Diversity of HIV-1 subtype E in semen and cervicovaginal secretion.

OBJECTIVES: To characterize human immunodeficiency virus type 1 (HIV-1) subtype E variants in blood and genital fluid of infected Thai couples. STUDY DESIGN/METHODS: Blood and genital fluid were collected from 30 asymptomatic healthy HIV-1 subtype E infected couples from Bangkok, Thailand from 1995 to 1998. RESULTS: All 60 viruses in blood samples were identified as subtype E by heteroduplex mobility assay. The biotype of viruses founded in blood was syncytium-inducing (SI), whereas M-tropic and non-syncytium-inducing (NSI) isolates were predominantly detected in genital fluid. HIV-1 proviral DNA was detected in 43.33% and 56.67%, and viral RNA was detected in 93.33% and 56.67%, of semen (n = 30) and cervicovaginal secretion (n = 30) samples tested, respectively. A higher intersample genetic distance and more positive charge of the V3 loop were found in blood strains composed of genital fluid strains (22.30 +/- 5.92% and 17.96 +/- 6.3%), which was statistically significant (P = 0.003). The env V1-V4 intraperson variation of the HIV-1 subtype E in the blood and genital fluid of each individual was in the range 3.0%-5.7%. We also determined the intrasample variation of HIV-1 from blood and genital fluid by heteroduplex mobility assay. The mean heteroduplex mobility of the HIV-1, V1-V4 region of env gene, in blood (n = 8) and genital fluid (n = 8) was 0.59 +/- 0.06 and 0.74 +/- 0.11 (t test, p = 0.001), respectively. CONCLUSIONS: There was genetic and phenotypic compartmentalization of HIV-1 subtype E in blood and genital fluid with the presence of SI and NSI phenotypic variants as a common property of subtype E isolates from blood and genital fluid, respectively.

Molecular and phenotypic characteristics of neurotropic HIV-1 subtype E.

Although HIV-1 subtype E associated with neurological dysfunction is common, the virological characteristics of HIV-1 isolated from the CNS for this subtype have not yet been identified. In this study, paired blood and CSF isolated from patients with AIDs-defining illnesses were cultured, sequenced and aligned. Phylogenetic tree and nucleotide-distances from both blood and CSF were investigated. Cytopathicity and co-receptor usage of paired blood and CSF isolates were compared to define the specific characteristics of CNS isolates. The results confirmed that CSF isolates showed less cytopathicity. It was found that both blood and CSF isolates used either CXCR4 or CXCR4 and CCR5 as co-receptors. Interestingly, one CSF isolate using CCR3 as a co-receptor was identified. By sequence analysis, the pair-wise distances of envelope gp 120 sequence and those of all variable regions (except V3 region) between blood and CSF isolates were significantly different. The genetic distances in V1/V2 regions of CSF isolates showed more diversity than those of blood isolates. These findings suggest that the evolution of V1/V2 regions of CSF isolates seems to be an advantage for HIV-1 in CNS infection. In contrast, the genetic distance in V4 and V5 regions of CSF isolates showed less diversity, suggesting that conservation in these regions might be necessary during the process of HIV-1 CNS infection.

Clostridium difficile infections in HIV-positive patients.

The prevalence of Clostridium difficile infections in HIV-positive patients with regard to the presence of its enterotoxin was investigated. Enzyme immunoassay (EIA, Meridian Diagnostic Inc) was used for the detection of C. difficile enterotoxin in stool specimens collected from 201 HIV-positive and 271 HIV-negative diarrheal patients. Culture was performed on cycloserine cefoxitin fructose agar. Chromosomal DNA types of C. difficile isolates were determined by pulsed-field gel electrophoresis (PFGE). In the HIV-positive group, C. difficile enterotoxin was found in 58.8% and 12.6% of diarrheal and non-diarrheal patients, repectively, whereas this toxin was found in 36.5% of HIV-negative-diarrheal patients. However, 13.6% of stool samples were negative by toxin assay, but were positive for C. difficile by culture and latex agglutination test. Among 11 isolates from both HIV-positive and HIV-negative patients, 6 patterns of PFGE type were observed: A, B, C, D, E and F.

Prevalences of human herpesvirus 6 and human herpesvirus 7 in normal Thai population.

Prevalences of human herpesvirus 6 (HHV-6) and human herpesvirus 7 (HHV-7) DNA were investigated in normal Thai population. Peripheral blood mononuclear cells (PBMC) and saliva were collected from 238 healthy adults in five provinces which might be a representative of each part of the country, and 120 normal children in one province. Prevalences of HHV-6 DNA PBMC were 45.5-74.3% in adults and 78.3% in children, and in saliva, very low prevalences were detected; 5.7-8.6% in adults and 15.0% in children, respectively. Additionally, all HHV-6 DNA detected in this study were variant B. Comparingly to those of HHV-7 DNA, the prevalences were significantly higher than those of HHV-6, ie, 82.9-91.4% in PBMC of adults, 85% in PBMC of children, 84.8-89.0% in saliva of adults and 92.5% in saliva of children. HHV-6 and HHV-7 isolation from saliva specimens were also performed. No HHV-6 could be isolated from any samples, whereas, in the present study, HHV-7 could be isolated as 90.0% from children and as 20.0-54.5% from adults.

Incidence of new Salmonella serovar (S. ratchaburi) in Thailand.

Eighteen strains of Salmonella group E from stool samples were confirmed as Salmonella new serovar. 3, 10 : Z35 : 1, 6 by Centre International des Salmonella, Institut Pasteur, Paris, WHO Collaborating Center for Salmonella, Atlanta, USA and Salmonella-Zentrale Hygienischen Institut, Hamburg, Germany. The name of this new serovar was proposed as S. ratchaburi according to the place of its first isolation in Ratchaburi province. The new serovar of Salmonella was sensitive to many antimicrobial agents except streptomycin and erythromycin.

The association of skin diseases with human herpesvirus 8 infection in HIV carriers.

The seroprevalence to Kaposi's sarcoma-associated herpesvirus (KSHV) or human herpesvirus type 8 (HHV-8) was surveyed in human immunodeficiency virus type 1 (HIV-1) carriers with or without skin diseases, and also in HIV-1 negative individuals in Thailand. Using an immunofluorescence assay, the seropositive rates to lytic antigens of HHV-8 in HIV-1 carriers with or without skin diseases were 25% and 7.4%, respectively, but none of HIV-1 negative individuals had antibody. The seroprevalence to HHV-8 antigens was high in HIV positive individuals with low CD4/CD8 ratio, suggesting that HHV-8 is reactivated during the immunosuppressive state. Several polypeptides with apparent molecular weights of 34-38,000 and 40,000, which were specific to HHV-8, were identified by the immunoprecipitation test using the seropositive sera. Our results suggested that HHV-8 co-existed with HIV in HIV-1 carriers and the existence of HHV-8 may be associated with clinical features in the skin.

HIV type 1 in Thailand, 1994-1995: persistence of two subtypes with low genetic diversity.

Extensive transmission of human immunodeficiency virus type 1 (HIV-1) in Thailand began in 1988, resulting in an estimated 800,000 cumulative infections by 1994. During 1994 and 1995, we collected blood specimens from 215 asymptomatic HIV-1-infected people with various risk behaviors from nine locations in all four regions of Thailand. HIV-1 subtypes and genetic heterogeneity were determined for 214 strains by a combination of direct DNA sequencing (n = 95), subtype-specific oligonucleotide probe testing (n = 201), and V3-loop peptide enzyme immunoassay (PEIA) (n = 214). All strains were either env subtype E (175; 81.8%) or B (39; 18.2%). Of the subtype B isolates, 37 (94.9%) were B' and 2 (5.1%) were more typical North American-like B strains (most subtype B strains in Thailand are part of a distinct subcluster within the subtype B branch on phylogenetic trees, termed B'; formerly Thai B or BB). Of 149 viruses from people with sexual risk behaviors from all regions, 146 (98.0%) were subtype E. Of 65 viruses from injecting drug users (IDUs), 29 (44.6%) were subtype E and 36 (55.4%) were subtype B, including 35 B' strains. There was regional variation in the proportions of subtypes E and B' among IDUs. The intrasubtype nucleotide divergence within the V3 and flanking regions of the env gene (mid-C2 to the start of the V4 region) was low (5.7% for subtype E and 3.1% for subtype B') compared with other HIV-1 group M subtypes from different countries. These findings of two subtypes with low heterogeneity indicate that Thailand may be a desirable setting for evaluating candidate HIV-1 vaccines. The mix of subtype E and B' strains among IDUs also offers the opportunity to study phenotypic differences between the two subtypes.

PIP: The extensive transmission of HIV-1 in Thailand which began in 1988 led to an estimated 800,000 cumulative infections in the country by 1994. The authors collected blood specimens during 1994 and 1995 from 215 asymptomatic HIV-1-infected people with various risk behaviors from 9 locations across Thailand. HIV-1 subtypes and genetic heterogeneity were then determined for 214 strains using a combination of direct DNA sequencing, subtype-specific oligonucleotide probe testing, and V3-loop peptide enzyme immunoassay. 175 strains were subtype E and 39 were subtype B. 37 of the subtype B isolates were B' and 2 were more typical North American-like B strains. Of 149 viruses from people with sexual risk behaviors from all regions of the country, 146 were subtype E. Of 65 viruses from IV drug users (IVDUs), 29 were subtype E and 36 were subtype B, including 35 subtype B' strains. Regional variation was observed in the proportions of subtypes E and B' among IVDUs. The intrasubtype nucleotide divergence within the V3 and flanking regions of the env gene was 5.7% for subtype E and 3.1% for subtype B'. The finding of 2 HIV-1 subtypes with low heterogeneity suggests that Thailand may be an appropriate setting in which to evaluate candidate HIV-1 vaccines. The mix of subtype E and B' strains among IVDUs will also allow the study of phenotypic differences between the 2 subtypes.

The latency rate of human herpesvirus 6 (HHV6) in positive and negative human immunodeficiency virus (HIV) infection of intravenous drug users (IVDU).

The seropositive and latency rates of HHV6 among IVDU with positive and negative HIV and control group were demonstrated. By immunofluorescent antibody test, no differences in the seropositive rates were found among these three groups. All groups had seropositive rate at the average 89% and GMT antibody 1:26. This meant that most of them had previous infection with HHV6. In addition, HHV6-DNA was determined and classified into subgroups: HHV6A and HHV6B, by polymerase chain reaction. The prevalence of HHV6-DNA indicated HHV6 latency in vivo. High latency rate of HHV6 was found in all three groups (the average 54%). Moreover, HHV6B (49%) had a higher frequency than HHV6A (5%); HHV6a was found only in IVDU with or without HIV infection. The result suggested that the HHV6 latency in IVDU with positive HIV may possibly transactivate HIV. The pathogenesis of HHV6 in AIDS patients should be further investigated. However, this research finding is useful for treatment, health care, prevention and control of AIDS in case of dual infections and latency of herpesvirus infection in AIDS.

Diagnosis of perinatal HIV-1 infection by in-house PCR.

A study on how to apply PCR as a diagnostic test for the infants born to HIV-1 infected mothers is described. All steps including clinical care, blood sampling, specimen processing and PCR analysis were carried out using native facilities and personnel. An open cohort of 130 children was evaluated at birth, 1, 6, 9, 15, and 18 months of age. Definite infection status was assessed by clinical and serological data during an 18 months of follow up period. PCR results were reported as positive or negative when at least 2 concordant data were denoted. This in-house PCR, compared to known infection status, gave 100% sensitivity and 94.4% specificity within 6 months after birth. On the other hand, clinical diagnosis could identify only the infected infants at 9 months of age. The HIV-1 transmission rate from mother to infant was 23.2%. Though this PCR was not at an optimal level of specificity, it was still beneficial to identify uninfected infants in the first year of their lives and avoid unnecessary medical care. Here, we report an in-house PCR that offers good performance at low cost for the diagnosis of HIV-1 vertical transmission.

No proof of HTLV-I/II in intravenous drug abusers with a high rate of HIV-1 infection in central Thailand.

Serum specimens of 1,074 intravenous drug abusers (IVDA) were examined for infection with HIV-1, HTLV-I and HTLV-II in central Thailand. Three hundred and sixty-two of the specimens were seropositive for HIV-1 (33.7%). The HIV-1 seropositive IVDAs exhibited increased seropositivity with age through group 40-44 and significantly decreased seropositivity over the age of 45. In contrast, no seropositivity to either HTLV-I or -II was detected in the samples tested by a particle-agglutination assay for HTLV followed by type-specific Western blotting for HTLV. Reference to previous reports suggested that the rate of HIV infection in IVDAs has decreased with no HTLV-I or HTLV-II in Thailand when compared with that of the HIV infection in 1992.

PIP: A major HIV-1 epidemic took off in Thailand in 1988 and spread throughout the country. Blood serum samples from 1036 male and 38 female IV drug users at Thanyarak Narcotics Hospital in Pathurmthani, Bangkok, in January 1995 were examined for infection with HIV-1, HTLV-I and HTLV-II. 362 specimens (33.7%) were HIV-1-seropositive, 49% of whom were 25-34 years old. At ages 45 years and older, the level of HIV seropositivity was significantly lower. No seropositivity of either HTLV-I or HTLV-II was detected in the samples tested by particle-agglutination assay for HTLV followed by type-specific Western blotting for HTLV. The identified level of HIV seroprevalence in this study population is lower than the 1992 level and the 38% determined in 1994 among IV drug users at the hospital. No other population in Thailand has been found to have an increased prevalence of HIV-1 infection. These findings suggest that HIV-1 infection in Thailand may be quite widely spread with prevalence decreasing only among IV drug users.

Distinct yearly change of serotype distribution of human rotavirus in Thailand as determined by ELISA and PCR.

A total of 241 group A rotavirus-positive stool samples collected from diarrhoeic patients in Thailand between July 1988 and June 1991 were characterized for their serotypes by enzyme-linked immunosorbent assay (ELISA) using serotype-specific monoclonal antibodies and by a polymerase chain reaction (PCR). In July 1988-June 1989, serotype 1 was the most prevalent (63.4%), followed by serotype 4 (11.0%) and serotype 2 (8.5%). In July 1989-June 1990, 59.8% were serotype 1, 24.3% were serotype 2, and 6.1% were serotype 3. In contrast, in July 1990-June 1991, serotype 3 was detected in the highest frequency (40.5%), 29.9% were serotype 1, and 27.3% were serotype 2. Thus, a distinct yearly change of serotype distribution of rotavirus in Thailand was observed in the three consecutive years. In particular, it was of note that the prevalence of serotype 3 greatly increased, in contrast to the previous studies in which almost no serotype 3 rotaviruses were detected in the years 1983-8 in Thailand.