Evaluation of the Nuclisens HIV-1 QT assay for quantitation of human immunodeficiency virus type 1 RNA levels in plasma

Nuclisens HIV-1 QT is a new version of the NASBA HIV-1 QT assay for quantitation of human immunodeficiency virus type 1 (HIV-1) RNA in plasma. The specificity of this assay was 100% in one laboratory and 99%-with nonrepeatability of the initial false positive-in another. The test was linear between 2.0 and 6.0 log RNA copies per ml. According to the input HIV-1 RNA concentration, accuracy varied from -0.11 to +0.10 log RNA copy per ml and precision varied from 0.66 to 0.14 log RNA copy per ml. Reproducibility decreased when the HIV-1 RNA level was near the lower limit of quantitation of the test. HIV-1 RNA could be quantitated by Nuclisens HIV-1 QT in 36% (laboratory 1) and 24% (laboratory 2) of clinical samples with HIV-1 RNA levels lower than the lower limit of quantitation by NASBA HIV-1 QT. Nuclisens HIV-1 QT was not suitable for measurement of RNA from clade G and group O HIV-1 strains.     

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Antepartum plasma hepatitis C virus (HCV) RNA was quantified in 155 mothers coinfected with HCV and human immunodeficiency virus type 1 (HIV-1), and HCV RNA was serially assessed in their infants. Of 155 singleton infants born to HCV antibody-positive mothers, 13 (8.4%) were HCV infected. The risk of HCV infection was 3.2-fold greater in HIV-1-infected infants compared with HIV-1-uninfected infants (17.1% of 41 vs. 5.4% of 112, P = .04). The median concentration of plasma HCV RNA was higher among the 13 mothers with HCV-infected infants (2.0 x 10(6) copies/mL) than among the 142 mothers with HCV-negative infants (3.5 x 10(5) copies/mL; P < .001), and there were no instances of HCV transmission from 40 mothers with HCV RNA concentrations of < 10(5) copies/mL. Women dually infected with HIV-1 and HCV but with little or no detectable HCV RNA should be reassured that the risk of perinatal transmission of HCV is exceedingly low.     

Detection of HIV-1 RNA by nucleic acid sequence-based amplification combined with fluorescence correlation spectroscopy

Nucleic acid sequence-based amplification (NASBA) has proved to be an ultrasensitive method for HIV-1 diagnosis in plasma even in the primary HIV infection stage. This technique was combined with fluorescence correlation spectroscopy (FCS) which enables online detection of the HIV-1 RNA molecules amplified by NASBA. A fluorescently labeled DNA probe at nanomolar concentration was introduced into the NASBA reaction mixture and hybridizing to a distinct sequence of the amplified RNA molecule. The specific hybridization and extension of this probe during amplification reaction, resulting in an increase of its diffusion time, was monitored online by FCS. As a consequence, after having reached a critical concentration of 0.1-1 nM (threshold for unaided FCS detection), the number of amplified RNA molecules in the further course of reaction could be determined. Evaluation of the hybridization/extension kinetics allowed an estimation of the initial HIV-1 RNA concentration that was present at the beginning of amplification. The value of initial HIV-1 RNA number enables discrimination between positive and false-positive samples (caused for instance by carryover contamination)-this possibility of discrimination is an essential necessity for all diagnostic methods using amplification systems (PCR as well as NASBA). Quantitation of HIV-1 RNA in plasma by combination of NASBA with FCS may also be useful in assessing the efficacy of anti-HIV agents, especially in the early infection stage when standard ELISA antibody tests often display negative results.     

Effects of specimen collection, processing, and storage conditions on stability of human immunodeficiency virus type 1 RNA levels in plasma

To define the optimal blood collection parameters for plasma human immunodeficiency virus type 1 (HIV-1) viral load testing, plasma HIV-1 RNA levels were quantitated with the NASBA HIV-1 RNA QT System from blood specimens that were collected, processed, and stored under a variety of conditions that might have affected HIV-1 RNA stability. We determined that when whole blood was processed within 2 h of specimen collection the levels of HIV-1 RNA detected in EDTA-, heparin-, and acid citrate dextrose (ACD)-anticoagulated plasma samples were comparable. The levels of HIV-1 RNA in serum specimens (mean = 4.126 log units) were significantly lower (P < 0.01) than the levels in corresponding plasma samples (mean = 4.501 log units). One cycle of freeze-thaw (-70 degrees C) did not significantly reduce the level of HIV-1 RNA detected in EDTA-, heparin-, or ACD-anticoagulated plasmas. The EDTA-anticoagulated plasmas showed the smallest decrease in HIV-1 RNA copies (0.050 log units). HIV-1 RNA levels decreased over a 6-month time period in serum as well as in EDTA-, ACD-, and heparin-anticoagulated plasmas stored at -70 degrees C. However, the only significant decreases were for serum (mean decrease = 0.317 log units) and heparin-anticoagulated samples (mean decrease = 0.384 log units). A comparison of the levels of HIV-1 RNA in cell-free plasma collected in VACUTAINER EDTA Plasma Preparation Tubes and in standard VACUTAINER EDTA tubes determined that HIV-1 RNA levels were stable for up to 30 h after collection when stored at either room temperature (mean standard deviation [SD] = +/- 0.101 log units) or at 4 degrees C (mean SD = +/- 0.102 log units) as cell-free plasma or as EDTA-anticoagulated whole blood (mean SD = +/- 0.109 log units). These data indicate that EDTA-anticoagulated plasma is the most suitable and stable matrix for HIV-1 RNA quantitation.     

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Some new commercial methods for the extraction of viral RNA have been introduced recently. In addition to the study published previously (Verhofstede, C., Reniers, S., Van Wanzeele. F., Plum J., 1996. AIDS 8, 1421-1427), seven different methods (four newly developed and three reference methods) for extraction of HIV-1 RNA from plasma have been evaluated. The RNA preparation method that gave the best results (acceptable reproducibility, highest sensitivity, reasonable price, fast and easy to perform), was the QIAamp Viral RNA kit from QIAgen. The High Pure Viral RNA Kit (Boehringer Mannheim) as well as the non-commercialised extraction kits were also very sensitive. The non-commercial tests seem less suitable for routine use and for the processing of large number of samples. Two methods, RNA Insta-Pure LS (Eurogentec) and PANext RNA extraction kit 1 (NTL, PANsystems GmbH) are not adapted for HIV plasma extraction. The single step methods using glass fibre or silica column are rapid (from 60 to 75 min depending on the number of wash steps) and although the price is high they are cheaper than the Boom extraction methods: High Pure Viral RNA Kit (Boehringer Mannheim) ($3.3/sample), QIAamp Viral RNA Kit (Qiagen) ($3.6/sample), Boom extraction ($5/sample). The Qiagen kit is the only kit that combines sensitivity with reproducibility, it is commercialised, rapid and affordable in price and can be automated. For most of the methods evaluated the inter-test variability was acceptable (mean variation coefficient between duplicate extractions varied between 26.4 and 48.6%).     

The effect of plasma drug concentrations on HIV-1 clearance rate during quadruple drug therapy

OBJECTIVE: To investigate the relationship between exposure to antiretroviral drugs and the initial decline of plasma HIV-1 RNA. DESIGN: Open-label study in antiretroviral-naive HIV-1 infected patients using a quadruple drug regimen [nelfinavir (NFV), saquinavir (SQV), stavudine, and lamivudine]. METHODS: The elimination rate constant (k) for HIV-1 clearance was calculated during the first 2 weeks of treatment in 29 patients. Exposure to NFV and SQV was quantified on each study visit. Observed NFV and SQV concentrations were related to those expected in a reference population and a concentration ratio was calculated. The median concentration ratios for NFV and SQV, the baseline CD4+ lymphocyte count and baseline log10 HIV-1 RNA were correlated with k. RESULTS: A significant positive correlation was observed between k and the median NFV (P = 0.001) or SQV concentration ratio (P = 0.016) in univariate analysis. In multivariate analyses, the median NFV concentration ratio remained significantly correlated with k. CONCLUSIONS: The variation in the rate of decline of plasma HIV-1 RNA between patients after the initiation of a quadruple drug regimen could be explained by differences in exposure to NFV or SQV. Determination of k could be used to optimise further antiretroviral drug therapy and may be a first tool to assess antiretroviral activities of new or increasing doses of drugs administered in combination regimens. Furthermore, our data suggest that exposure to antiretroviral drugs should be incorporated in mathematical models to describe HIV-1 dynamics in more detail.     

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HIV-1 group O serological screening or confirmation strategies so far have not proved 100% sensitive and specific, indicating a lack of antibody reactivity or cross-reactivity with group O antigens. Therefore, genetic analysis currently represents the only method by which confirm presumed HIV-1 group O or group O/M infections. We have optimized the sensitivity (100%) and specificity (100%) of an HIV-1 group O/M-specific PCR of a pol gene fragment. In addition, we report on a highly sensitive (97.2%) and specific (100%) method for differentiation between HIV-1 group O and group M viruses, using PCR and PstI enzyme restriction fragment analysis of a pol fragment. Compared with sequencing, these methods are fast, inexpensive, and simple.     

Comparison of NucliSens and Amplicor monitor assays for quantification of human immunodeficiency virus type 1 (HIV-1) RNA in plasma of persons with HIV-1 subtype A infection in Abidjan, C?te d'Ivoire

We compared the sensitivity and accuracy of the NucliSens assay and those of both the standard and modified (addition of a new primer set, primer mix 1, supplied by Roche) Amplicor HIV Monitor assays to quantify human immunodeficiency virus type 1 (HIV-1) RNA in persons infected with HIV-1 subtype A in Abidjan, C?te d'Ivoire. Seventy-one plasma samples from HIV-1-seropositive persons at different stages of HIV infection and 15 samples from HIV antibody-negative persons were analyzed. The HIV-1 genetic subtype was determined either by DNA sequencing or by a restriction fragment length polymorphism assay. Of the 71 samples, 70 (98%) were subtype A and 1 was subtype G. Of the 70 subtype A samples, the proportion of RNA-positive plasma samples and mean HIV-1 RNA levels were significantly higher by the modified HIV Monitor assay (n = 67 [96%]; mean RNA levels, 5.2 log10 HIV-1 RNA copies/ml) than the NucliSens assay (n = 56 [80%]; 4.3 log10 HIV-1 RNA copies/ml) or the standard HIV Monitor assay (n = 44 [63%]; mean RNA levels, 3.8 log10 HIV-1 RNA copies/ml) (all P values were <0.05). The HIV-1 RNA levels by the modified HIV Monitor assay correlated significantly with those by the NucliSens assay (r = 0.76; P < 0.001) and the standard HIV Monitor assay (r = 0.57; P < 0.001), as did the RNA levels by the NucliSens and the standard HIV Monitor assays (r = 0.60; P < 0. 001). Lower CD4 cell counts were significantly correlated with higher HIV-1 RNA levels by all three assays (r = -0.47 for the NucliSens assay, -0.45 for the standard HIV Monitor assay, and -0.62 for the modified HIV Monitor assay). These results indicate that the modified HIV Monitor assay has the highest sensitivity and efficiency at quantifying the levels of RNA in persons infected with HIV-1 subtype A and thus constitutes a valuable tool for the monitoring of RNA levels in areas of Africa were HIV-1 subtype A is predominant.