The new REGA subtyping tool, developed using Java programming and PERL scripts, combines phylogenetic analyses with boot-scanning methods for the genetic subtyping of full-length and subgenomic fragments of HIV-1. When used to investigate the subtype of previously published reference datasets that were analysed using manual phylogenetic methods, the automated method correctly identified 97.5-100% of non-recombinant and circulating recombinant forms of HIV-1, including 108 full-length, 108 gag and 221 env sequences downloaded from the Los Alamos database.

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Microbial translocation is a major force driving chronic inflammation in HIV-infected Africans receiving cART. Prevention of monocyte activation may be especially effective at enhancing therapeutic outcomes.

To investigate transmission of human herpesvirus (HHV)-8, 2546 mother-child pairs were recruited from rural clinics in South Africa and were tested for antibodies against lytic and latent HHV-8 antigens. The prevalence of antibodies in children increased with increasing maternal antibody titer (lytic, chi 21=26, and P<.001; latent, chi 21=55, and P<.001). HHV-8 DNA was detectable in 145 of 978 maternal saliva samples (mean virus load, 488,450 copies/mL; range, 1550-660,000 copies/mL) and in 12 of 43 breast-milk samples (mean virus load, 5800 copies/mL; range, 1550-12,540 copies/mL). The prevalence of HHV-8 DNA in maternal saliva was unrelated to latent anti-HHV-8 antibody status but was higher in mothers with the highest titers of lytic antibodies than in other mothers (34% vs. 8%; P<.001). The prevalence of lytic anti-HHV-8 antibodies in children was 13% (70/528) if the mother did not have HHV-8 in saliva and was 29% (8/28) if the mother had a high HHV-8 load (>50,000 copies/mL) in saliva (odds ratio, 2.6; 95% confidence interval, 1.1-6.2). The presence of HHV-8 DNA in maternal saliva was unrelated to latent antibodies in children. Saliva could be a route of transmission of HHV-8 from person to person, although other routes cannot be ruled out.

Eleven laboratories evaluated the use of dried blood and plasma spots for quantitation of human immunodeficiency virus (HIV) RNA by two commercially available RNA assays, the Roche Amplicor HIV-1 Monitor and the bioMerieux NucliSens HIV-1 QT assays. The recovery of HIV RNA was linear over a dynamic range extending from 4,000 to 500,000 HIV type 1 RNA copies/ml. The Monitor assay appeared to have a broader dynamic range and seemed more sensitive at lower concentrations. However, the NucliSens assay gave more consistent results and could be performed without modification of the kit. HIV RNA was stable in dried whole blood or plasma stored at room temperature or at ؊70°C for up to 1 year. Dried blood and dried plasma spots can be used as an easy and inexpensive means for the collection and storage of specimens under field conditions for the diagnosis of HIV infection and the monitoring of antiretroviral therapy.Human immunodeficiency virus (HIV) RNA levels in blood plasma are used to monitor the response to antiretroviral drug therapy in developed countries. Since the International AIDS Conference held in Durban, South Africa, in 2000, there has been a global effort to provide access to antiretroviral drugs to all infected individuals, including those in the resource-poor countries which have been hit the hardest by the AIDS pandemic. Studies have demonstrated that the provision of treatment is not enough, however. In order for the drugs to be most effective, both for an individual and for public health reasons, it will be necessary to monitor the responses to the therapy. HIV RNA assays typically use plasma, which implies access to laboratory equipment that may not be readily available in all field settings. Therefore, alternative specimens must be considered for use in these situations.Whole blood dried on filter paper (dried blood spots [DBSs]) has been used to qualitatively detect HIV antibodies (13, 15), HIV DNA (1, 3, 5-7, 9, 10, 11, 12, 16, 18) and HIV RNA (4, 14, 17; J. L. Gerstel and A. M. Comeau, Proc. 10th Natl. Neonatal Screening Symp., abstr. P-19, p. 64, 1994). In addition, dried plasma spots (DPSs) have also been used to quantitate the HIV load (8). Although the stabilities of antibodies and DNA in DBSs have been determined (2, 6, 13), there has been some question regarding the stability of the HIV RNA when it is dried and stored at room temperature (14, 17).The purpose of this study was to compare two different methods for quantitatively measuring HIV RNA in DBSs and DPSs and to assess their long-term stability at room temperature and at Ϫ70C.(This study was presented in part at the 12th World AIDS Conference, Geneva, Switzerland, 28 June to 3 July 1998 [S. Cassol, A. Comeau, S. Fiscus, G. Aldrovandi, J. Sullivan, J. Bremer, and B. Jackson, Abstr. 12th World AIDS Conf., abstr. 33166, 1998].) MATERIALS AND METHODS Specimens.Three different panels were prepared by the Virus Quality Assurance Laboratory by using filter paper (903; Schleicher & Schuell, Keene, N.H.) spotted with 50 l of plasma or whol...

Human immunodeficiency virus type-1 (HIV-1), hepatitis B and C and other rapidly evolving viruses are characterized by extremely high levels of genetic diversity. To facilitate diagnosis and the development of prevention and treatment strategies that efficiently target the diversity of these viruses, and other pathogens such as human T-lymphotropic virus type-1 (HTLV-1), human herpes virus type-8 (HHV8) and human papillomavirus (HPV), we developed a rapid high-throughput-genotyping system. The method involves the alignment of a query sequence with a carefully selected set of pre-defined reference strains, followed by phylogenetic analysis of multiple overlapping segments of the alignment using a sliding window. Each segment of the query sequence is assigned the genotype and sub-genotype of the reference strain with the highest bootstrap (>70%) and bootscanning (>90%) scores. Results from all windows are combined and displayed graphically using color-coded genotypes. The new Virus-Genotyping Tools provide accurate classification of recombinant and non-recombinant viruses and are currently being assessed for their diagnostic utility. They have incorporated into several HIV drug resistance algorithms including the Stanford (http://hivdb.stanford.edu) and two European databases (http://www.umcutrecht.nl/subsite/spread-programme/ and http://www.hivrdb.org.uk/) and have been successfully used to genotype a large number of sequences in these and other databases. The tools are a PHP/JAVA web application and are freely accessible on a number of servers including:http://bioafrica.mrc.ac.za/rega-genotype/html/http://lasp.cpqgm.fiocruz.br/virus-genotype/html/http://jose.med.kuleuven.be/genotypetool/html/.

Introduction of subtype C of HIV-1 in Brazil was likely a single event of one or a mixture of similarly related strains. Recombination between subtype C and B viruses is an ongoing process in the country. Primary and secondary drug resistance mutations were observed, although some of the secondary mutations could be associated with subtype C molecular signatures. Subtype-specific polymorphisms of PR and RT sequences found in this subtype C Brazilian variant might influence this emergence and have an impact on HIV treatment and on vaccine development in the country.

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