Episodes of low-level viremia (LLV), with plasma human immunodeficiency virus type 1 (HIV-1) RNA levels ranging from 50 to 400 copies (c)/ml, occur commonly during highly active antiretroviral therapy (HAART). LLV has been associated with virologic failure of HAART in some studies, while in others LLV did not appear to affect the clinical outcome. To understand the processes leading to LLV, genetic analyses were used to determine whether plasma virions emanated from archived or from newly evolved viral genomes. Episodes of LLV (plasma HIV-1 RNA, 50 to 379 [median, 77] c/ml) were detected in 21/37 (57%) HIV-1-infected children with median plasma HIV-1 RNA levels of <50 c/ml during 79 patient years of HAART. Viral sequences were derived by direct sequencing of PCR products from 21 plasma specimens diluted to end point. In phylogenetic analysis, LLV viral sequences grouped with virus from early in the course of infection in 8/11 subjects. Six specimens had multiple identical viral sequences, suggesting origin from clonally expanded infected cells. LLV plasma virus evolved over time, indicating viral replication, in 3/11 subjects. Two of these had frequent LLV, including the selection of drug-resistant mutants. In summary, plasma virus from episodes of LLV during effective HAART appeared to originate from two distinct processes, (i) clonal outgrowth from long-lived HIV-1-infected cells, presumably following activation and proliferation of these cells, and (ii) ongoing viral replication that included the selection of new drug-resistant mutants. These observations provide a plausible explanation for the divergent clinical outcomes previously associated with LLV.Suppressive combination highly active antiretroviral therapy (HAART) has improved the health and life span of human immunodeficiency virus type 1 (HIV-1)-infected children (34) and adults (23). The long-term efficacy of HAART, however, has been limited in many individuals (11, 16), often due to the selection of drug-resistant viral mutants (2, 48). Transient lowlevel viremias (LLV), with plasma HIV-1 RNA levels in the range of 50 to 400 copies (c)/ml, have been reported to occur in 25 to 40% of adults in whom viral replication appeared to have been suppressed by HAART (21,24,45) and up to 90% of subjects with very frequent determinations of plasma HIV-1 RNA levels (35). LLV have been associated with selection of drug-resistant virus in several (8, 21, 22), but not all, studies (25,35,45).LLV appeared to be clinically insignificant in two studies, as subjects with and without LLV had similar rates of virologic failure of HAART (24,45). Detailed studies of LLV have demonstrated viral sequences similar to that found early in the course of infection (25,35,38). These observations support the hypothesis that activation of latently infected cells is the source of LLV (7,17,25,38). A modification of this hypothesis proposes that there is continuous low-level expression of virus from a stable proviral reservoir, with detectable virus occurring when random biological pr...

To evaluate human immunodeficiency virus type 1 (HIV-1) replication and selection of drug-resistant viruses during seemingly effective highly active antiretroviral therapy (HAART), multiple HIV-1 env and pol sequences were analyzed and viral DNA levels were quantified from nucleoside analog-experienced children prior to and during a median of 5.1 (range, 1.8 to 6.4) years of HAART. Viral replication was detected at different rates, with apparently increasing sensitivity: 1 of 10 by phylogenetic analysis; 2 of 10 by viral evolution with increasing genetic distances from the most recent common ancestor (MRCA) of infection; 3 of 10 by selection of drug-resistant mutants; and 6 of 10 by maintenance of genetic distances from the MRCA. When four-or five-drug antiretroviral regimens were given to these children, persistent plasma viral rebound did not occur despite the accumulation of highly drug-resistant genotypes. Among the four children without genetic evidence of viral replication, a statistically significant decrease in the genetic distance to the MRCA was detected in three, indicating the persistence of a greater number of early compared to recent viruses, and their HIV-1 DNA decreased by >0.9 log 10 , resulting in lower absolute DNA levels (P ‫؍‬ 0.007). This study demonstrates the variable rates of viral replication when HAART has suppressed plasma HIV-1 RNA for years to a median of <50 copies/ml and that combinations of four or five antiretroviral drugs suppress viral replication even after short-term virologic failure of three-drug HAART and despite ongoing accumulation of drug-resistant mutants. Furthermore, the decrease of cellular HIV-1 DNA to low absolute levels in those without genetic evidence of viral replication suggests that monitoring viral DNA during HAART may gauge low-level replication.

To determine the potential role of methylation in the regulation of interferon-gamma (IFN-gamma) gene transcription by T cells, primary T-lineage cell populations were analyzed for the extent of methylation of three CpG sites within or near transcriptional activator elements in the 5' flank and first intron of the human IFN-gamma gene. A striking correlation was observed between the capacity of the IFN-gamma gene to be expressed and the degree of hypomethylation. The IFN-gamma gene was virtually completely methylated at all sites in thymocytes, neonatal T cells, and adult CD45RAhiCD45R0lo (antigenically naive) CD4 T cells, cell types that all have a low or undetectable capacity to express the IFN-gamma gene. In contrast, there was substantial hypomethylation in T-lineage cell types with relatively high capacities to express the IFN-gamma gene, including adult CD8 T cells and adult CD45RAloCD45R0hi (memory/effector) CD4 T cells. These results suggest that hypomethylation of the IFN-gamma genetic locus may be an important determinant of IFN-gamma gene expression in vivo by T-lineage cells.

The detection of virus is used to diagnose human immunodeficiency virus type 1 (HIV-1) infection in infants due to the persistence of maternal antibodies for a year or more. An HIV-1 DNA PCR assay with simple specimen collection and processing was developed and evaluated. Whole blood was collected on filter paper that lysed cells and bound the DNA, eliminating specimen centrifugation and extraction procedures. The DNA remained bound to the filter paper during PCR amplification. Assays of copy number standards showed reproducible detection of 5 to 10 copies of HIV-1 in 5 l of whole blood. The sensitivity of the assay did not decrease after storage of the standards on filter paper for 3 months at room temperature or after incubation at 37 or 45°C for 20 h. The primers used for nested PCR of the HIV-1 pol gene amplified templates from a reference panel of multiple HIV-1 subtypes but did not amplify a subtype A or a subtype C virus from children living in Seattle. The assay had a sensitivity of 98.4% and a specificity of 98.3% for testing of 122 specimens from 35 HIV-1-infected and 16 uninfected children and 43 seronegative adults living in Washington. The assay had a sensitivity of 99% and a specificity of 100% for testing of 102 HIV-1-positive (as determined by enzyme immunoassay) Peruvian women and 6 seropositive and 34 seronegative infants. This assay, with adsorption of whole blood to filter paper and no specimen processing, provides a practical, economical, sensitive, and specific method for the diagnosis of HIV-1 subtype B infection in infants.

Opportunistic infections and deaths are less common among HIV-infected youth in the US in the cART era, but the mortality rate remains elevated. Deaths were associated with poor HIV control and older age. Emerging complications, such as psychiatric, inflammatory, metabolic, and genital tract diseases, need to be addressed.