Marcia L. Kalish

Independent introduction of two major HIV-1 genotypes into distinct high-risk populations in Thailand.

To investigate the genetic heterogeneity and epidemiological distribution of human immunodeficiency virus type 1 (HIV-1) in Thailand, we determined proviral sequences for 63 HIV-1-infected patients in various risk groups from all over the country between April and July, 1991. Two distinct genotypes of HIV-1, A and B, were found to segregate by mode of transmission. Of 29 sexually infected patients, 25 (86%) had HIV-1 of genotype A and 4 (14%) had genotype B. Among 29 injecting drug users, probably parenterally infected, only 7 (24%) had genotype A and 22 (76%) had genotype B. This segregation is unlikely to have arisen by chance (p < 0.001). No patient was found to have dual infection. Nucleotide divergence averaged 3.4% among genotype-A-infected patients and 3.5% among genotype-B-infected patients, but 22.0% between the genotypes. 37 of 40 isolates (both genotypes) had the GPGQ tetrapeptide at the tip of the V3 loop, which is common in African HIV-1 strains but rare in North American and European strains, where the GPGR motif predominates. These findings suggest that the waves of HIV-1 infection in injecting drug users and in sexually infected patients in Thailand may not be epidemiologically linked. The nucleotide divergence data point to the separate introductions of the two genotypes in Thailand. Further studies in Thailand and neighbouring countries will be useful in the design and selection of candidate HIV vaccines.

Near full-length clones and reference sequences for subtype C isolates of HIV type 1 from three different continents.

Among the major circulating HIV-1 subtypes, subtype C is the most prevalent. To generate full-length subtype C clones and sequences, we selected 13 primary (PBMC-derived) isolates from Zambia, India, Tanzania, South Africa, Brazil, and China, which were identified as subtype C by partial sequence analysis. Near full-length viral genomes were amplified by using a long PCR technique, sequenced in their entirety, and phylogenetically analyzed. Amino acid sequence analysis revealed 10.2, 6.3, and 17.3% diversity in predicted Gag, Pol, and Env protein sequences. Ten of 13 viruses were nonmosaic subtype C genomes, while all three isolates from China represented B/C recombinants. One of them was composed primarily of subtype C sequences with three small subtype B portions in gag, pol, and nef genes. Two others exhibited these same mosaic regions, but contained two additional subtype B portions at the gag/pol overlap and in the accessory gene region, suggesting ongoing B/C recombination in China. All subtype C genomes contained a prematurely truncated second exon of rev, but other previously proposed subtype C signatures, including three potential NF-kappa B-binding sites in the viral promoter-enhancer regions, were found in only a subset of these genomes.

Maternal viral load and timing of mother-to-child HIV transmission Bangkok Thailand.

OBJECTIVES To determine the proportion of HIV-1-infected infants infected in utero and intrapartum, the relationship between transmission risk factors and time of transmission, and the population-attributable fractions for maternal viral load. DESIGN Prospective cohort study of 218 formula-fed infants of HIV-1-infected untreated mothers with known infection outcome and a birth HIV-1-positive DNA PCR test result. METHODS Transmission in utero was presumed to have occurred if the birth sample (within 72 h of birth) was HIV-1-positive by PCR; intrapartum transmission was presumed if the birth sample tested negative and a later sample was HIV-1-positive. Two comparisons were carried out for selected risk factors for mother-to-child transmission: infants infected in utero versus all infants with a HIV-1-negative birth PCR test result, and infants infected intrapartum versus uninfected infants. RESULTS Of 49 infected infants with an HIV-1 birth PCR result, 12 (24.5%) [95% confidence interval (CI), 14 -38] were presumed to have been infected in utero and 37 (75.5%) were presumed to have been infected intrapartum. The estimated absolute overall transmission rate was 22.5%; this comprised 5.5% (95% CI, 3-9) in utero transmission and 18% (95% CI, 13-24) intrapartum transmission. Intrapartum transmission accounted for 75.5% of infections. High maternal HIV-1 viral load (> median) was a strong risk factor for both in utero [adjusted odds ratio (AOR) 5.8 (95% CI, 1.4-38.8] and intrapartum transmission (AOR, 4.4; 95% CI, 1.9-11.2). Low birth-weight was associated with in utero transmission, whereas low maternal natural killer cell and CD4(+) T-lymphocyte percentages were associated with intrapartum transmission. The population-attributable fraction for intrapartum transmission associated with viral load > 10 000 copies/ml was 69%. CONCLUSIONS Our results provide further evidence that most perinatal HIV-1 transmission occurs during labor and delivery, and that risk factors may differ according to time of transmission. Interventions to reduce maternal viral load should be effective in reducing both in utero and intrapartum transmission.

Determination of HIV-1 subtypes in injecting drug users in Bangkok, Thailand, using peptide-binding enzyme immunoassay and heteroduplex mobility assay : evidence of increasing infection with HIV-1 subtype E

ObjectivesTo evaluate the sensitivity, and specificity of peptide-binding enzyme immunoassay (PEIA), and heteroduplex mobility assay (HMA) for the determination of HIV-1 subtypes B, and E; to determine the proportions of infections due to subtypes B, and E over time;, and to generate data on DNA sequences of the C2-V3 region of the env genes. MethodsHIV-1 subtyping was conducted by PEIA, and HMA on blood specimens obtained from 97 injecting drug users (IDU) infected with HIV between 1988, and 1993. Genetic sequencing was performed on 84 specimens. ResultsBoth laboratory methods were highly sensitive, and specific for the determination of HIV-1 subtypes B, and E. The two tests were complementary; samples which could not be typed by HMA were correctly typed by PEIA, and vice versa. While subtype B accounted for 80.4% (78 out of 97) of infections overall, the proportion of new infections due to subtype E increased from 2.6% (one out of 38) in 1988–1989 to 25.6% (11 out of 43) in 1990–1991, and to 43.8% (seven out of 16) in 1992–1993 (4cH2 for linear trend, P< 0.001). ConclusionsHMA, and PEIA are practical, sensitive, and specific laboratory methods for the determination of HIV-1 subtypes in Thailand, and may be useful in other geographic areas to define the molecular epidemiology of the global HIV-1 pandemic. Data suggest that the proportion subtype E infections have increased among Bangkok IDU from 1988 through 1993.

U.S. Human Immunodeficiency Virus Type 1 Epidemic: Date of Origin, Population History, and Characterization of Early Strains

ABSTRACT Human immunodeficiency virus (HIV) type 1 subtype B sequences (whole envelope and the p17 region of gag) were obtained from peripheral blood mononuclear cell samples collected in 1981 from seven HIV-infected U.S. individuals and in 1982 from one infected Canadian resident. Phylogenetic and nucleotide distance analyses were performed by using database sequences representing North American strains collected from 1978 to 1995. The estimated phylogeny was starlike, with early strains represented on different lineages. When sequences were grouped by years of collection, nucleotide distance comparisons demonstrated an increase in diversity over time and indicated that contemporary strains are more closely related to early epidemic strains than to each other. Using a recently developed likelihood ratio reduction procedure, the date of origin of the U.S. epidemic was estimated to be 1968 ± 1.4 years. A coalescent approach was also used to estimate the population history of the U.S. subtype B epidemic. Our analyses provide new information that implies an exponential growth rate from the beginning of the U.S. HIV epidemic. The dating results suggest a U.S. introduction date (or date of divergence from the most recent common ancestor) that precedes the date of the earliest known AIDS cases in the late 1970s. Furthermore, the estimated epidemic growth curve shows a period of exponential growth that preceded most of the early documented cases and also indicates a leveling of prevalence rates in the recent past.

Association of Human Immunodeficiency Virus (HIV) Load Early in Life with Disease Progression among HIV-Infected Infants

The utility of RNA virus load to predict progression of human immunodeficiency virus (HIV)-1 disease was assessed in 89 HIV-1-infected children. Of 22 virus load values during week 1 of life, 17 were below the detection threshold. Geometric mean virus load increased to approximately 7 x 10(5) copies/mL by week 4, was sustained throughout the first 6 months of life, and then declined to 1.6 x 10(5) copies/mL during the third year. Samples from week 1 of life had little predictive value, but virus load during days 7-30 strongly predicted progression to CDC-3 classification or death (P = .024; risk ratio = 1.6), and virus load during months 2-3 predicted progression to CDC-C or death within the first 6 months of life (P = .002, risk ratio = 11). Virus load was highly associated with imminent vulnerability to CDC-C or death (P = .002) during the first 18 months of life. Except for values from the first week of life, virus load at any age through 18 months is strongly associated with risk of HIV disease progression.

Maternal Virus Load and Perinatal Human Immunodeficiency Virus Type 1 Subtype E Transmission, Thailand

To determine the rate and risk factors for human immunodeficiency virus (HIV)-1 subtype E perinatal transmission, with focus on virus load, pregnant HIV-infected women and their formula-fed infants were followed prospectively in Bangkok. Of 281 infants with known outcome, 68 were infected (transmission rate, 24.2%; 95% confidence interval, 19.3%-29.6%). Transmitting mothers had a 4.3-fold higher median plasma HIV RNA level at delivery than did nontransmitters (P<.001). No transmission occurred at <2000 copies/mL. On multivariate analysis, prematurity (adjusted odds ratio [AOR], 4.5), vaginal delivery (AOR, 2.9), low NK cell percentage (AOR, 2.4), and maternal virus load were associated with transmission. As RNA quintiles increased, the AOR for transmission increased linearly from 4.5 to 24.8. Two-thirds of transmission was attributed to virus load>10,000 copies/mL. Although risk is multifactorial, high maternal virus load at delivery strongly predicts transmission. This may have important implications for interventions designed to reduce perinatal transmission.

The effect of maternal viral load on the risk of perinatal transmission of HIV-1

Objective:To determine the effect of maternal viral load at delivery on the risk of perinatal transmission of HIV-1. Design:A nested case–control study within a prospectively followed cohort of HIV- 1-infected pregnant women and their infants. Setting:The multicenter New York City Perinatal HIV Transmission Collaborative Study. Participants:Fifty-one women who gave birth to HIV-1-infected infants were frequency-matched within CD4+ cell count quintiles with 54 non-transmitting mothers. Main outcome measures:Maternal quantity of HIV-1 viral RNA was assayed in plasma obtained near delivery using the nucleic acid sequence-based amplification assay system. Results:Viral RNA was detected in 73 (70%) out of 105 women and the median viral load was 16 000 RNA copies/ml in transmitters and 6600 in non-transmitters (P < 0.01). When adjusted for maternal CD4+ count near delivery, women with measurable viral load were nearly sixfold more likely to transmit HIV-1 than women with viral load below detection [adjusted odds ratio (AOR), 5.8; 95% confidence interval (CI), 2.2–15.5]. The odds ratio for perinatal transmission of log10 viral load, adjusted for CD4 count was 2.7 (95% CI, 1.5–5.1). When stratified by the stage of HIV-1 disease, the only group with significant association between log10 viral load and transmission were AIDS-free women with CD4+ count > 500 x 106/l (AOR, 9.1; 95% CI, 2.6–31.5). Conclusions:High maternal viral load increases the likelihood of perinatal transmission of HIV-1 in women without AIDS and advanced immunosuppression. HIV-1-infected pregnant women without advanced disease, shown by others to have the lowest risk of perinatal transmission, may benefit the most from efforts to identify and decrease viral load at delivery.

Chimpanzee adenovirus antibodies in humans, sub-Saharan Africa.

Human sera from the United States, Thailand, and sub-Saharan Africa and chimpanzee sera were tested for neutralizing antibodies to 3 chimpanzee adenoviruses. Antibodies were more common in humans residing in sub-Saharan Africa than in humans living in the United States or Thailand. This finding suggests cross-species transmission of chimpanzee adenoviruses.

Identification of Single and Dual Infections with Distinct Subtypes of Human Immunodeficiency Virus Type 1 by Using Restriction Fragment Length Polymorphism Analysis

The simultaneous presence of multiple HIV-1 subtypes has become common in communities with the growth of the pandemic. As a consequence, the potentiality for an increased frequency of HIV-1 mixed infections caused by viruses of distinct subtypes could be expected. Thus, there is a need to estimate the prevalence and geographic distribution of infections caused by viruses of a singular subtype as well as coinfections caused by two or more HIV-1 strains of distinct subtypes. To address this need, we have developed a genetic method based on restriction fragment length polymorphism (RFLP) to screen for these two types of infections within infected populations. In this assay, restriction enzymes may be used to predict the phylogroup of HIV-1 infected samples. A 297 bp pol fragment spanning the entire viral protease gene and a 311 bp fragment of the p24 gag region are used for this analysis. The viral regions are amplified by nested PCR using DNA templates from uncultured peripheral blood mononuclear cells (PBMC) or virus culture. Classification of HIV-1 strains to well defined subtypes B, D, F, and A/C is done by sequential endonuclease restriction analysis of a PCR amplified-protease gene followed by analysis of the p24 gag region. The electrophoretic migration patterns visualized by ethidium bromide staining or by radiolabeled probes are then determined on a 10% polyacrylamide gel. In infections caused by viruses of a singular subtype, a single restriction pattern is detected, whereas in multiple infections caused by two or more viral strains of different subtypes, the combination of different digestion patterns are observed in infected individuals. Using this methodology we have screened for genetic variations in HIV-1 proviral DNA from thirty-three Brazilian samples. Our RFLP procedure classified thirty-two samples as single infections caused by viruses of subtypes B (31) and F (1), and one sample as dual infection caused by distinct viral strains. Subsequent sequence and phylogenetic analysis of the viral protease gene in lymphocytes of all these patients confirmed our RFLP findings in single infections, and demonstrated the existence of two distinct HIV-1 strains of subtypes F and D in a patient which lymphocytes showed the simultaneous presence of two different digestion patterns. As up to now, single infections caused by subtype D variants were not identified in Brazil, our data provide the first evidence of subtype D HIV-1 in this country. Because sequencing of HIV proviral DNA is not particularly practical for large-scale molecular epidemiological studies, the protease/gag-based RFLP screening method will be useful to predict the phylogroup of HIV-1, and to identify multiple infections caused by HIV-1 strains of distinct subtypes. We believe that this information is crucial for both evaluation of the HIV-1/AIDS pandemic and intervention strategies.