Sensitive and reproducible quantitation of mucosal HIV-1 RNA and DNA viral burden in patients with detectable and undetectable plasma viral HIV-1 RNA using endoscopic biopsies

Mucosal tissue is the main portal of entry for HIV-1 infection and, in macaques, has been demonstrated to be a significant compartment for viral replication and CD4+ T lymphocyte depletion. Quantitating tissue viral burden in addition to plasma viral load provides insights into HIV-1 pathogenesis and an additional means to gauge antiretroviral response. The aim of this study was to develop reliable, reproducible, and sensitive assays to quantitate tissue viral burden of HIV-1 RNA and DNA using 1-3 endoscopically acquired, rectosigmoid biopsies. Total DNA and RNA were simultaneously extracted following homogenization from the same tissue samples. Quantitative polymerase chain reaction (PCR) assay in the HIV-1 LTR region was used to detect viral DNA and RT-PCR for viral RNA. It was determined that HIV-1 RNA and DNA can be reproducibly quantified from a single rectosigmoid biopsy with minimal intra-assay or intra-patient variability. These results reflect high recovery of extracted nucleic acids with calculated results accurately reflecting in vivo levels. The techniques outlined differ from currently available approaches by incorporating control standards to identify loss or degradation of RNA and DNA from acquisition through the in vitro assay and permit extraction with high yields of RNA and DNA from the same tissue sample.     

Simple determination of human immunodeficiency virus type 1 syncytium-inducing V3 genotype by PCR.

Human immunodeficiency virus type 1 (HIV-1) phenotype variability plays an important role in the pathogenesis of AIDS. The presence of syncytium-inducing (SI) HIV-1 isolates in infected individuals is associated with a rapid decline of CD4+ T cells, rapid disease progression, and reduced survival time after AIDS diagnosis. The strong association between the SI capacity of HIV-1 and the presence of positively charged amino acid residues at positions 306 and/or 320 in the third variable domain (V3) of gp120 could here be confirmed in 97% of 402 primary HIV-1 isolates, indicating that the V3 genotype may be useful for prediction of the viral phenotype. The V3 DNA sequences revealed a remarkably limited codon usage for the amino acid residues that are responsible for virus phenotype. On the basis of this limited SI-specific DNA sequence variation, four SI-specific oligonucleotides were designed for selective amplification of V3 from SI but not non-SI HIV-1 isolates. This PCR analysis allowed the prediction of the biological phenotype of HIV-1 isolates on the basis of the V3 genotype and may prove to be useful for monitoring SI capacity of HIV-1 isolates in infected individuals.     

Human immunodeficiency virus type 1 detected in all seropositive symptomatic and asymptomatic individuals.

Between February 1987 and October 1988, peripheral mononuclear blood cells (PBMC) from 409 adult individuals antibody positive by Western (immuno-)blot for human immunodeficiency virus type 1 (HIV-1) (56 acquired immunodeficiency syndrome [AIDS] patients, 88 patients with AIDS-related complex, and 265 asymptomatic individuals) were consecutively cultured for HIV-1 or tested for the presence of HIV-1 DNA sequences by a polymerase chain reaction assay (PCR). We isolated HIV-1 or detected HIV-1 DNA sequences from the PBMC of all 409 HIV-1 antibody-positive individuals. None of 131 healthy HIV-1 antibody-negative individuals were HIV-1 culture positive, nor were HIV-1 DNA sequences detected by PCR in the blood specimens of 43 seronegative individuals. In addition, HIV-1 PCR and HIV-1 culture were compared in testing the PBMC of 59 HIV-1 antibody-positive and 20 HIV-1 antibody-negative hemophiliacs. Both methods were found to have sensitivities and specificities of at least 97 and 100%, respectively. In contrast, the sensitivities of serum HIV-1 antigen testing in AIDS patients and asymptomatic seropositive patients were 42 and 17%, respectively. Our ability to directly demonstrate HIV-1 infection in all HIV-1 antibody-positive individuals provides definitive support that HIV-1 antibody positivity is associated with present HIV-1 infection. Moreover, the sensitivities and specificities of PCR and culture for the detection of HIV-1 appear to be equivalent, and both methods are superior to testing for HIV-1 antigen in serum for the direct detection of HIV-1.     

Detection of human immunodeficiency virus type 1 (HIV-1) antibody by western blotting and HIV-1 DNA by PCR in patients with AIDS.

The human immunodeficiency virus type 1 (HIV-1) Western blotting (immunoblotting) band patterns and the sensitivity of an HIV-1 DNA PCR assay were determined by testing the blood of patients with AIDS. Plasma and cell pellets processed from the peripheral blood of 199 patients with absolute CD4 cell counts of less than 200 cells per mm3 were tested by a licensed enzyme immunoassay (EIA; Abbott HIV-1) and Western blot assay (Cambridge-Biotech) for HIV-1 antibody. The Roache HIV-1 AMPLICOR DNA PCR assay was used to test cell pellets from 125 of the 199 patients for HIV-1 gag DNA sequences. All plasma samples from these 199 sequential patients were reactive for HIV-1 antibody by EIA and were positive by Western blot assay using the criteria recommended by the Centers for Disease Control and Prevention. The majority of samples (192 of 199; 96.5%) displayed at least six of nine bands characteristic of the virus by Western blotting, with the lowest number of bands characteristic of the virus displayed by any sample being three. However, 39 and 48% of all patients exhibited no bands to p17 and p55 antigens, respectively, whereas 0 to 7.5% of all patients exhibited no bands to the other antigens. HIV-1 gag DNA sequences were detected in 117 (93.6%) of 125 cell pellets processed from the peripheral blood of these same patients. All eight patients initially negative by PCR tested positive when a second pellet which had been produced from the same blood sample was tested. Despite a decrease in antibody reactivity to HIV Gag and Pol proteins, patients with advanced HIV-1 infection remained positive for HIV-1 antibody by EIA and Western blot testing. Confirmation by the HIV-1 Western blot assay still appears to be the more sensitive assay for the diagnosis of HIV-1 infection in those individuals with advanced HIV-1 infection in the United States.     

Genetic composition of replication competent clonal HIV-1 variants isolated from peripheral blood mononuclear cells (PBMC), HIV-1 proviral DNA from PBMC and HIV-1 RNA in serum in the course of HIV-1 infection

The HIV-1 quasispecies in peripheral blood mononuclear cells (PBMC) is considered to be a mix of actively replicating, latent, and archived viruses and may be genetically distinct from HIV-1 variants in plasma that are considered to be recently produced.Here we analyzed the genetic relationship between gp160 env sequences from replication competent clonal HIV-1 variants that were isolated from PBMC and from contemporaneous HIV-1 RNA in serum and HIV-1 proviral DNA in PBMC of four longitudinally studied therapy naïve HIV-1 infected individuals.Replication competent clonal HIV-1 variants, HIV-1 RNA from serum, and HIV-1 proviral DNA from PBMC formed a single virus population at most time points analyzed. However, an under-representation in serum of HIV-1 sequences with predicted CXCR4 usage was sometimes observed implying that the analysis of viral sequences from different sources may provide a more complete assessment of the viral quasispecies in peripheral blood in vivo.     

Measurement of HIV-1 p24 antigen by signal-amplification-boosted ELISA of heat-denatured plasma is a simple and inexpensive alternative to tests for viral RNA

Assessment of HIV-1 RNA concentration is widely used for monitoring antiretroviral therapies. Tests are, however, expensive and require technically advanced equipment and highly trained personnel. Increasing availability of antiretroviral treatment in resource-poor settings calls for simple and inexpensive virus tests. HIV-1 p24 antigen tests were frequently used before the availability of nucleic acid tests (NAT). Two simple modifications, heat-mediated destruction of test-interfering antibodies and increased sensitivity achieved by signal amplification, have shaped the p24 antigen test into a tool that rivals NAT. This improved p24 antigen test, for which all reagents are available from Perkin Elmer Life Sciences, was evaluated in clinical studies in comparison with the most sensitive PCR methods available at a given time. In a prospective study over 4 years, HIV-1 infection among 859 samples from 307 infants born to HIV-positive mothers in Switzerland was detected as sensitively by p24 antigen assay as by PCR for viral DNA or RNA: 100% sensitivity of all methods after 10 days of age; 99.2% diagnostic specificity of p24 after neutralization (RNA, 98.6%). A study conducted in Dar es Salaam (Tanzania) found 123 of 125 samples from 76 PCR-positive infants positive for p24 antigen (sensitivity = 98.7%). In 169 infected Swiss adults with a median CD4+ T-cell count of 140 cells/microliter followed for a median of 2.7 years, p24 at baseline correlated as well as or better than HIV-1 RNA with the ensuing CD4+ T-lymphocyte decline and was independently predictive of progression to clinical AIDS (P = 0.043) and survival (P = 0.032). RNA predicted AIDS (P < 0.005), but not survival (P = 0.19). Another study of first-visit samples from 496 mostly black IVDU in the U.S. with a median CD4+ count of 518 cells/microliter showed equally strong prediction of progression to clinical AIDS for p24 antigen, HIV-1 RNA, and CD4+ T-lymphocyte concentrations at baseline. Treatment-associated changes in p24 and RNA levels in adults and children correlated well in three Swiss studies. The half-life of p24 antigen in the first phase of effective treatment was 1.6 +/- 0.4 days (RNA, 1.7 +/- 0.8). A second, slower decay phase had a half-life of 42 +/- 16 days. One study suggested that a strategy involving a somewhat more frequent testing for p24 antigen permitted detection of viral failures significantly earlier than tests for HIV-1 RNA conducted at 3-month intervals, while at the same time significantly saving on costs. Experience from three studies indicates that the p24 antigen test recognizes viruses of subtypes A-G and O, as well as some recombinant isolates, but leaves open the possibility that some non-B p24 antigens may be suboptimally detected. This improved p24 antigen test provides diagnosis of pediatric HIV infection, prediction of prognosis and treatment monitoring in quality comparable to tests for HIV-1 RNA, but at much lower costs. There is no problem with sample instability and no need for cumbersome nucleic acid extraction. The test is validated for subtype B, but requires further studies for non-B subtypes.     

Human immunodeficiency virus isolates from asymptomatic homosexual men and from AIDS patients have distinct biologic and genetic properties.

Human immunodeficiency virus type 1 (HIV-1) isolates from asymptomatic homosexual men and AIDS patients were compared for their in vitro biologic and genetic properties. Most of the HIV-1 isolates from asymptomatic men, but not from AIDS patients, failed to infect CD4+ H9 cells and phytohemagglutinin-stimulated peripheral blood lymphocytes. In a longitudinal study, serial HIV-1 isolates obtained from men who seroconverted to HIV-1 and later developed AIDS were able to infect H9 cells. In contrast, longitudinal isolates from men who remained asymptomatic did not infect H9 cells. HIV-1 isolates from AIDS patients in general exhibited increased production of intracellular viral DNA, RNA, and protein as compared to isolates from asymptomatic men. Cells infected with HIV-1 isolates from asymptomatic men produced very little gp120, p24, and p55 proteins as compared to those from AIDS patients. The overall restriction patterns of HindIII, Sac-1, Pst-1, EcoR1, and BamH1 were very similar between HIV-1 isolates from asymptomatic men and those from AIDS patients. However, the restriction endonuclease pattern of BglII was quite distinct for isolates from asymptomatic men as compared to AIDS patients. Preliminary studies mapped a unique BglII site in the gag region of most of the isolates from asymptomatic men, approximately 2.0 kb from the 5' end. Thus, HIV-1 isolates from asymptomatic subjects and from AIDS patients have distinct biologic and genetic properties which may be related to the various clinical outcomes of HIV-1 infection.     

Evidence of productively infected CD8+ T cells in patients with AIDS: implications for HIV-1 pathogenesis

CD8+ T lymphocytes play an important protective role against HIV infection. The onset of AIDS is associated with a decline in both the number of CD8+ T lymphocytes and anti-HIV cytotoxic activity in CD8+ T cells. The reason for this progressive failure of CD8+ T cells in HIV-1 infection remains unknown. Earlier reports have shown presence of viral DNA in CD8+ cells of HIV-1-infected patients; under some conditions, CD8+ T cells have been shown to express CD4 in vitro and can be susceptible to infection with HIV-1. However, whether CD8+ lymphocytes in vivo can be productively infected with HIV-1 remains unclear. In this study, we generated multiple CD8+ T-cell clones from two patients with AIDS. These clones were CD8+/CD3+ but did not express CD4. Several of these CD8+ clones from both patients were found to be endogenously infected with HIV-1 and spontaneously produced these viruses. CD8+ cell-produced HIV-1 was biologically competent because viruses produced by most of these clones could efficiently infect and replicate in peripheral blood lymphocytes from HIV-negative donors. In addition, some of these viruses were able to form syncytia in MT-2 cells indicating syncytium-inducing phenotype. Comparison of the sequences in V3 loop areas among different viruses showed changes in some of the clones from both patients. For the first time, this report provides direct evidence that mature CD8+ T cells can be productively infected with HIV-1 in patients with AIDS. Direct infection of CD8+ T lymphocytes may play a role in the eventual failure of these cells in HIV-1 infection.     

False negative HIV-1 proviral DNA polymerase chain reaction in a patient with primary infection acquired in Thailand.

BACKGROUND: Qualitative HIV-1 proviral DNA PCR tests have three main diagnostic applications. These include direct detection of viral sequences in the pre-seroconversion window period which may be positive up to 8 days prior to the development of HIV specific antibodies; resolution of indeterminate HIV serological tests and in the diagnosis of neonates born to seropositive mothers where maternal antibodies may be detectable for up to 15 months past partum. METHODS: A total of three serial specimens from a single patient following symptomatic primary HIV-1 infection were assessed by commercially available serological assays for antibodies to HIV and HIV-1 antigen and nucleic acid amplification assays for proviral DNA (Amplicor HIV-1 test, Roche Molecular Systems, CA, USA) and RNA (HIV MONITOR ver1.5, Roche Molecular Systems) according to manufacturers recommendations. A subsequent modified PCR protocol for HIV proviral DNA was performed which included modified primers (SK145/SKCC1B) and altered thermal cycling parameters. RESULTS: We describe a case of HIV infection acquired in Thailand by heterosexual transmission, where a commercially available HIV proviral DNA PCR assay remained negative despite a typical evolving serological profile consistent with seroconversion. CONCLUSION: These data support the use of HIV DNA PCR tests only as a second line supplemental test to licensed standard HIV diagnostic testing strategies, and should be used with care in cases where non-B subtype infection is a possibility.     

Abbott RealTime HIV-1 m2000rt viral load testing: manual extraction versus the automated m2000sp extraction

The Abbott RealTime HIV-1 assay is a real-time nucleic acid amplification assay available for HIV-1 viral load quantitation. The assay has a platform for automated extraction of viral RNA from plasma or dried blood spot samples, and an amplification platform with real time fluorescent detection. Overall, this study found no clinically relevant differences in viral load, if samples were extracted manually.     

Human immunodeficiency virus type 2 infection associated with AIDS in West Africa

We recently reported the isolation of a new retrovirus, termed human immunodeficiency virus type 2 (HIV-2), from two West African patients with the acquired immunodeficiency syndrome (AIDS). This virus is related to but distinct from the well-characterized AIDS retrovirus, human immunodeficiency virus type 1 (HIV-1). We report here evidence of infection with HIV-2 in 30 patients, almost all from West Africa. Seventeen of them had a clinical syndrome indistinguishable from AIDS (7 of these 17 died). Others had either the AIDS-related complex or no HIV-related symptoms. All patients had serum antibodies reacting with HIV-2 in an indirect immunofluorescence assay. All serum tested contained antibodies reacting with the envelope glycoprotein of the virus in an immunoprecipitation assay. Cross-reactivity of serum antibodies with HIV-1 was detected in a minority of patients and varied according to the assay used. Retroviral isolates were obtained from the blood lymphocytes of 11 patients and were all identified as HIV-2 by nucleic acid hybridization; none hybridized with an HIV-1 probe. These findings indicate that some cases of AIDS in West Africa may be caused by HIV-2, but the extent of the spread of this virus and its clinical correlates will require careful epidemiologic investigation.     

Diagnosis of human immunodeficiency virus infection by a polymerase chain reaction assay evaluated in patients harbouring strains of diverse geographical origin

Since the development of the highly sensitive polymerase chain reaction, PCR has been increasingly used for the diagnosis of viral infections, including the detection of human immunodeficiency virus (HIV), the causative agent of AIDS. In our laboratory a diagnostic PCR is carried out on proviral HIV-1 DNA using a standardised algorithm based on three HIV-1 primer sets. The three primer sets, amplifying a fragment in the LTR-gag gene, in the pol gene and in the env gene, are situated within conserved regions of the HIV-1 genome. These primers allow us to detect not only HIV strains from Belgian patients but also from African patients, who are, for historical reasons, a substantial part of the HIV-positive patients in Belgium. We are able to detect 1–5 copies of proviral HIV-1 DNA with each of the three nested primer sets. A sensitivity and specificity of 92 and 100%, respectively, were achieved when testing 24 Belgian and African HIV-1 seropositive samples. In our lab, the same PCRs are also used for the detection of viral RNA in cases of a doubtful undetectable viral load when using a commercial HIV-1 viral load assay. This is because present-day commercial assays are not entirely reliable with divergent strains. Both our ‘in-house’ diagnostic DNA and RNA-PCR can also be used semiquantitatively with limiting dilutions.     

A new cell-based assay for measuring the forward mutation rate of HIV-1

Over 20 years into the ever-worsening AIDS pandemic, genetic variation remains the greatest obstacle for treating and preventing HIV-1 infection. Mutation rate assays for HIV-1 have been reported; however, none measure directly the forward mutation rate during replication of the virus in cell culture while still retaining the ability to propagate and further study mutant proviruses. Therefore, the objective of the current study was to develop such a phenotypic cell-based assay for measuring the forward mutation rate of HIV-1. Conventional recombinant DNA techniques and polymerase chain reaction were used to create a replication defective HIV-1 vector, pNL4-3Delta+cass, which is based on the NL4-3 strain and contains the thymidine kinase gene from human herpes virus type 1 as the mutational target. A series of transfection and infection steps were used to introduce the vector into 143B cells, which are negative for thymidine kinase function, and produce vector virus for a single cycle of replication. Viral titers were measured by counting the number of drug resistant colonies on the assay plates, and forward mutation rates were calculated from the viral titers. Mutant proviruses were sequenced to determine the types of genetic alterations that occurred. The average forward mutation rate for HIV-1 was 2.2 x 10(-5)mutations/base/cycle. The majority of mutations were base substitutions, including high frequencies of C-->U and G-->A transitions. Single adenosine insertions were also observed frequently. The new assay is economical and provides a direct measurement of the mutation rate during a single cycle of viral replication. Target cells containing mutant proviruses survive the drug selection process and may be propagated for further analysis. The new assay is novel and has many advantages over previous mutation rate assays and thus will be very useful in future studies on genetic variation of HIV-1.     

Reliable and reproducible LightCycler qPCR for HIV-1 DNA 2-LTR circles

Highly active anti-retroviral therapy (HAART) has reduced the plasma load of HIV-1 to undetectable levels. It has however failed to eliminate the virus from other body compartments. Current methods for monitoring persistent viral replication in HIV-1+ patients require a large amount of blood and/or repeated tissue biopsies. Furthermore, some of the viral reservoirs, such as brain and eye, are inaccessible for sampling. The detection of episomal HIV-1 DNA 2-LTR circles in CD4+ cells is indicative of recent, acute infection events. This paper describes a reliable and reproducible LightCycler-based assay for the quantitative measurement of HIV-1 DNA 2-LTR circles in human peripheral blood mononuclear (PBMN) cells. It details the modifications to the DNA extraction procedure and to the LightCycler PCR procedure that were required to achieve this. This new surrogate marker of persistent viral replication can now be reliably, reproducibly and robustly used to study the clinical progress of large numbers of patients whose plasma HIV-1 RNA has been reduced to undetectable levels by anti-retroviral drugs.