Gerald Schochetman

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.

Use of the polymerase chain reaction for early detection of the proviral sequences of human immunodeficiency virus in infants born to seropositive mothers

Abstract The early diagnosis of infection with human immunodeficiency virus (HIV) in infants born to infected mothers is essential for early treatment, but current tests cannot detect HIV infection in newborns because of the presence of maternal antibodies. We used the polymerase chain reaction, a new technique that amplifies proviral sequences of HIV within DNA, to detect HIV infection in peripheral-blood mononuclear cells obtained from infants of seropositive women during the neonatal (age less than 28 days) and postneonatal periods. In blood obtained during the neonatal period, the polymerase chain reaction was positive in five of seven infants in whom the acquired immunodeficiency syndrome (AIDS) later developed (a mean of 9.8 months after the test). The test was also positive in one of eight newborns who later had nonspecific signs and symptoms suggestive of HIV infection (mean follow-up, 12 months). No proviral sequences were detected in neonatal samples from nine infants who remained well (mean fol...

DURATION OF HUMAN IMMUNODEFICIENCY VIRUS INFECTION BEFORE DETECTION OF ANTIBODY

To estimate the duration and frequency of the period of HIV infection without detectable antibody, modelling techniques were applied to results of detection of HIV DNA by means of the polymerase chain reaction (PCR) and to data from cases in published reports. PCR was carried out with gag and env region primers on samples from 27 homosexual and 12 haemophilic men for whom stored samples were available from before and after seroconversion; serum was also tested for p24 antigen by antigen-capture enzyme immunoassay. HIV DNA was detectable before seroconversion in 4 men; in all 4 PCR was positive only in the seronegative sample taken closest to the time of seroconversion. In 3 men antigen was detected before seroconversion; in each case HIV DNA was also detected. By a Markov model, the time from infection with HIV (as assessed by detection of HIV DNA) to first detection of HIV antibody was estimated to be 2.4 (SE 2.1) months for the median individual. Modelling of cases of HIV infection with known exposure in published reports gave a median estimate of 2.1 (0.1) months from exposure to antibody detection, and 95% of cases would be expected to seroconvert within 5.8 (0.6) months. HIV infection for longer than 6 months without detectable antibody seems uncommon.

Transmission of Human Immunodeficiency Virus in a Dental Practice

OBJECTIVE To determine if patients of a dentist with the acquired immunodeficiency syndrome (AIDS) became infected with human immunodeficiency virus (HIV) during their dental care and, if so, to identify possible mechanisms of transmission. DESIGN Retrospective epidemiologic follow-up of the dentist, his office practice, and his former patients. SETTING The practice of a dentist with AIDS in Florida. PARTICIPANTS A dentist with AIDS, his health care providers and employees, and former patients of the dentist, including eight HIV-infected patients. MEASUREMENTS Identification of risks for HIV transmission (if present), degree of genetic relatedness of the viruses, and identification of infection control and other office practices. RESULTS Five of the eight HIV-infected patients had no confirmed exposures to HIV other than the dental practice and were infected with HIV strains that were closely related to those of the dentist. Each of the five had invasive dental procedures, done by the dentist after he was diagnosed with AIDS. Four of these five patients shared visit days (P greater than 0.2). Breaches in infection control and other dental office practices to explain these transmissions could not be identified. CONCLUSION Although the specific incident that resulted in HIV transmission to these patients remains uncertain, the epidemiologic evidence supports direct dentist-to-patient transmission rather than a patient-to-patient route.

Sensitivity of United States HIV antibody tests for detection of HIV-1 group O infections.

Infections by highly divergent strains of HIV-1, first detected in central Africa and grouped provisionally as group O, have not been reliably detected by certain European HIV screening tests. Serum specimens from eight probable group O infections from Cameroon were tested by ten HIV assays licensed by the US Food and Drug Administration. All assays based on synthetic peptides or recombinant antigens failed to detect at least one of the infections; assays based on whole-virus lysates performed better. Divergent HIV strains may be undetected by current HIV tests. Thus active surveillance for and characterisation of HIV variants to evaluate and, when necessary, modify current tests is urgently needed.

Highly specific V3 peptide enzyme immunoassay for serotyping HIV-1 specimens from Thailand.

ObjectiveTo develop and evaluate a simple V3 peptide-based enzyme immunoassay (EIA) for large-scale serotyping of HIV-1 specimens from Thailand. DesignSerologic reactivities with synthetic peptides derived from the V3 loop of gp120 were used for typing HIV-1 specimens. MethodsSynthetic peptides PND-A and PND-B, derived from the consensus amino-acid sequences of the V3 loop of gp120 from two major genomic variants of HIV-1 in Thailand (A and B), were evaluated in an EIA on 61 Thai HIV-1 sera for which genotypes had been determined by polymerase chain reaction. The peptide EIA was then applied to sera from 188 HIV-1-infected patients, selected in non-random, convenience samples of known risk groups from four geographic regions of Thailand. ResultsThe sensitivities and specificities of PND-A and PND-B were 86% (30 out of 35) and 96% (25 out of 26) and 92% (24 out of 26) and 94% (33 out of 35), respectively, with 100% predictive values of a monoreactive positive test for both peptides. The assay classified 101 specimens as serotype A, 39 as serotype B, eight as serotype AB (dually reactive), and 40 as untypable (non-reactive). Excluding dual reactors and non-reactors, 92% (77 out of 84) of specimens from patients probably infected by sexual contact were serotype A; conversely, 76% (28 out of 37) of injecting drug users were serotype B. ConclusionThe serologic results corroborated previous findings, in a smaller subset of samples, of an apparent segregation of viral subtypes by mode of transmission, suggesting two separate HIV-1 epidemics in Thailand. This peptide EIA could be a valuable epidemiologic tool in determining the dynamics of the rapid spread of HIV-1 in Thailand.

DIRECT DETECTION OF HIV RNA EXPRESSION IN SEROPOSITIVE SUBJECTS

The polymerase chain reaction (PCR) and reverse transcription were used to assess human immunodeficiency virus type 1 (HIV1) RNA expression in peripheral blood mononuclear cell samples from seropositive subjects. HIV RNA was detected from seropositive subjects who had no symptoms, lymphadenopathy syndrome, and acquired immunodeficiency syndrome. DNA PCR of the samples used for RNA extraction showed that seventeen of eighteen (94%) contained HIV proviral DNA. Eleven (65%) of the seventeen DNA-positive samples were also positive for HIV RNA, including samples from four patients undergoing antiviral drug treatment. Serum HIV antigen assays detected only six (32%) of the nineteen PCR-positive samples. Owing to the speed and high sensitivity of PCR for HIV detection, this technique will be suitable for monitoring antiviral therapy and the virus load of people with HIV infections.

No Evidence of Murine-Like Gammaretroviruses in CFS Patients Previously Identified as XMRV-Infected

Chronic fatigue syndrome patients reported previously to be XMRV-infected show no signs of the virus in an independent evaluation. Members of the gammaretroviruses—such as murine leukemia viruses (MLVs), most notably XMRV [xenotropic murine leukemia virus (X-MLV)–related virus—have been reported to be present in the blood of patients with chronic fatigue syndrome (CFS). We evaluated blood samples from 61 patients with CFS from a single clinical practice, 43 of whom had previously been identified as XMRV-positive. Our analysis included polymerase chain reaction and reverse transcription polymerase chain reaction procedures for detection of viral nucleic acids and assays for detection of infectious virus and virus-specific antibodies. We found no evidence of XMRV or other MLVs in these blood samples. In addition, we found that these gammaretroviruses were strongly (X-MLV) or partially (XMRV) susceptible to inactivation by sera from CFS patients and healthy controls, which suggested that establishment of a successful MLV infection in humans would be unlikely. Consistent with previous reports, we detected MLV sequences in commercial laboratory reagents. Our results indicate that previous evidence linking XMRV and MLVs to CFS is likely attributable to laboratory contamination.

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.