V3 loop region based on seroreactivity and amino acid sequences in Thailand.

The third variable (V3) domain of the envelop (env) protein has been used for determining genetic subtype and phenotypic characteristics of human immunodeficiency virus type 1 (HIV-1) isolates. Based on the seroreactivity of the HIV-1 subtype by V3 peptide binding enzyme immunoassay (EIA) of 351 samples obtained in 1998 from HIV-1 infected individuals and AIDS patients, we found that 283 (80.6%) were subtype E, 20 (5.7%) were subtype B, 28 (8.0%) were cross-reactive between both types and 20 (5.7%) were non-typeable. The degree of seroreactivity of HIV-1 subtype E decreased significantly when the amino acid at the crown of the V3 loop was substituted from a GPGQ motif to GPGR motif. Interestingly, AIDS patients who had V3 sequences of subtype E as GPGR motif had a stronger immunoreactivity to GPGQ motif peptides than to GPGR motif peptides, in contradiction for their proviral sequences. The results suggested that mutations in the V3 loop may lead to a changed immunoreactivity that makes HIV-1 mutants unrecognizable or allow escape from the primary immune response by means of neutralizing sensitivity. In connection with vaccine development, it should be pointed out that the combination of V3 sequencing and peptide EIA could provide a novel approach to obtain a primarily infected virus sequence as a target for a preventive AIDS vaccine.

Recombinant envelope protein of HIV-1 subtype E as antigen in HIV-1 antibody detection enzyme immunoassay.

In order to develop a reliable and inexpensive serodiagnostic method to be used for anti-HIV antibody detection in Thailand, recombinant envelope (TM or gp41 subunit) protein of HIV-1 subtype E was produced from prokaryotic cell (Escherichia coli) as the source of antigen in enzyme immunoassay (TE diagnostic EIA kit). HIV-1 gp41 subunit of subtype E was successfully expressed in E. coli in the form of polyhistidine-tagged proteins, comprising of rgp41A (601 bases N-terminal half of TM or 25kDa) and rgp41B (560 bases C-terminal half of TM or 24 kDa) by using an expression vector, pBAD/His C. The amount of protein, dilution of sera, and anti-human IgG labeled HRP used in the EIA test optimized by a checker board titration of the protein and seropositive or seronegative sera, were 5.0 microg/ml, 1:300, and 1:4,000, respectively. The blinded test evaluation of TE-diagnostic EIA in 500 seropositive and 500 seronegative sera which have been simultaneously tested by two available commercial kits and compared with our TE diagnostic EIA, gave 99.6% sensitivity and specificity. The other known genetic subtypes sera such as subtype A (n=5), B (n=9), C (n=4) and D (n=5) were also positive with this EIA. The estimated manufacturer cost per test of rgp41 based anti-HIV antibody detection EIA or TE-diagnostic EIA was about 15 baht. This recombinant envelope (gp41 or TM) protein from HIV-1, which can be produced in large quantities without any hazards from growing the virus and has lower cost to produce anti-HIV antibody serological diagnostic kit, should be considered as an HIV screening test in Thailand.

Cryptococcus neoformans in Siriraj Hospital 1994-1995.

One-hundred twenty strains of C.neoformans were cultured from the 104 patients (25 female, 79 male patients) admitted in Siriraj Hospital, Mahidol University, Bangkok, during January 1994 – December 1995. All patients were suffering from cryptococcal meningitis. Four female patients were diagnosed as systemic lupus erythematosus and one girl as acute lymphoblastic leukemia. The other 99 patients were diagnosed as having AIDS. The average duration of hospitalization for female and male patients was 19.3 and 23.7 days, respectively. Four female patients (16%) and sixteen male patients (20%) died, and the rest were discharged after improvement. The median initial latex agglutination test was 1:864. Biochemical testing of C.neoformans cultured from 104 patients by using both canavanine-glycinebromthymol blue agar and glycine cycloheximide medium identified 103 C.neoformans var neoformans and 1. C.neoformans var gattii. PCR fingerprinting using two primers, the single-oligonucleotide primer (GACA)4 and the phage M13 core sequence (5’ GAGGGTGGXGGXTCT 3’), revealed 102 serotype A, 1 serotype D (from AIDS patient) and 1 serotype B (from systemic lupus erythematosus patient). The phage M13 core sequence primer revealed more bands than the (GACA)4 primer. We could not differentiate by fingerprinting serotype A affecting various underlying diseases patients. Patients suffered from only one serotype of C.neoformans. The fingerprint pattern was stable under the stress of various drug therapy regimens and after subculturing in the laboratory; thus PCR fingerprinting has emerged as an important method for use in epidemiological studies. C.neoformans is susceptible to various antifungal agents, namely amphotericin B, flucytosine, fluconazole and itraconazole, in the therapeutic range.