Amount Of HIV DNA Research Articles

After 18 months of antiretroviral therapy, total HIV DNA decreases more pronouncedly in patients infected by CRF01_AE than in those infected by subtype B and CRF07_BC.

Whether the amount of HIV DNA is associated with the subtype of HIV-1 after antiretroviral therapy (ART) has not been reported. In the present study, the amount of HIV DNA and RNA and CD4+T counts in blood and semen prior to and after 18 months of ART were compared in 48 patients infected by CRF01_AE, subtype B or CRF07_BC of HIV-1. Viral RNA was suppressed and CD4 cell count recovery achieved in all patients. The level of HIV DNA were similar before ART; however, patients with CRF01_AE had less HIV DNA after ART than those with subtype B and CRF07_BC infection. According to prediction of co-receptor usage by Geno2Pheno and PSSM in combination, more than 35.6% of clones for CRF01_AE were predicted as CXCR4-using before ART, whereas less than 6% of those for subtype B and CRF07_BC were predicted as CXCR4-using. After 18 months of ART, no CXCR4-using clones were predicted in any of the subtypes. Despite more HIV RNA and fewer CD4 + T cells in patients with CRF01_AE before therapy, no significant differences (P > 0.05) in viral RNA or CD4 cell counts were observed between the subtypes after 18 months of ART. Thus, 18 months of antiretroviral therapy was more efficient in patients with CRF01_AE. Considering that successful ART dramatically reduces the viral load in both blood and semen, risks of sexual transmission of HIV were reduced, contributing to prevention of rapid spread of HIV among men who have sex with men in the region.

Biomarker reveals HIV's hidden reservoir.

Determining the total amount of HIV DNA in people undergoing antiretroviral therapy could accelerate the development of novel therapies and potential cures for HIV infection.

The prevalence of HIV-1 DNA in AIDS-related lymphoma and Kaposi Sarcoma throughout the AIDS epidemic

Background Chronic inflammation is linked to tumorigenesis for many cancer types and likely contributes to tumor development in the HIV-infected patient population. AIDSrelated lymphoma (ARL) and Kaposi Sarcoma (KS), two AIDS-defining cancers, are associated with the tumor viruses EBV and KSHV, respectively. However, EBV is only detectable in ~40% of ARLs and KSHV alone is not sufficient for KS development. Recent studies have shown that HIV is localized to tumor associated macrophages (TAM), not malignant B cells, in a portion of EBV-negative ARLs suggesting, that HIV infected TAM may play ar ole in tumorigenesis. The goal of this research was to determine the prevalence of HIV+ ARL and KS throughout the AIDS epidemic and examine tumor associated HIV for unique genetic signatures. Material and methods Whole genomic amplified DNA from ARL and KS biopsies was used for quantitative HIV gag gene amplification. The 3’ env-LTR segment of HIV-1 genomes from tumor and non-tumor tissues from two patients that died of ARL were sequenced and Bayesian phylogenies were inferred using BEAST. All specimens were provided by the AIDS and Cancer Specimen Resource. Results Of the 119 ARL and 91 KS biopsies, 45% and 40% contained detectable HIV-1 DNA, respectively. There was a significant decrease in the prevalence of HIV-1 DNA positive ARL and KS cases in the post-HAART era (after 1996; ARL=39%, KS=16.7) as compared to preHAART (ARL=54%, KS=45%). Our data suggest the overall amount of HIV DNA is less in tumor biopsies from the post-HAART era. A subset of ARL contained extremely high levels of HIV-1 DNA (~1 copy/cell). In addition, visceral KS had a higher prevalence of HIV-1 DNA (51.9%) as compared to skin KS (30.7%). HIV sequence evolution analysis of metastatic ARLs revealed that HIV was compartmentalized within sites of lymphoma and was distinct from HIV present in nonlymphoma sites. Conclusions The prevalence of HIV-1 DNA positive ARLs declined in the post-HAART era, but not to the same extent as KS, consistent with the incidence of both tumor types in the post-HAART era. Higher prevalence of HIV-1 DNA in visceral sites of KS and lymphoma specific-HIV sequences in sites of metastatic lymphoma suggests that HIV, especially HIV infected macrophages, may play a role in the pathogenesis of KS and ARL disease progression. Additionally, HIV-infected macrophages are a source of chronic inflammation that may further enhance tumorigenesis. Our data suggest a tumor specific form of HIV may be evolving within individuals who develop ARL.

HIV-1 and HIV-2 produce different amounts of 2-long terminal repeat circular DNA in vitro

We assessed HIV-1 and HIV-2 2-long terminal repeat (LTR) circular DNA production in peripheral blood mononuclear cells, MT4-CXCR4 cells and HeLa-CXCR4-CCR5 cells in vitro, relative to the respective total amounts of HIV DNA. Whatever the cell type, HIV-2 produced a smaller total amount of DNA than HIV-1 between 6 and 96 h; HIV-2 2-LTR DNA appeared later than HIV-1 2-LTR DNA, but rapidly became more abundant. This accumulation of HIV-2 2-LTR DNA points to less efficient host cell integration relative to HIV-1.

Significant differences in DNA viral load between HIV-1 and HIV-2 infected patients

Gottlieb et al.[1] suggest that the significance of the lower viral load in HIV-2-infected patients compared with HIV-1-infected patients could be biased by different distributions of CD4 cell counts and that we failed to adjust for CD4 cell count and other potential confounding factors like age, sex and the duration of infection. In fact, our analysis of DNA levels was stratified by CD4 cell counts (fig. 1 in reference [2]) and showed lower levels of DNA viral load in HIV-2 compared with HIV-1 in the higher CD4 strata (≥300 cells/μl), but not in the lower CD4 stratum (<300 cells/μl). To complete this analysis, we have performed a multivariate linear regression analysis including age, sex and HIV RNA within each CD4 cell count stratum (Table 1). After adjustment for age the difference of HIV DNA between the two types of HIV persisted in the higher strata (≥300 cells/μl), but it was only at the limit of significance in both strata after adjustment for age and sex. The relationship between viral type and DNA load was not significant any more after adjustment for HIV RNA, suggesting that the difference was mainly explained by the relation between viral replication and amount of HIV DNA. Therefore, at a given level of replication, no difference in the DNA level is found between HIV-1 and HIV-2. These results were also confirmed when we took into account the difference in sensitivity of our PCR method by imputing DNA values of 5 copies/μg for HIV-1 and 10 copies/μg for HIV-2 when the sample was below the respective detection limits.Table 1: Multivariate linear regression analysis of factors associated with HIV DNA: ANRS CO5 HIV-2 cohort.Given the low detection rate of HIV-2 RNA in plasma, Gottlieb et al.[1] also questioned whether our population of HIV-2-infected patients was representative. Studies in Africa generally overrepresent patients with advanced HIV disease and detectable plasma viral load, as in the study by Gottlieb et al.[1] in Senegal, taken as an example. We recently published viro-immunological follow-up data among a subset of incident HIV-2 infected patients from the French ANRS CO5 HIV-2 cohort compared with a matched HIV-1-infected patient incident group. The HIV RNA load was detectable in 86% and 15% of HIV-1-patients and HIV-2-patients, respectively, with 4.11 log10 copies/ml for HIV-1 and 2.09 log10 copies/ml for HIV-2, in full agreement with the values reported in this HIV DNA load comparative study involving prevalent cases [3]. Furthermore, the 29% overall rate of detectable HIV-2 RNA in prevalent cases of the French HIV-2 cohort, with the limit of detection of 100 copies/ml, ranges from 8% for a CD4 cell count greater than 500 cells/μl, to 35% when in the range 300–500 cells/μl, and 62% when below 300 cells/μl. However, the main conflict between our data and those of Gottlieb et al.[1] concerns the level of HIV-1 DNA. Gottlieb et al.[1] report median HIV DNA of 1.45 log10/μg which is lower than that reported in other studies quantifying HIV-1 DNA in antiretroviral-naïve patients, using the same quantification method (Amplicor HIV-1 Monitor, Roche Molecular Systems, Inc., Branchburg, New Jersey, USA) [4–6]. Indeed, these studies report median values between 2.0 and 2.6 log10 of total HIV-1 DNA/μg. Assuming that the amount of target DNA initially present in the PCR tube follows Poisson's law, the claimed sensitivity of the Monitor test (1 copy/μg) is controversial as it is highly unlikely to detect 1 copy/PCR in 100% of cases [7]. If a sample with 1 copy/μg is systematically detectable, then the calibration standard is underestimated. Moreover, HIV-1 strains circulating in Senegal are highly divergent [8] and sometimes difficult to quantify with primers and probes corresponding to less highly conserved genome regions than those selected for our PCR method [9]. Our work shows that significant differences in total DNA levels exist between HIV-1 and HIV-2 and that they are highly related to viral replication and CD4+ cell count as expected. These differences should be considered as one of the many factors contributing to the clinical and epidemiological differences between these two infections.

Quantitative assessment of cell-associated and cell-free virus in cervicovaginal samples of HIV-1-infected women.

To examine the amount of cell-free and cell-associated virus in cervicovaginal secretions (CVS) of HIV-infected women. Paired cervicovaginal and blood samples from 61 seropositive women were quantitatively evaluated by competitive polymerase chain reaction (cPCR) and reverse transcription-PCR (cRT-PCR) for: (1) genomic RNA from plasma and cell-free CVS, and (2) unspliced (u/s) RNA transcripts and proviral DNA in cells from secretions. HIV DNA was detected in 42.6%, u/s transcripts in 32.7% and cell-free HIV RNA in 31.1% of 61 cervicovaginal samples. The median copy numbers of HIV DNA, u/s transcripts, and cell-free RNA were 125 copies/10(5) cells, 40 copies/10(5) cells, and 300 copies/mL of secretion, respectively. Nineteen of 26 (73.1%) and 17 of 26 (65.3%) women positive for DNA were also positive for RNA transcripts and cell-free RNA, respectively (P<0.001). A significant correlation between the amounts of cell-free and u/s transcripts was also found (Spearman Rho 0.618, P=0.014). The prevalences of u/s transcripts and cell-free RNA were 42.6% and 53.8% respectively among patients with detectable blood RNA, and 22.9% (P=0.09) and 14.3% (P=0.0017) among patients with undetectable blood RNA. In stepwise logistic regression, cell-free RNA was independently associated with the presence of detectable blood viremia. The amount of HIV DNA was lower among subiects currently under treatment (50 copies/10(5) cells) than in untreated subjects (250 copies/10(5) cells) (P=0.037). Both cell-free and cell-associated HIV could be detected and quantitated in CVS, providing a means to examine the level of viral activity in the female genital tract.

Increased production of nitric oxide correlates with viral load and activation of mononuclear phagocytes in HIV-infected patients.

The objective of our study was to determine the production of nitric oxide (NO) in patients infected with human immunodeficiency virus (HIV) and its relation to cellular immunity, activation of mononuclear phagocytes and the amount of circulating virus. Therefore, serum nitrate, the stable metabolite of NO, the number of peripheral CD4+ T-lmphocytes, serum neopterin, plasma HIV-RNA and HIV-DNA in peripheral blood mononuclear cells of afebrile HIV-infected patients were determined. Serum nitrate levels were significantly (p = 0.002) increased in HIV-infected patients (median 37 microM, range 13-137 microM, n = 77) in comparison to healthy subjects (median 28 microM, range 21-40 microM, n = 17). Serum nitrate levels did not correlate with the number of CD4+ T-lymphocytes (r = 0.05, p > 0.05). Serum nitrate levels were positively correlated with neopterin (r = 0.36, p = 0.05, n = 30), the amount of HIV-DNA in peripheral blood mononuclear cells (r = 0.63, p < 0.001, n =27) and plasma HIV-RNA levels (r = 0.35, p = 0.08, n = 27). A possible explanation of our findings is that HIV induces the production of NO by means of activated mononuclear phagocytes.

Quantitation of HIV viral burden by PCR in HIV seropositive Navy personnel representing Walter Reed stages 1 to 6.

To quantitate the amount of human immunodeficiency virus type 1 (HIV-1) DNA in peripheral blood mononuclear cells (PBMC) of 78 infected individuals, we have developed a polymerase chain reaction (PCR) assay that is both quantitative and sensitive. Quantitation was based on incorporation of a 32P end-labelled primer (SK39) in the PCR reaction and on comparison after electrophoresis with known amounts of HIV DNA. A linear relationship was obtained between the natural logarithms of the radioactive counts detected and the number of HIV-1 DNA copies (10-1000 copies) from the standard DNA. HIV copy numbers from patient samples were then extrapolated from the standard curves. This sensitive and reproducible method was compared with virus isolation which is a semiquantitative evaluation of viral burden. HIV DNA levels correlated with virus isolation, i.e., high viral burden (100-1000 HIV copies) were found in most samples from which virus was isolated after only 7 days in culture; low viral burden (less than 100 HIV copies) was observed in samples from which virus was isolated after 14 to 21 days in culture. These estimates of viral burden were then compared with the clinical stage of the individuals.

Solid phase capture method for the specific amplification of microbial nucleic acids—avoidance of false-positive and false-negative reactions

DNA amplification assays such as the polymerase chain reaction are being developed for the amplification of small quantities of microbial nucleic acids. These assays offer the potential for a great deal of sensitivity. However, the high level of sensitivity increases the likelihood of cross-contamination of amplified products and the generation of false-positive reactions. In addition, substances in body fluids can inhibit the efficient performance of the amplification reactions. We have developed an assay format in which microbial nucleic acids are specifically bound to a solid phase surface. Contaminating DNA and enzyme inhibitors present in the sample are removed by washing prior to the performance of the amplification reaction. We could use this system to amplify and detect small amounts of HIV DNA diluted in whole blood. The assay system could distinguish target DNA from contaminating DNA fragments generated by prior amplification reactions.

Quantitative Analysis of HIV-1 Proviral DNA in Peripheral Blood Mononuclear Cells from Patients with AIDS or ARC: Decrease of Proviral DNA Content Following Treatment with 2′,3′-Dideoxyinosine (ddI)

A rapid and simple method for determining the proviral DNA content in peripheral blood mononuclear cells (PBM) from patients with human immunodeficiency virus type 1 (HIV-1) infection was established by using the polymerase chain reaction (PCR) technique. We found that the majority of HIV proviral DNA copies detectable in unfractionated PBM resided in T cells, while B cells/monocytes contained lesser amounts of HIV DNA (93.9 +/- 3.5% for T cells vs. 6.1 +/- 3.5% for B cells/monocytes: p less than 0.05). When we compared the amount of HIV proviral DNA in PBM from 13 patients with AIDS or AIDS-related complex (ARC) before and during antiretroviral therapy with 2',3'-dideoxyinosine (ddI) which was given as an escalating dose in a Phase I clinical study, a significant decrease was observed in 9 of 12 evaluable patients receiving the drug for 8 to 14 weeks (p less than 0.02). The decrease appeared more pronounced in patients receiving relatively high doses of the drug. These data suggest that the quantitation of HIV viral DNA in PBM by PCR is feasible and may theoretically contribute to an overall monitoring of patients receiving experimental therapy. However, larger studies will be required to determine the sensitivity and specificity of this assay and further longitudinal studies will be essential.

Heterogeneity of the ARV-2 strain and natural isolates of the human immunodeficiency virus.

To study heterogeneity of HIV isolates ARV-2 and strains isolated from lymphocytes of asymptomatic carriers were cloned using limiting dilutions of virus and infected CEM cells. All viral stocks turned out to be biologically heterogeneous giving clones with cytotoxic and noncytotoxic type of infection. Coinfection of CEM cells with cytotoxic and noncytotoxic variants resulted in interference of their biological effects. This interference ended with distinct cytotoxicity. No correlation between the production of virus and the amount of HIV DNA was found pointing out that nonfunctional DNA copies exist.