Chi-Cheng Luo

Molecular Epidemiology of HIV Transmission in a Dental Practice

Human immunodeficiency virus type 1 (HIV-1) transmission from infected patients to health-care workers has been well documented, but transmission from an infected healthcare worker to a patient has not been reported. After identification of an acquired immunodeficiency syndrome (AIDS) patient who had no known risk factors for HIV infection but who had undergone an invasive procedure performed by a dentist with AIDS, six other patients of this dentist were found to be HIV-infected. Molecular biologic studies were conducted to complement the epidemiologic investigation. Portions of the HIV proviral envelope gene from each of the seven patients, the dentist, and 35 HIV-infected persons from the local geographic area were amplified by polymerase chain reaction and sequenced. Three separate comparative genetic analyses—genetic distance measurements, phylogenetic tree analysis, and amino acid signature pattern analysis—showed that the viruses from the dentist and five dental patients were closely related. These data, together with the epidemiologic investigation, indicated that these patients became infected with HIV while receiving care from a dentist with AIDS.

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.

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.

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.

A molecular epidemiologic survey of HIV in Uganda.

Objective:Previous data, based on a small sampling of convenience, reported subtypes A, B, C, D, and G in Uganda, but neither the extent nor the proportion of these subtypes could be evaluated. To establish correctly the prevalence and distribution of HIV-1 subtypes, we analysed viral clades in 739 HIV-1-seropositive specimens from different areas of Uganda. Methods:Blood specimens from 1100 patients were collected in five districts of Uganda. Within this collection, 929 HIV-1-seroreactive samples underwent analysis of viral DNA, and 739 were selected for further subtyping in env or pol regions. Results:Using a combination of subtype A- and D-specific probes to C2–V3 region and DNA sequencing, HIV-1 env subtypes were determined in 594 specimens: 341 were of subtype A (57.4%), 250 of subtype D (42.1%), and three of subtype C (0.5%). Sixty-two samples showed reactivity with both probes, suggesting potential mixed infections, cross-reactivity to probes, or possibly other subtypes. Subsequent sequence analysis of 19 randomly selected specimens revealed subtypes A (n = 4), D (n = 12), and C (n = 3). Sequence analysis of the 27 samples chosen from the remaining 83 samples, which could be amplified only with viral gp41 or protease gene primers, classified them as subtypes A (n = 13) and D (n = 14). No significant clinical, demographic, or geographic differences were found between HIV-1 infections with viruses of subtypes A and D, despite considerable genetic diversity within these clades. Conclusions:This is the first major population-based study of the prevalent HIV-1 strains in an African country selected for vaccine trials. The subtyping methods we describe should be of use to investigators seeking to conduct large-scale screening for HIV variants in other populations.

Horizontal and Vertical Transmission of Human Immunodeficiency Virus Type 1 Dual Infections Caused by Viruses of Subtypes B and C

This article describes a case of horizontal (heterosexual) and subsequent vertical (mother to infant) transmission of 2 human immunodeficiency viruses type 1 (HIV-1) subtypes. Dual infection in a husband, his wife, and their child was initially detected by use of a restriction fragment length polymorphism assay of the proviral protease in peripheral blood mononuclear cells. The simultaneous presence of highly similar sets of HIV-1 subtypes B and C infecting the 3 family members was confirmed by DNA sequence analysis of pol, gag, and env genes. These data, together with available epidemiologic information, may indicate that the husbands high-risk sexual behavior was the source of dual infections. Because his wife did not report such activities, it was likely that he passed HIV-1 strains to his spouse, who subsequently transmitted them to their child.

Genetic Analysis of Human Immunodeficiency Virus in Abidjan, Ivory Coast Reveals Predominance of HIV Type 1 Subtype A and Introduction of Subtype G

To better understand the molecular epidemiology of HIV genetic diversity in Abidjan, Ivory Coast, we performed a genetic analysis of 170 HIV-1-seropositive specimens representing newly diagnosed tuberculosis patients (n = 143) and women monitored in a mother-to-child transmission cohort study (n = 27). Preliminary screening with RFLP presumptively classified 162 (95.3%) of these as subtype A. The envelope region of 108 specimens was subtyped by sequence analysis: 102 (94.4%) were subtype A, 2 (1.9%) were subtype D, and 4 (3.7%) were subtype G. Subtyping gag and env regions of the genome suggested that five of the six nonsubtype A isolates exhibited a potentially mosaic structure. A comparative phylogenetic analysis of HIV-1 subtype A C2V3 from 27 Ivory Coast and 21 Ugandan sequences revealed a striking clustering among Ivory Coast variants, and an independent segregation from Ugandan subtype A. Despite independent clustering with other subtype A specimens, limited variability of the V3 loop apex was observed; the globally predominant V3 motif, GPGQ, represented 90.1% of the HIV-1 strains. This study demonstrates that clade A is the predominant HIV-1 subtype in HIV-seropositive individuals in Abidjan, Ivory Coast and that these strains are phylogenetically distinct from other subtype A strains observed in East Africa.

HIV Type 1 Subtypes in Guangxi Province, China, 1996

81 HIV-1 INFECTION WAS FIRST DOCUMENTED IN China in 1985; by 1997, the Joint United Nations Programme on HIV/AIDS (UNAIDS) estimated that there were 200,000 to 400,000 HIV-infected persons living in China among a population of 1.2 billion.3 To date, most infections have occurred among injection drug users (IDUs), primarily in ethnic minority communities in Yunnan, a southwestern province bordering Myanmar (Burma), Laos, and Vietnam. This province can be seen as an extension of the a Golden Triangleo region of northern Thailand, Myanmar, and Laos, one of the world’ s foremost opiumand heroin-producing areas. In recent years, HIV-1 transmission has extended to new areas of China, and sexual transmission has increased in importance. The HIV-1 epidemic among IDUs in Yunnan province was primarily due to env subtype B strains, both typical North American/European-like subtype B strains and strains that clustered on phylogenetic analyses with subtype B strains found among IDUs in Thailand, termed B 9 (formerly Thai B).9±11 Subsequently, subtype C viruses were identified among IDUs in Yunnan. More recently, both subtype C and subtype E strains have been reported among geographically separate groups of IDUs in Guangxi province.13 There also appear to have been multiple other introductions of subtype E strains in China10,14 and subtype A has also been identified.10 We investigated the molecular epidemiology of HIV-1 in Guangxi, a southwestern Chinese province bordering Yunnan, Guizhou, Hunan, and Guangdong provinces and northern Vietnam. Guangxi, a mountainous, subtropical area, has a population of approximately 45 million, of whom about a third are ethnic minorities. From April through July 1996, blood samples were collected from HIV-seropositive persons identified at blood donation centers, drug treatment centers, and other testing locations. Specimens were obtained from 44 persons: 25 commercial blood donors (mean age, 24.5 years [range, 18±36]; 21 male and 4 female), 16 IDUs from drug treatment centers (mean age, 22.8 years [range, 16±38]; 14 male and 2 female), 2 men (aged 29 and 35 years) who had traveled to Thailand and reported sex with female sex workers, and 1 female (aged 36) who did not inject drugs and whose husband had AIDS. Fifteen of the 16 IDUs were identified in Pingxiang, a city near the border with Vietnam; the other IDU was located in Nanning, in central Guangxi. The other 28 persons were identified from throughout the province. All specimens were HIV-1 positive by enzyme immunoassay (EIA) and Western blot when tested at the Guangxi AIDS Surveillance and Testing Center, Nanning. Unlinked serum specimens were transferred to Nonthaburi, Thailand, and then to Atlanta, Georgia for further characterization. In Thailand, specimens were tested with 14-amino acid V3loop peptide enzyme immunoassays (PEIAs) highly specific for subtypes B 9 and E from Thailand.15±17 Serum specimens were later sent to the Centers for Disease Control and Prevention (Atlanta, GA) for genetic characterization. RNA was prepared with a blood kit from Qiagen (Chatsworth, CA). RNA from each sample was reverse transcribed (primer JH35R) and subsequently amplified by nested PCR (outer primers JH44F/JH35R and inner primers JH33F/JH48R). The 525-bp nested C2±V4 PCR product encompasses the V3 loop. The sequences of these HIV-1 group M env (gp120) gene-based degenerate/inosine primers with coordinates on HIV-MNCG sequence (in parentheses) are as follows:

Transmission of drug-resistant HIV after an occupational exposure despite postexposure prophylaxis with a combination drug regimen.

We documented a case of occupational human immunodeficiency virus (HIV) despite postexposure prophylaxis (PEP) with a combination drug regimen after percutaneous injury with a needle from a sharps disposal container in the hospital room of an HIV-infected patient. This failure of PEP with a combination drug regimen may have been related to antiretroviral drug resistance, other factors, or both. This case highlights the importance of preventing injury to prevent occupational transmission of HIV.