Toxoplasma gondii antibodies in HIV and non-HIV infected Thai pregnant women

Serological evidence for Toxoplasma gondii infection in Thai pregnant women was investigated. One thousand six hundred and sixty-nine blood specimens were collected from 838 HIV-seropositive and 831 HIV-seronegative pregnant women attending the antenatal-care clinic at Siriraj Hospital, Bangkok, Thailand, during a two-year period. Toxoplasma IgG antibody was detected, using a solid-phase enzyme-linked immunosorbent assay in which the membrane protein p-30 was the predominant antigen. IgG positive sera were subsequently examined for IgM antibody by the capture antibody enzyme immunoassay. The IgG antibody was found in 450 (53.7%) HIV seropositive women and 44 (5.3%) non-HIV infected women, with a statistically significant difference (p < 0.0001). Three of the 450 HIV-seropositive and 2 of the 44 HIV-seronegative sera with IgG antibody were positive for IgM antibody against T. gondii. This result suggested that HIV seropositive pregnant women had a higher risk of Toxoplasma infection with increase exposure to their offspring.     

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.     

Epstein-Barr virus serological markers for nasopharyngeal carcinoma in Thailand.

The present study reports on the prevalence of specific IgA and IgG antibodies to EBV viral capsid antigen in nasopharyngeal carcinoma (NPC) patients with different histological types of carcinoma and their age-matched controls by the indirect immunofluorescence test, using the B-95-8 lymphoblastoid cell line as source of viral capsid antigen. EBV specific IgG was found in almost all the study cases, and antibody titers were significantly higher in the NPC patients than in non-cancer controls. GMT of anti-EBV IgG in NPC patients, patients with other malignant diseases, and those with non-malignant diseases were 371.5, 97.7 and 35.5, respectively. Anti-EBV specific IgA was more specific to NPC than was IgG, and was present in 86.5% (83 of 96) cases of NPC patients, 6.6% (2 of 30) of patients with other cancers, and 3.1% (3 of 97) cases of non-malignant diseases. A weak correlation between level of anti-EBV IgA in NPC patients was observed (r = 0.3). EBV IgA was found in all histological types of NPC, ie, WHO types 1, 2 and 3, but WHO type 1 was rare among NPC patients in Thailand. Use of anti-EBV IgA for monitoring cancer therapy is to be further investigated.     

Survey of reverse transcriptase from the heterosexual epidemic of human immunodeficiency virus type 1 CRF01_AE in Thailand from 1990 to 2000

Genetic diversity of the HIV-1 envelope gene has shown a steady increase over time in the Thai and other regional epidemics. A serial survey of subtype CRF01_AE polymerase gene (RT) diversity in Thailand was performed, using 48 novel and 15 reported sequences covering the period 1990--2000. These sequences were gathered from individuals whose sole risk factor for infection was heterosexual contact. By contrast to envelope, diversity was low and, despite a 40% increase early in the epidemic, has remained static since 1996. These results indicate that epidemic HIV-1 may be constrained within defined limits of genetic diversity at least in some genomic regions.     

HIV-1 drug resistance in Thailand: before and after National Access to Antiretroviral Program.

BACKGROUND: The Ministry of Public Health (Thailand), MoPH, has had a program called National Access to Antiretroviral Program for People who have AIDS (PHA) or "NAPHA", to offer free antiretroviral drugs (ARV), which are locally produced in Thailand, to any HIV-1 infected patients with CD4<200 since 2002. This program may increase usage of ARV therapy and the emergence of HIV-1 drug resistance. OBJECTIVES: To monitor HIV-1 ARV drug resistant codon mutation in Thailand before and after the "NAPHA" program. MATERIALS AND METHODS: EDTA blood samples were collected from 542 HIV-1 infected subjects, who received ARV therapy in 1999 and 2001-2003, and perinatal chemoprophylaxis in 1998 and 2000. HIV-1 pol nucleotide sequences were analyzed. RESULTS: The percentage of drug resistant detection from the ARV therapy group in 1999 and 2001-2003 were 12.14 (34/280), 10.23 (9/88), 86.96 (20/23) and 57.55 (61/106), respectively. Of 332 NRTI drug resistant codon mutation, 226 (68.07%) were thymidine analogue mutations (TAMs). The percentage of TAMs detection in 1999 and 2001-2003 were 7.14 (20/280), 9.09 (8/88), 56.52 (13/23) and 43.34 (46/106), respectively. Of 105 NNRTI drug resistant codon mutation, 95 (90.48%) were related to nevirapine drug resistance. CONCLUSION: Thailand may need more appropriate monitoring of drug resistance in the free ARV therapy program to protect the future usage of drugs by minimizing the emergence of drug resistance.     

Usage of dried blood spots for molecular diagnosis and monitoring HIV-1 infection

The usage of dried blood spots as specimens for diagnosis and monitoring of HIV-1 infection in Thailand was evaluated. EDTA blood samples, which were collected from 100 HIV seronegative and 109 HIV seropositive individuals, were tested on dried blood spots; Whatman, Schleicher and Schuell (S&S) No. 903 and S&S IsoCode filter paper. Nucleic acid was extracted and used as a template for HIV-1 proviral DNA detection by an "in-house" multiplex PCR and a commercial Amplicor HIV-1 PCR test (Roche, version 1.0). HIV-1 RNA qualitative (QL) and quantitative (QT) detection was determined by Nucleic Acid Sequence Based Amplification (NASBA). The average DNA per blood spot recovered from Whatman and S&S IsoCode was not statistically different (p = 0.512) with a range of 218.9+/-46.84 and 225.63+/-88.33 microg, respectively. The concordance of HIV-1 proviral DNA detection by PCR from dried blood spots Whatman and S&S IsoCode was 94% versus 89.4% for sensitivity and 100% versus 100% for specificity. The sensitivity and specificity of HIV-1 RNA QL detection in dried blood spots was 89.7 and 97.5%, respectively. The HIV-1 RNA QT from dried blood spots showed a good correlation in paired dried blood spots and plasma with Pearson correlation, r = 0.817 (R2 = 0.667, P < 0.05). The data showed that dried blood spots could be used for the diagnosis and monitoring of HIV-1 infection.     

Primary infection of human herpesvirus 6 in children with vertical infection of human immunodeficiency virus type 1.

The role of human herpesvirus 6 (HHV-6) infection in 227 children born to human immunodeficiency virus (HIV)-seropositive mothers was investigated. Of 41 HIV-uninfected infants, 3 (7%) were positive for HHV-6 DNA in the first month of life, suggesting possible intrauterine infection. The cumulative infection rates of HHV-6 at 6 and 12 months of age were significantly lower in HIV-infected children (11% and 33%, respectively) than in uninfected children (28% and 78%, respectively; P<.001). There was an association between high CD4+ cell numbers (>15%) before HHV-6 infection and high HHV-6 infection rate. Twenty-two infants with HIV classed as Centers for Disease Control and Prevention stages N1 or N2 were studied for an association of HHV-6 infection with progression of HIV disease. Ten of the infants had HHV-6, and 12 did not. In 5 of the infants without HHV-6 (42%), HIV disease had not progressed by 1 year of age; however, HIV disease had progressed in all 10 children with HHV-6 infection. These results suggest an association of HHV-6 infection and progression of HIV disease in the study children with vertical HIV-1 infection (P<.05).     

p24 Antigen detection assay modified with a booster step for diagnosis and monitoring of human immunodeficiency virus type 1 infection

We modified a p24 antigen enzyme-linked immunosorbent assay as a method for diagnosis and monitoring of human immunodeficiency virus type 1 (HIV-1) subtype E infection. This modified assay is based on the use of preheated immune complex dissociation combined with a booster step using a regular Vironostika HIV-1 p24 antigen assay (bioMerieux) to decrease the lower limit of p24 antigen detection from 10 pg/ml (lower limit achievable when using a regular p24 antigen assay) to 0.5 pg/ml (100 virions/ml) by the new method. The correlation between the values obtained by the HIV-1 RNA (Amplicor HIV-1 Monitor) assay and the p24 antigen assay modified with a booster step antigen assay in 160 frozen plasma samples with known viral load and 80 blind fresh plasma samples by Spearman rank were 0.671 (R(2) = 0.450; P < 0.01) and 0.782 (R(2) = 0.612; P < 0.01). During antiretroviral treatment, the change of p24 antigen level at >/=0.5 log correlated well with the level of HIV-1 in plasma. In order to improve the early diagnosis of HIV-1 infection in 121 infants born to HIV-1-infected mothers, a heat-denatured plasma p24 antigen assay modified with a booster step was compared with DNA-PCR and HIV RNA (nucleic acid sequence-based amplification) assays. The sensitivity of the antigen test modified with a booster step was similar to that of the HIV-1 RNA (NASBA QL) assay and better than that of the DNA-PCR assay (100 versus 61.90%) for subjects 1 to 2 months old. The overall results from this study might renew interest in p24 antigen detection as an easily affordable alternative method for diagnosis of HIV-1 infection and monitoring of disease progression in developing countries.     

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.