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Biomedical subjects

M Holodniy

Publications and source records attributed to M Holodniy.

At least 55 records · Page 3Linked to original sources
PubMed1994

Deficiency in antibody response to human cytomegalovirus glycoprotein gH in human immunodeficiency virus-infected patients at risk for cytomegalovirus retinitis.

Human immunodeficiency virus (HIV)-infected patients at risk for symptomatic human cytomegalovirus (CMV) infection were studied for serum antibody to CMV glycoproteins gH and gB. Antibody titers to gB in HIV-seropositive patients, irrespective of CD4 cell counts or presence of CMV retinitis, were significantly higher than titers in HIV-seronegative, CMV-seropositive patients but were comparable to titers detected in HIV-seronegative patients with CMV mononucleosis. In contrast, antibody to gH was rarely detected in HIV-seropositive patients with CD4 cell counts > 100/mm3 compared with patients with counts > 100/mm3. The inability to detect gH antibody at a time of high risk for symptomatic CMV retinitis suggests that immune intervention with either gH-specific vaccine or passive immunotherapy may benefit HIV-infected persons at risk for symptomatic CMV disease.

AIDS-Related Opportunistic Infections↗
PubMed1994

Stabilities of quantitative plasma culture for human immunodeficiency virus, RNA, and p24 antigen from samples collected in VACUTAINER CPT and standard VACUTAINER tubes.

We evaluated the stability of human immunodeficiency virus (HIV) load markers from blood samples collected in VACUTAINER CPT or standard VACUTAINER brand tubes using sodium heparin or sodium citrate as anticoagulants. Quantitative plasma culture and p24 antigen concentrations were determined, and HIV RNA levels in plasma were measured by both reverse transcription-PCR-enzyme-linked immunosorbent assay (RT-PCR-ELISA) and branched DNA methods. All tubes were stored at room temperature for analysis at 2, 24, 48, and 72 h after the blood samples were drawn. No difference was seen between tube types with respect to the HIV titer in plasma or the positivity rate for all samples that demonstrated a fall in titer over time. Unbound p24 antigen levels in plasma decreased during the initial 48-h period in both tube types. Immune complex-dissociated p24 antigen levels decreased in CPT tubes but not in standard VACUTAINER tubes. The HIV RNA copy number in plasma measured by RT-PCR-ELISA was stable in most subjects and was significantly higher in CPT tubes than in standard VACUTAINER tubes at 24 and 72 h after the blood samples were drawn. The branched DNA probe assay detected a significant decline in HIV RNA equivalent in plasma over 72 h in both collection tubes, the decline being more dramatic in the standard VACUTAINER tube than the CPT tube. Overall, interday variability suggests that samples collected for a particular assay should be processed at the same time after blood is drawn and that a particular tube type be used throughout a given study.

Anticoagulants↗
PubMed1994

Clinical application of reverse transcription-polymerase chain reaction for HIV infection.

This article describes the current knowledge of the various RT-PCR assays and their clinical application in HIV disease. The importance of assay precision and sample handling is emphasized. A review of quantification techniques is also presented. Finally, the relevant literature describing the application of RT-PCR for both virion RNA in plasma and cellular mRNA is reviewed.

HIV↗
PubMed1994

Diurnal and short-term stability of HIV virus load as measured by gene amplification.

To determine whether human immunodeficiency virus (HIV) viral load has short term stability, eight clinically stable subjects infected with HIV and having CD4 counts ranging between 10-600/mm3, had blood samples taken at 0800 and 1700 on 3 consecutive days and then weekly at 0800 for 1 month (8-10 observations/subject). Plasma HIV RNA, peripheral blood mononuclear cell (PBMC) proviral DNA, serum p24 antigen levels, and mononuclear cell subsets were measured at each time point. Mean plasma HIV RNA, PBMC HIV DNA, and p24 antigen [both regular and immune complex dissociated (ICD)] levels did not change significantly between mornings and afternoons or on successive days or weeks. CD4+, CD8+, and CD56+ number demonstrated a diurnal variation in those subjects with > 200 CD4 cells/mm3. We conclude that HIV viral load demonstrates short-term stability in clinically stable subjects. This stability has important implications for monitoring HIV disease progression or antiretroviral therapy.

Adult↗
PubMed1993

Detection of human immunodeficiency virus type 1 in semen: effects of disease stage and nucleoside therapy.

The effects of clinical stage of infection and antiviral therapy on the detection of human immunodeficiency virus type 1 (HIV-1) nucleic acids in semen were investigated by the polymerase chain reaction. HIV-1 was detected in 45 (87%) of 52 semen specimens from 29 (81%) of 36 men. Seventeen (77%) of 22 stage II or III subjects and 12 (86%) of 14 stage IV subjects had positive specimens. The CD4+ lymphocyte count was not significantly different comparing subjects with positive and negative semen. Moreover, 6 (67%) of 9 untreated men had positive specimens compared with 23 (85%) of 27 men treated with zidovudine, 2',3'-dideoxyinosine, or both for a mean of 20 months. Thus, the detection of HIV-1 in semen was independent of both stage of infection and long-term treatment. In a semiquantitative analysis of 6 men followed for 8 weeks after the start of nucleoside therapy, a decrease in HIV-1 RNA in seminal plasma was demonstrated in 2.

Anti-Bacterial Agents↗
PubMed1993

Measurement of HIV virus load and genotypic resistance by gene amplification in asymptomatic subjects treated with combination therapy.

Quantification of viral load in HIV disease has become increasingly important as a marker of antiviral efficacy. We applied gene amplification techniques in vivo to asses antiretroviral activity of combination therapy. Five HIV-infected subjects, four of whom were drug naive, were administered combination therapy with zidovudine (ZDV) and didanosine (ddI). Plasma and peripheral blood mononuclear cells (PBMC) were obtained twice at baseline and then at 1, 3, 6, 9, and 12 months after the initiation of therapy. Results show that plasma HIV RNA copy number fell from 2,170 +/- 660/ml to undetectable at 1 month, with continued suppression at 12 months. HIV proviral DNA copy number decreased from 3.9 to 3.0 log10/10(6) CD4+ T cells at 12 months. Cell dilution cultures were positive in 4 of 5 subjects at baseline and in only 1 of 5 after 12 months. CD4+ T-cell count increased from 390 +/- 30/mm3 pretherapy, to 505 +/- 66/mm3 after 6 months of therapy, but returned to baseline levels after 12 months of therapy. No mutations were detected from PBMC DNA for codon 215 and 74 in the HIV pol gene from the drug-naive subjects. These findings suggest that gene amplification techniques can be used to study changes in viral load or genotype and can be applied in real time to samples from patients involved in clinical trials.

CD4-Positive T-Lymphocytes↗
PubMed1992

Quantitative RNA and DNA gene amplification can rapidly monitor HIV infection and antiviral activity in cell cultures.

We have developed a quantitative gene amplification procedure to assess the replication of human immunodeficiency virus (HIV) in cell cultures and evaluate the effect of drugs on viral replication. Increases in HIV gag RNA and DNA in phytohemagglutinin-stimulated normal peri-pheral blood mononuclear cells (PBMC) infected with HIV at very low multiplicity of infection paralleled the production of HIV p24 antigen in culture supernatants. Quantitative gene amplification was able to monitor the accumulation of viral nucleic acids in control cultures and demonstrate the effect of various concentrations of azidothymidine (AZT) on the replication of both AZT-sensitive and -resistant strains of HIV. The sensitivity of patient-derived virus strains to AZT could also be successfully measured by these procedures. The results of our studies suggest that quantitative measurement of HIV gag RNA and DNA can be used to monitor the kinetics of viral replication, antiviral activity, viral drug resistance, and mechanism of drug action.

Cells, Cultured↗
PubMed1992

Correlation of morphologic diagnosis of Pneumocystis carinii with the presence of pneumocystis DNA amplified by the polymerase chain reaction.

We compared the presence of P. carinii in clinical specimens as detected by standard cytomorphologic techniques with amplification of P. carinii-specific DNA by the polymerase chain reaction (PCR). Results correlated in 33 of 37 instances (89%): nine specimens were positive by both PCR and morphology; 24 specimens were negative by both techniques. Two specimens from one patient were obtained 3 days apart. The first specimen was both cytologically and PCR negative, while the second specimen was both cytologically and PCR positive for P. carinii. At least in some instances, therefore, PCR is no more sensitive than morphology, and other factors such as specimen adequacy are more important. Twelve of the 24 negative specimens were from patients with prior histories of P. carinii pneumonia, suggesting that recurrent disease may be from reacquisition of organisms in previously exposed individuals, rather than reactivation of latent organisms. Discrepant results included three morphologically negative specimens that were positive by PCR. It remains to be determined whether the increased sensitivity of PCR in these cases is real or artifactual. One morphologically positive specimen was negative by PCR. Polymerase chain reaction correlates well with cytomorphologic diagnosis of P. carinii pneumonia and may be a valuable diagnostic and epidemiologic tool.

Bronchoalveolar Lavage Fluid↗
PubMed1992

Detection and quantification of gene amplification products by a nonisotopic automated system.

We describe in this report the ability to determine human immunodeficiency virus proviral copy number by an automated nonisotopic method. Our system utilizes a FACStarPLUS cell sorter, the GeneAmp PCR System 9600 and a Biomek 1000 robotic workstation. Linking these three machines allows cell populations to be sorted and the DNA amplified and quantitated with minimal technical effort. We have developed this system to quantitate proviral DNA copy number in sorted subpopulations of peripheral blood cells in one day.

Autoanalysis↗
PubMed1992

Plasma viremia in human immunodeficiency virus infection: relationship to stage of disease and antiviral treatment.

Quantitative culture of human immunodeficiency virus (HIV) was performed on 121 plasma samples from 76 HIV-infected individuals to determine the sensitivity of the assay at different stages of disease and to measure the effect of antiviral therapy on plasma viremia. Plasma virus was detected in 49 of 76 (64%) of patients, primarily those with AIDS and AIDS-related complex (36 of 38) versus asymptomatic subjects (13 of 38) (p less than 0.001, chi 2). Similarly, plasma cultures were more often positive in patients with less than 250 CD4+ T cells per microliter (38 of 40) than in those with greater than 250 CD4+ T cells per microliter (11 of 36) (p less than 0.001, chi 2). Plasma virus cultures were also more likely to be positive in patients with detectable serum p24 antigen (24 of 26) than in those without detectable p24 antigen (25 of 50) (p = 0.0023, chi 2). An effect of zidovudine (ZDV) treatment on plasma viremia was seen in a comparison of treated and untreated patients with less than 250 CD4+ T cells per microliter. Geometric mean titers of plasma viremia from 16 patients treated with ZDV for more than 3 months were significantly lower than titers from 24 untreated patients (10(1.3) versus 10(2.1), p less than 0.05, Student's t test. A comparison of pre- and posttherapy titers in 33 patients receiving antiviral treatment showed that plasma virus was not detectable at either time in 17 patients; there was a fall in plasma virus titer in 12; and titers were unchanged or increased in 4. In patients with advanced disease, plasma viremia is a potential marker of antiviral drug activity.

AIDS-Related Complex↗
PubMed1992

Decrease in HIV provirus in peripheral blood mononuclear cells during zidovudine and human rIL-2 administration.

Quantification of human immunodeficiency virus (HIV) proviral DNA in peripheral blood mononuclear cells (PBMC) was performed in 13 HIV-seropositive asymptomatic individuals during 10-24 months by polymerase chain reaction amplification of multiple half-log dilutions of cellular DNA. At enrollment, subjects had a geometric mean titer of 100 copies of HIV provirus per 10(6) PBMC (mean +/- SD, 2 +/- 0.9 log10). In four untreated individuals there was no significant change in provirus levels during a mean period of 13.3 months. In eight patients treated with zidovudine (ZDV) and human recombinant interleukin 2 (rIL-2), HIV provirus copies declined to 13 per 10(6) cells (1.1 +/- 0.8 log10) at the end of the first course of ZDV and rIL-2 at week 20 (p less than 0.01), and to 40 per 10(6) cells (1.6 +/- 0.9 log10) after 12 months of treatment (p less than 0.04). Subsequent courses, which included 12 weeks of ZDV alone or 4 weeks of IL-2 alone, did not significantly change the already depressed provirus copy numbers. Proviral copy number also remained depressed during drug-free "washout periods" between courses. Finally, we observed a return to a geometric mean of 400 copies per 10(6) cells (2.6 +/- 0.3 log10) a mean of 7.9 months after discontinuation of therapy. Measurement of changes in HIV provirus should provide a direct marker for defining antiviral activity of drugs, biologics, and combination therapy.

DNA, Viral↗
PubMed1991

Detection and quantification of human immunodeficiency virus RNA in patient serum by use of the polymerase chain reaction.

Human immunodeficiency virus (HIV) RNA was detected and quantified in the serum of HIV-seropositive individuals using the polymerase chain reaction (PCR) and a nonisotopic enzyme-linked affinity assay. Of 55 HIV-infected patients who were not receiving therapy, serum HIV RNA was detected in 9 of 19 who were asymptomatic, 11 of 16 with AIDS-related complex (ARC), and 18 of 20 with AIDS, with copy numbers ranging from 10(2) to greater than or equal to 5 x 10(4) 200 microliters of serum based on a relationship between absorbance and known copy number of gag gene RNA. Linear regression analysis demonstrated a correlation between infectious titer in 42 patient sera cocultured with donor peripheral blood mononuclear cells (PBMC) and PCR product absorbance (r = .70, P less than .01). Serum HIV RNA detected by PCR also correlated with serum p24 antigen positivity, CD4 counts less than 400/mm3, and the presence of HIV-related symptoms or disease. Quantification of infectious HIV RNA in cell-free serum by PCR may be useful as a marker for for disease progression or in monitoring antiviral therapy.

AIDS-Related Complex↗
PubMed1991

Detection of human immunodeficiency virus DNA and RNA in semen by the polymerase chain reaction.

Peripheral blood mononuclear cells (PBMC) and semen of 23 men infected with human immunodeficiency virus (HIV) were examined for the presence of HIV DNA and RNA using the polymerase chain reaction (PCR) and a nonisotopic detection assay. None of the men was receiving antiretroviral therapy at the time of collection. Semen samples were separated into cell-free seminal fluid, nonspermatozoal mononuclear cells (NSMC), and spermatozoa. All of the PBMC samples, 17 (74%) of 23 NSMC samples, and none of the spermatozoal samples were positive for HIV gag gene DNA. Of 23 cell-free seminal fluid samples, 15 (65%) were positive for HIV gag gene RNA by PCR. Cell-free HIV RNA was more likely to be present in the semen of men with less than 400 than in those with greater than or equal to 400 cells/mm3 (P less than .04) and was present in all patient with p24 antigen in serum. The presence of HIV DNA in NSMC samples was not related to CD4 cell count, disease status, or the presence of p24 antigen in the serum. This study shows that HIV nucleic acid can be detected by PCR in either the cell-free seminal fluid or NSMC of 87% of semen samples but not in the DNA of spermatozoa from HIV-infected men.

AIDS-Related Complex↗
PubMed1991

Inhibition of human immunodeficiency virus gene amplification by heparin.

Gene amplification of virus-specific sequences is widely used as a method to detect or confirm human immunodeficiency virus (HIV) infection. In this study we used an enzyme-linked affinity assay to quantify polymerase chain reaction products from whole blood, plasma, and separated mononuclear cells collected in the presence of four common anticoagulants: acid citrate dextrose, sodium EDTA, potassium oxalate, and sodium heparin. Attenuation of the product signal was observed after amplification of nucleic acid extraction from whole blood, washed mononuclear cells, and plasma from specimens collected in sodium heparin. These inhibitory effects on gene amplification could be reversed with heparinase. The addition of as little as 0.05 U of heparin completely inhibited amplification of an HLA-DQa sequence from placental DNA. We conclude that heparin can cause attenuation or inhibition of gene amplification. Acid citrate dextrose and EDTA, which lack inhibitory activity, are the most appropriate anticoagulants for clinical blood samples when polymerase chain reaction amplification is anticipated.

Citric Acid↗
PubMed1991

Reduction in plasma human immunodeficiency virus ribonucleic acid after dideoxynucleoside therapy as determined by the polymerase chain reaction.

Cell-free HIV RNA in plasma was detected and quantitated after antiviral therapy by the polymerase chain reaction. RNA was extracted from plasma, reverse transcribed to cDNA, amplified by polymerase chain reaction, and quantitated by absorbance based on an enzyme-linked affinity assay. 72 HIV antibody-positive subjects had one plasma sample taken. 39 who were not receiving antiretroviral therapy at the time had a mean plasma HIV RNA copy number of 690 +/- 360 (mean +/- SEM) per 200 microliters of plasma, while 33 subjects who had been receiving zidovudine therapy for a minimum of 3 mo had a mean copy number of 134 +/- 219 (P less than 0.05). 27 additional HIV antibody-positive patients had two plasma samples taken before and 1 mo after initiating dideoxynucleoside therapy. Plasma HIV RNA copy number fell from 540 +/- 175 to 77 +/- 35 (P less than 0.05). Finally, nine of these subjects had two baseline samples obtained before initiating therapy and two posttreatment samples 1 and 2 mo after therapy was begun. Mean plasma RNA copy number declined from 794 +/- 274 to less than 40 (below the lower limit of sensitivity) after 1 mo of therapy, with suppression maintained after 2 mo of therapy. These results suggest that gene amplification can be used to detect and quantitate changes in plasma HIV RNA after dideoxynucleoside therapy. Plasma HIV polymerase chain reaction may be a more sensitive marker to monitor antiviral therapy, particularly in asymptomatic patients where measurement of p24 antigen or quantitative plasma cultures are negative.

CD4-Positive T-Lymphocytes↗