Automated procedure for improving the RNA isolation step in viral load testing for human immunodeficiency virus

An automated RNA isolation procedure on the Qiagen BioRobot is described for performing viral load tests for human immunodeficiency virus (HIV) with the Amplicor HIV type 1 test. The new procedure improves the precision of the assay and requires significantly less labor than the presently used manual RNA isolation procedure.     

Improved sensitivity of human immunodeficiency virus type 2 subtype B plasma viral load assay

We developed a new assay for human immunodeficiency virus type 2 plasma RNA quantification based on a previous format. The new version performed significantly better than the original regarding the detection of subtype B, allowing the detection of 14 out of 36 plasma RNAs in the subtype B-infected patients not detected with the original version.     

Interactions between beta-herpesviruses and human immunodeficiency virus in vivo: evidence for increased human immunodeficiency viral load in the presence of human herpesvirus 6

In vitro, beta-herpesviruses can stimulate or inhibit HIV replication under particular circumstances. In order to investigate the effects of beta-herpesvirus infection on HIV replication and vice versa at an organ level, we determined the quantitative relationships between cytomegalovirus (CMV), human herpesviruses (HHV) 6 and 7, and HIV-1 proviral DNA using quantitative competitive PCR methods in 141 organs collected at autopsy from 11 AIDS patients. The presence of HHV-6 DNA in an organ was significantly associated with elevated HIV-1 proviral DNA (difference in HIV median loads, 1.3 log10 genomes; P = 0.004). Consistent with this, there was a trend for the presence of HIV-1 proviral DNA to be associated with an elevated HHV-6 load (0.44 log10 difference; P = 0.07). In contrast, there were no significant differences between viral loads in the combinations of either CMV or HHV-7 with HIV-1 proviral DNA load. Pairwise combinations of the beta-herpesviruses revealed that the quantity of HHV-7 was increased in the presence of HHV-6 (difference in median loads, 1.3 log10; P = 0.001) and the quantity of HHV-6 was increased in the presence of HHV-7 (difference in median loads, 0.7 log10; P=0.002). These results demonstrate that the presence of HHV-6 in an organ is significantly associated with an elevated HIV-1 proviral load and have implications for understanding HIV pathogenesis in the human host and the role that beta-herpesviruses, especially HHV-6, might play as cofactors in the HIV disease process.     

Plasma RNA viral load in human immunodeficiency virus type 2 subtype A and subtype B infections

Human immunodeficiency virus type 2 (HIV-2) is much less pathogenic than HIV-1, and HIV-2 infection is associated with plasma viral loads significantly lower than those found in HIV-1 infection. We have developed a real-time quantitative PCR method for measuring the HIV-2 RNA load that covers the range of genetic diversity of HIV-2 isolates and that detects extremely low viral loads. Samples from 49 patients were studied. Proviral DNA was first detected and quantified. The strains that were detected were then genotyped: 21 patients were infected with HIV-2 subtype A and 15 patients were infected with HIV-2 subtype B; 1 patient was infected with a highly divergent strain. Env PCR failed for the remaining 12 patients, so subtypes could not be determined. For viral RNA quantification, a stock of HIV-2 strain NIHZ, which was counted by electron microscopy, was used as the standard. Several primer sets targeting the highly conserved gag region were evaluated. Various primer combinations failed to amplify subtype B strains. With the final primer pair selected, which detected both subtype A and subtype B strains, the sensitivity of the assay was 100% at a viral load of 250 copies/ml and 66% at a viral load of 125 copies/ml. We found a correlation between the CD4(+)-cell count, the clinical stage, and the plasma HIV-2 RNA level. The median plasma HIV-2 RNA value for the 33 asymptomatic patients was 2.14 log(10), whereas it was 3.1 log(10) for the 16 patients with AIDS (P < 0.01). Proviral DNA was detectable in 18 symptom-free patients with high CD4(+)-cell counts, in whom viral RNA was undetectable.     

Complement-dependent control of viral dynamics in pathogenesis of human immunodeficiency virus and simian immunodeficiency virus infection

Since the first contact with the host, human immunodeficiency virus (HIV) exploits the complement system to reach maximal spread of infection. HIV has adapted many strategies to avoid complement-mediated lysis and uses the opsonization with complement fragments for attachment to complement receptors (CR). From the pathogen's perspective, binding to CR-expressing cells is remarkably beneficial, bringing together virus and activated target cells that are highly susceptible to infection. Moreover, complement-mediated trapping on CR+ cells permits HIV to infect surrounding cells even in the presence of an excess of neutralizing antibodies. Thus, complement activation initiates the assumption of power over the host's immune system by HIV and thus augments viral spread and replication throughout the body. On the other hand, natural hosts of primate lentiviruses, such as sooty mangabeys, African green monkeys and chimpanzees, are generally considered to be resistant to the development of AIDS, despite persistent viral replication. This review focuses on the possible link between the resistance to disease and species-specific diversity in function of human and monkey complement system.     

Comparison of three current viral load assays for the quantitation of human immunodeficiency virus type 1 RNA in plasma

The LCx HIV RNA quantitative assay (Abbott Laboratories, Delkenheim, Germany) was compared with the Versant HIV-1 RNA 3.0 (bDNA) assay (Bayer, Tarrytown, NY) and the COBAS Amplicor HIV-1 Monitor v1.5 test (Roche Diagnostics, Branchburg, NJ), using plasma samples of various viral load levels from HIV-1-infected patients. Considering the lower limit of the linear range of 50 copies/ml of both assays, the detection range of the LCx was 127/151 (84.1%) versus the 131/151 (86.8%) of the bDNA 3.0 assay, while overall agreement between the two assays was 93.4% (141/151). LCx and bDNA 3.0 results were found to be strongly correlated (r = 0.96). The fitted regression line was described by the equation log10(LCx copies/ml) = 0.05 + 1.06 x log10(bDNA 3.0 copies/ml) with 95% CI for the estimated slope and intercept at 1.01, 1.12 and -0.16, 0.26, respectively. Similarly, the detection range of the LCx was 115/148 (77.7%) versus the 128/148 (86.5%) of the Monitor v1.5 test. A 91.2% concordance (135/148) was observed between these two assays at a cut-off of 50 copies/ml. LCx and Monitor v1.5 results were highly correlated (r = 0.96). The fitted regression line was described by the equation log10(LCx copies/ml) = 0.06 + 1.03 x log(10)(Monitor v1.5 copies/ml) with 95% CI for the estimated slope and intercept at 0.97, 1.09 and -0.16, 0.28, respectively.     

Resistant viral variants in cellular reservoirs of human immunodeficiency virus infection

Since the advent of antiretroviral therapy (ART), morbidity and mortality rates in those infected with human immunodeficiency virus type 1 (HIV-1) have been significantly reduced. However, HIV-1 is known to persist in several types of cells and tissues, and will usually return to pretreatment levels when therapy is stopped, even in those individuals who have been on suppressive ART for a long time. The discovery of drug sanctuaries and viral reservoirs in the body, in which HIV may persist, has helped to explain why therapeutic eradication of HIV-1 has proved so difficult. Several studies have indicated that the latent reservoir is an archive, composed of a mixture of wild-type and drug-resistant strains. Archived variants are assumed to remain life-long, thereby precluding the successful recycling of any drug towards which resistance has arisen. Several studies have underlined the value of pro-viral DNA as an additional source of information on the total burden of resistance in an individual. The HIV mutation patterns detected in plasma do not necessarily reflect those found in the cell-associated compartment, and may not be the same as those in different anatomical compartments. Although assessment of drug resistance in plasma is of direct and immediate importance for treatment, examination of the genotypic pattern of HIV-1 in cellular compartments might also provide information allowing a more sustainable response to therapy and better disease management.     

The role of viral load determination for the management of human immunodeficiency virus, hepatitis B virus and hepatitis C virus infection.

During the last 5 years, considerable scientific and financial efforts have been made in the development of quantitative nucleic acid detection technology. For detection of human immunodeficiency virus (HIV), quantitative culture is time consuming, cumbersome and requires appropriate laboratory safety equipment. Quantitative determination of p24 antigen by enzyme immunoassay is of limited value due to its relatively poor sensitivity. Therefore, quantitative determination of viral load using nucleic acid amplification techniques is the most accurate, prognostic marker for HIV type 1 infection, independently of the CD4+ cell count. Hepatitis B virus (HBV) is not cultivable in vitro. Serological assays allow an accurate diagnosis and follow-up of acute or chronic infection. Quantification of HBV DNA is used for the monitoring of antiviral therapy, determination of infectivity and for resolution of unclear serological profiles, e.g. isolated anti-HBc reactivity, as well as for patients in which HBV mutants are suspected. Hepatitis C virus (HCV) can only be detected by molecular based assays because no cell culture system, which permits a reliable isolation of clinical specimens, is currently available. Furthermore, early diagnosis and follow-up of infection cannot be achieved with antibody serology. The prognostic relevance of quantitative HCV RNA determination is of limited value for the long-term prognosis of chronic hepatitis C. However, viral load may predict the outcome of antiviral therapy. Genetic diversity is another challenge for HCV RNA quantification.     

Simplified testing for antibodies to human immunodeficiency virus.

Test strips for the detection of antibodies to human immunodeficiency virus type 1 were investigated using specimens from risk groups in Thailand (141 reactive; 445 nonreactive) in a local Thai laboratory. The diagnostic sensitivity and specificity were both 100%. Using a set of seroconversion panels, the sensitivity of the test strips was within the range of sensitivities obtained with enzyme immunoassays. The test was developed for performance at decentralized settings under nonlaboratory conditions.     

Evaluation of rapid prenatal human immunodeficiency virus testing in rural cameroon

Pregnant women (n = 859) in rural Cameroonian prenatal clinics were screened by two rapid human immunodeficiency virus (HIV) antibody tests (rapid tests [RT]) (Determine and Hema-Strip) using either whole blood or plasma. One additional RT (Capillus, HIV-CHEK, or Sero-Card) was used to resolve discordant results. RT results were compared with HIV-1 enzyme immunoassay (EIA) and Western blot (WB) results of matched dried blood spots (DBS) to assess the accuracy of HIV RTs. DBS EIA/WB identified 83 HIV antibody-reactive, 763 HIV antibody-nonreactive, and 13 indeterminate specimens. RT results were evaluated in serial (two consecutive tests) or parallel (two simultaneous tests) testing algorithms. A serial algorithm using Determine and Hema-Strip yielded sensitivity and specificity results of 97.6% and 99.7%, respectively, whereas a parallel RT algorithm using Determine plus a second RT produced a sensitivity and specificity of 100% and 99.7%, respectively. HIV RTs provide excellent alternatives for identifying HIV infection, and their field performance could be monitored using DBS testing strategies.     

Rheumatic symptoms in patients with human immunodeficiency virus are related to levels of tumor necrosis factor-alpha but not to viral load

Infection with human immunodeficiency virus (HIV) can lead to osteoarticular involvement, usually in the late stages. The pathogenesis of these symptoms has usually been attributed to viral load or to dysregulated cytokine production. We evaluated the presence of rheumatic symptoms and levels of tumor necrosis factor (TNF)-alpha viral load and CD4 count in 46 patients with HIV from southern Italy. The prevalence of rheumatic symptoms was 23.9%; CD4 count and viral load presented no statistically significant differences between patients with rheumatic symptoms and patients without osteoarticular involvement, whereas TNF-alpha levels were increased in HIV patients with arthralgias compared with those in patients without arthralgias (p = 0.02). Evidence that TNF-alpha is increased in patients with osteoarticular or soft tissue involvement is a clear index of the pivotal role this cytokine plays in the pathogenesis of these manifestations.     

Use of calibrated viral load standards for group M subtypes of human immunodeficiency virus type 1 to assess the performance of viral RNA quantitation tests

Optimal management of human immunodeficiency virus type 1 (HIV-1) disease requires accurate quantitation of viral RNA concentrations in plasma. Evidence for increasing geographic intermixing of HIV-1 subtypes makes equivalent quantitation of all subtypes essential. The performances of six quantitative viral RNA tests are described, for the first time, with calibrated viral isolates of diverse subtypes.     

Segregation of human immunodeficiency virus type 1 subtypes by risk factor in Australia

The aim of this study was to determine which human immunodeficiency virus type 1 (HIV-1) subtypes were circulating in Australia and to correlate the subtypes with risk factors associated with the acquisition of HIV-1 infection. DNA was extracted from peripheral blood mononuclear cells, and HIV-1 env genes were amplified and subtyped using heteroduplex mobility analysis, with selected samples sequenced and phylogenetic analysis performed. The HIV-1 env subtypes were determined for 141 samples, of which 40 were from female patients and 101 were from male patients; 13 samples were from children. Forty-seven patients were infected by homosexual or bisexual contact, 46 were infected through heterosexual contact, 21 were infected from injecting drug use (IDU), 13 were infected by vertical transmission, 8 were infected from nosocomial exposure, and 6 were infected by other modes of transmission, including exposure to blood products, ritualistic practices, and two cases of intrafamilial transmission. Five subtypes were detected; B (n = 104), A (n = 5), C (n = 17), E (CRF01_AE; n = 13), and G (n = 2). Subtype B predominated in HIV-1 acquired homosexually (94% of cases) and by IDU (100%), whereas non-subtype B infections were mostly seen in heterosexually (57%) or vertically (22%) acquired HIV-1 infections and were usually imported from Africa and Asia. Subtype B strains of group M viruses predominate in Australia in HIV-1 transmitted by homosexual or bisexual contact and IDU. However, non-B subtypes have been introduced, mostly acquired via heterosexual contact.