Competitive polymerase chain reaction assay for quantitation of HIV-1 DNA and RNA.

A quantitative PCR assay for the detection of HIV-1 nucleic acids is described. The assay is based on a competitive internal standard nucleic acid which can be discriminated from target sequences by the presence of a new restriction enzyme site. The method was used to quantitate plasmid molecules containing HIV-1 sequences, HIV-1 DNA and HIV-1 RNA purified from HIV-1-infected tissue culture cells as well as HIV-1 DNA present in the peripheral blood mononuclear cells of an AIDS patient. The assay will be valuable for assessing viral load in AIDS patients and for the study of viral gene expression.     

Examination of real-time PCR for HIV-1 RNA and DNA quantitation in patients infected with HIV-1 BF intersubtype recombinant variants

The impact of HIV-1 genetic diversity on the performance of laboratory testing is an issue that has to be monitored continuously. An “in-house” real-time PCR assay was developed by the Agence Nationale de Recherche sur le SIDA (ANRS) in France for viral load (VL) quantitation based on the amplification of the HIV-1 long terminal repeat (LTR) region. This technology has not been used in Argentina yet and considering the HIV-1 diversity in the country, a comparative analysis of this assay was undertaken versus the Versant HIV-1 RNA 3.0 Assay (b-DNA). The performance was assessed on 30 drug-naïve HIV-1 infected patients who were characterized previously by phylogenetic analysis of the pol and vpu gene. The results showed that there is a significant linear correlation between values of transformed viral load logarithms measured by both, bDNA and real-time PCR assay and that this assay can be used to quantify viral load in samples from BF-infected patients with the same accuracy and reliability as for B subtype samples. The use of “in-house” real-time PCR to measure DNA in PBMCs correlated strongly with the HIV-1 RNA levels in all specimens.     

Plasma viral RNA assay in HIV-1 group O infection by real-time PCR

HIV-1 group O infections remains essentially restricted to central Africa, and particularly Cameroon, although isolated cases have been reported in Western countries. Genomic differences explain why commercial tests used to quantify HIV-1 group M plasma load are unsuitable for HIV-1 group O. This lack of a quantitative tool hinders the clinical management of HIV-O-infected patients. We have therefore developed a real-time PCR assay, based on LightCycler technology, to quantify HIV-1 group O RNA in plasma. The primers were selected in the LTR 3' region. Forty-eight plasma samples containing strains belonging to the different HIV-1 type O clades (O:A, O:B and O:C) were tested. RNA was quantifiable in 40 of these samples. RNA was always detected in samples from untreated patients, except for one patient infected by a highly divergent strain. The kinetics of plasma viral load were also examined in seven patients for whom clinical and immunologic follow-up data were available. HIV-1 group O plasma load was high in the absence of treatment and correlated negatively with the CD4 cell count. Serial samples obtained during treatment allowed us to compare viral load changes with immunologic outcome. Despite the high initial cost of acquiring the required cycling device, the per-sample cost of this real-time quantitative PCR assay for HIV-1 group O is low, making it suitable for use in endemic zones.     

Coamplification of HIV-1 Proviral DNA and Viral RNA in Assays Used for Quantification of HIV-1 RNA

Elevated HIV-1 viral load (VL) observed in specimens frozen in situ in plasma preparation tubes (PPTs) compared to EDTA plasma specimens may affect therapeutic monitoring of HIV-infected patients. The increase in viral load is cell associated and minimized when plasma from the PPT is aspirated or recentrifuged prior to freezing. This study investigates the contribution of integrated HIV-1 proviral DNA to elevated VL in the quantification of HIV-1 RNA in plasma. Fifty paired specimens collected in EDTA tubes and PPTs frozen in situ were used for analysis. HIV-1 VL was measured using the COBAS Amplicor Monitor ultrasensitive test version 1.5. Contaminating proviral DNA was detected using a nested PCR targeting the Alu repeat in human genomic DNA and HIV pol gene simultaneously. Treatment of the specimen with DNase resulted in significantly lower quantifiable HIV-1 RNA in specimens from PPTs compared to the corresponding EDTA tubes (P = 0.004). After the RNA was destroyed by heat treatment, the mean HIV-1 RNA VL decreased by 79% in the EDTA tube compared to 65% in the PPT. The nested PCR amplified integrated proviral DNA in nucleic acid extracted from plasma in PPT and EDTA specimens with high viral load values. Likewise, a semiquantitative densitometric analysis revealed that the total amount of genomic DNA in the PPT was higher than that in the EDTA tube. Our investigation clearly shows that both proviral DNA and intracellular RNA are amplified simultaneously in the COBAS Amplicor HIV-1 Monitor assay and that proviral DNA contributes to the elevated VL in plasma frozen in PPTs.Discrepancies in viral load (VL) measurements obtained in different plasma collection tubes have underscored the importance of specimen collection and handling in the determination of accurate results in HIV viral load assays. Plasma preparation tubes (PPTs) are Vacutainer tubes that contain dehydrated K₂EDTA and a gel separator which, upon centrifugation, forms a barrier that separates blood cells from plasma, allowing for collection, storage, freezing, and shipment of the plasma within the same tube. Use of the PPT also reduces the time required to process specimens, eliminates potential error in specimen labeling, and reduces the risk of HIV exposure associated with the transfer of plasma to secondary tubes for shipping.Elevated HIV-1 viral loads in plasma specimens collected and frozen in PPTs and quantified in the standard and ultrasensitive Roche COBAS Amplicor HIV-1 Monitor assays (2, 4, 13) have led investigators to believe that it may have an impact on therapeutic management of HIV-infected patients (1, 8). Some reports attribute the observed VL blips to random biological and statistical variations, with limited clinical significance (6), while others have implied that the elevated HIV-1 VL may interfere in the assessment of virological suppression. Subsequent studies show that aspiration and transfer of plasma from a PPT to a storage tube after centrifugation or recentrifugation of the PPT after transportation reduces or eliminates the artificial elevation of VL observed in the PPT (9, 10, 11). Salimnia et al. (11) imply that the elevation may be due to nonparticulate genetic material released from cells associated with the gel in the PPT after the plasma was thawed. Additional investigations report that samples collected in PPTs can be centrifuged up to 6 h after phlebotomy and stored in situ at 4°C for up to 72 h without any effect on the measured HIV-1 VL (9).In a recent prospective study, Kran et al. (5) observed a direct correlation between the number of cells present in the plasma, measured in the Advia 60 cell counter, and the amount of HIV-1 that was quantified using the Roche COBAS AmpliPrep-COBAS TaqMan (CAP/TAQ) HIV-1 test. Using paired specimens that were collected and centrifuged in PPTs, they found lower cell counts and reduced HIV-1 VL in the PPTs that were recentrifuged prior to HIV-1 quantification versus the PPTs that were centrifuged once, after phlebotomy. Using the Roche proviral DNA assay, the authors were able to detect HIV-1 proviral DNA in the specimens with higher cell counts and corresponding elevated VLs.The aim of our investigation is to further examine the source of nucleic acid responsible for the elevated VL in the PPT. Our protocol specifically explores integrated HIV-1 proviral DNA as the major contributor to the elevation in viral load observed in PPT. In addition, we show that the Roche Amplicor HIV-1 RNA assay coamplifies both RNA and proviral DNA simultaneously in plasma that is frozen in PPT.     

Performance characteristics of HIV-1 culture and HIV-1 DNA and RNA amplification assays for early diagnosis of perinatal HIV-1 infection

A plasma HIV-1 RNA amplification assay (RNA assay), a quantitative peripheral blood mononuclear cell (PBMC) microculture (culture), and a PBMC HIV-1 DNA amplification assay (DNA assay) were compared for diagnosis of HIV-1 infection in infants receiving zidovudine in Pediatric AIDS Clinical Trials Group protocol 185; assays were performed for all 24 infected and 100 uninfected infants. HIV-1 infection was defined as >or=2 positive cultures or positive antibody to HIV-1 at >or=18 months. Cultures were performed at birth and 6 and 24 weeks of age; DNA and RNA assays were performed on cryopreserved specimens. The sensitivity of culture and DNA and RNA assays at birth was 20.8%, 10.5%, and 26.7%, respectively. At older ages, sensitivity typically exceeded 80%, remaining highest for the RNA assay (>85%). Assay specificity was >99%. Positive predictive values exceeded 93% for each assay at each age; negative predictive values were highest (>90%) for the RNA assay. At birth (P < 0.005) and age 6 weeks (P < 0.001), a significantly larger proportion of infected infants were identified by means of the RNA assay than by the other assays. The diagnostic performance of the RNA assay matched or exceeded that of culture and the DNA assay. Given that RNA assays require less blood volume and yield rapid results, our study adds to existing data suggesting that RNA assays may be used for early diagnosis of HIV-1 infection in infants.     

Comparison of the QUANTIPLEX HIV-1 RNA 2.0 Assay with the AMPLICOR HIV-1 MONITOR 1.0 Assay for Quantitation of Levels of Human Immunodeficiency Virus Type 1 RNA in Plasma of Patients Receiving Stavudine-Didanosine Combination Therapy

We compared the QUANTIPLEX HIV-1 RNA 2.0 assay with the AMPLICOR HIV-1 MONITOR 1.0 assay for quantitation of human immunodeficiency virus type 1 (HIV-1) RNA in plasma in the Stadi trail, which evaluated a stavudine plus didanosine combination therapy in 52 patients. HIV-1 RNA baseline values measured with AMPLICOR HIV-1 MONITOR 1.0 were significantly higher than those measured with QUANTIPLEX HIV-1 RNA 2.0, and decreases in HIV-1 RNA levels from baseline were also found to be significantly higher when measured with the AMPLICOR HIV-1 MONITOR 1.0 assay. The frequency of HIV-1 RNA levels below the lower limit of quantitation was significantly higher with QUANTIPLEX HIV-1 RNA 2.0 than with AMPLICOR HIV-1 MONITOR 1.0. Reanalysis of these results by an ultrasensitive procedure of AMPLICOR HIV-1 MONITOR 1.0 or by a modified version of the test that included additional primers adapted for non-B HIV-1 clades yielded greater differences between the QUANTIPLEX HIV-1 RNA 2.0 assay and the AMPLICOR HIV-1 MONITOR 1.0 assay. Our results indicate that a valid comparison of the virological efficacies obtained with different antiretroviral drug regimens requires the use of the same viral load quantitation procedure; further standardization between the different HIV-1 RNA quantitation kits is therefore needed.     

Detection of human immunodeficiency virus type 1 (HIV-1) DNA and RNA sequences in HIV-1 antibody-positive blood donors in Uganda by the Roche AMPLICOR assay.

The ability of commercially available PCR-based assays to accurately detect or quantitate human immunodeficiency virus type 1 (HIV-1) DNA or RNA in individuals predominantly infected with HIV-1 subtypes A and D is not known. Therefore, peripheral leukocytes from 43 individuals in Kampala, Uganda, positive for HIV by the Western blot (immunoblot) assay were tested by using the Roche AMPLICOR HIV-1 assay for the detection of DNA gag sequences. Plasma from these same individuals was tested by using the Roche HIV-1 AMPLICOR MONITOR HIV-1 assay for the quantitation of HIV-1 RNA gag sequences. In addition, peripheral leukocytes were tested for HIV-1 DNA by using a lower annealing temperature or a different primer pair for the HIV-1 pol region. The proportions of individuals with detectable HIV-1 DNA and RNA gag sequences by the Roche assays were 74 and 90%, respectively. The proportions positive for HIV-1 DNA sequences by using a 50 degrees C annealing temperature or the pol primer pair were 71 and 98%, respectively. In summary, the standard Roche assay did not detect HIV-1 DNA sequences in a significant number of HIV-1-infected individuals in Uganda. However, use of a pol primer pair increased the sensitivity of the assay to 98%. The sensitivity of the Roche AMPLICOR MONITOR assay for the detection and quantitation of HIV-1 RNA sequences was significantly higher than that of the DNA-based assay, but the efficiency of the assay, and hence, the accuracy of the values obtained with RNA, is not known. Modifications to existing assays are needed to enhance the sensitivities and accuracies of these commercially available assays for use in developing countries where non-B HIV-1 subtypes predominate.     

Diagnosing acute HIV infection: The performance of quantitative HIV-1 RNA testing (viral load) in the 2014 laboratory testing algorithm

New recommendations for laboratory diagnosis of HIV infection in the United States were published in 2014. The updated testing algorithm includes a qualitative HIV-1 RNA assay to resolve discordant immunoassay results and to identify acute HIV-1 infection (AHI). The qualitative HIV-1 RNA assay is not widely available; therefore, we evaluated the performance of a more widely available quantitative HIV-1 RNA assay, viral load, for diagnosing AHI. We determined that quantitative viral loads consistently distinguished AHI from a false-positive immunoassay result. Among 100 study participants with AHI and a viral load result, the estimated geometric mean viral load was 1,377,793 copies/mL.     

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.     

Detection of HIV-1 RNA/DNA and CD4 mRNA in feces and urine from chronic HIV-1 infected subjects with and without anti-retroviral therapy

HIV-1 infects gut associated lymphoid tissues (GALT) very early after transmission by multiple routes. The infected GALT consequently serves as the major reservoir for HIV-1 infection and could constantly shed HIV-1 and CD4⁺ T cells into the intestinal lumen. To examine this hypothesis, we monitored HIV-1 RNA/DNA and CD4 mRNA in fecal samples of chronically infected subjects with and without antiretroviral therapy (ART). We compared this to levels of HIV-1 RNA/DNA in urine and blood from the same subjects. Our results show that HIV-1 DNA, RNA and CD4 mRNA were detected in 8%, 19% and 31% respectively, of feces samples from infected subjects with detectable plasma viral load, and were not detected in any of subjects on ART with undetectable plasma viral load. In urine samples, HIV-1 DNA was detected in 24% of infected subjects with detectable plasma viral load and 23% of subjects on ART with undetectable plasma viral load. Phylogenetic analysis of the envelope sequences of HIV-1 revealed distinct virus populations in concurrently collected serum, feces and urine samples from one subject. In addition, our study demonstrated for the first time the presence of CD4 mRNA in fecal specimens of HIV-1 infected subjects, which could be used to assess GALT pathogenesis in HIV-1 infection.     

Impact of human immunodeficiency virus type 1 (HIV-1) genetic diversity on performance of four commercial viral load assays: LCx HIV RNA Quantitative, AMPLICOR HIV-1 MONITOR v1.5, VERSANT HIV-1 RNA 3.0, and NucliSens HIV-1 QT

Human immunodeficiency virus type 1 (HIV-1) evolution and changing strain distribution present a challenge to nucleic acid-based assays. Reliable patient monitoring of viral loads requires the detection and accurate quantification of genetically diverse HIV-1. A panel of 97 HIV-1-seropositive plasma samples collected from Cameroon, Brazil, and South Africa was used to compare the performance of four commercially available HIV RNA quantitative tests: Abbott LCx HIV RNA Quantitative assay (LCx), Bayer Versant HIV-1 RNA 3.0 (bDNA), Roche AMPLICOR HIV-1 MONITOR v1.5 (Monitor v1.5), and bioMérieux NucliSens HIV-1 QT (NucliSens). The panel included group M, group O, and recombinant viruses based on sequence analysis of gag p24, pol integrase, and env gp41. The LCx HIV assay quantified viral RNA in 97 (100%) of the samples. In comparison, bDNA, Monitor v1.5, and NucliSens quantified viral RNA in 96.9%, 94.8%, and 88.6% of the samples, respectively. The two group O specimens were quantified only by the LCx HIV assay. Analysis of nucleotide mismatches at the primer/probe binding sites for Monitor v1.5, NucliSens, and LCx assays revealed that performance characteristics reflected differences in the level of genetic conservation within the target regions.     

Quantitative detection of human immunodeficiency virus type 1 (HIV-1) viral load by SYBR green real-time RT-PCR technique in HIV-1 seropositive patients

HIV-1 viral load represents a basic marker for evaluation of the rate and severity of HIV-1 related disease and to monitor the effectiveness of treatment. An SYBR green-based real-time RT-PCR (SYBR green real-time RT-PCR) revealed by Light Cycler technology was evaluated for quantitation of HIV-1 RNA viral load in plasma of HIV-1 seropositive patients. The performance of the SYBR green real-time PCR was assessed on 56 HIV-1 seropositive patients under highly active retroviral therapy (HAART) and 25 blood donors. The results demonstrated that this technique detected 50 HIV-1 RNA copies per millilitre of plasma. Moreover, we compared real-time RT-PCR with the b-DNA technique considered widely a reference technique for HIV-1 RNA viral load measurement. The parallel quantitative analysis of HIV-1 positive samples showed a high correlation (r=0.908) between the two methods. Although b-DNA and the real-time-based method gave similar sensitivity, the assay determined quantitatively HIV-1 RNA copies in 4 out of 16 samples shown as undetectable by b-DNA. The SYBR green real-time RT-PCR represents a good alternative to b-DNA assay in HIV-1 viral load determination especially during the monitoring of HAART treatment.     

Comparative analysis of HIV-1 viral load assays on subtype quantification: Bayer Versant HIV-1 RNA 3.0 versus Roche Amplicor HIV-1 Monitor version 1.5

Quantification of HIV-1 subtypes is essential for appropriate clinical management. Whereas viral load assays were initially developed to accurately quantify subtype B, the recent worldwide spread of non-B subtypes and the introduction of treatment programs in regions with non-B subtypes have prompted adaptations of these assays. The Bayer Versant HIV-1 RNA 3.0 Assay (branched DNA [bDNA] 3.0) and the Roche Amplicor HIV-1 Monitor version 1.5 (Amplicor 1.5) assays are reported to quantify all subtypes in group M; however, evaluation of performance characteristics remains limited. In this study, we evaluated the accuracy and reliability of bDNA 3.0 and Amplicor 1.5 on multiple serially diluted viral isolates from HIV-1 group M, subtypes A through F. Testing was conducted on both assay systems in two independent laboratories. Comparative pansubtype quantification from regression analysis showed that quantification by bDNA 3.0 was approximately 0.3 log-fold lower than that by Amplicor 1.5. Comparative pansubtype accuracy analysis showed data points more closely distributed about their respective regression lines and thus showing greater reliability by bDNA 3.0 than by Amplicor 1.5.     

Quantification of genital human immunodeficiency virus type 1 (HIV-1) DNA in specimens from women with low plasma HIV-1 RNA levels typical of HIV-1 nontransmitters

Studies of human immunodeficiency virus type 1 (HIV-1) transmission suggest that genital HIV-1 RNA and DNA may both be determinants of HIV-1 infectivity. Despite its potential role in HIV-1 transmission, there are limited quantitative data on genital HIV-1 DNA. Here we validated an in-house real-time PCR method for quantification of HIV-1 DNA in genital specimens. In reactions with 100 genomes to 1 genome isolated from a cell line containing one HIV-1 provirus/cell, this real-time PCR assay is linear and agrees closely with a commercially available real-time PCR assay specific for a cellular housekeeping gene. In mock genital samples spiked with low numbers of HIV-1-infected cells such that the expected HIV-1 DNA copy number/reaction was 100, 10, or 5, the average copy number/reaction was 80.2 (standard deviation [SD], 28.3), 9.1 (SD, 5.4), or 3.1 (SD, 2.1), respectively. We used this method to examine genital HIV-1 DNA levels in specimens from women whose low plasma HIV-1 RNA levels are typical of HIV-1 nontransmitters. The median HIV-1 DNA copy number in endocervical secretions from these women (1.8 HIV-1 DNA copies/10,000 cells) was lower than that for women with higher plasma HIV-1 RNA levels (16.6 HIV-1 DNA copies/10,000 cells) (P=0.04), as was the median HIV-1 DNA copy number in vaginal secretions (undetectable versus 1.0 HIV-1 DNA copies/10,000 cells). These data suggest that women with low plasma HIV-1 RNA and thus a predicted low risk of HIV-1 transmission have low levels of genital HIV-1 cell-associated virus. The assay described here can be utilized in future efforts to examine the role of cell-associated HIV-1 in transmission.     

TaqMan RT-PCR and VERSANT HIV-1 RNA 3.0 (bDNA) assay Quantification of HIV-1 RNA viral load in breast milk.

BACKGROUND: Transmission of HIV via breast milk is a primary cause of pediatric HIV infection in developing countries. Reliable methods to detect breast milk viral load are important. OBJECTIVE: To correlate the ability of the VERSANT HIV 3.0 (bDNA) assay to real-time (RT) TaqMan PCR in quantifying breast milk HIV-1 RNA. STUDY DESIGN: Forty-six breast milk samples that had been spiked with cell-free HIV-1 and eight samples spiked with cell-associated HIV-1 were assayed for HIV-1 RNA by both VERSANT HIV 3.0 and TaqMan RNA assays. RESULTS: Only assays on the cell-free samples were statistically compared. Both a Deming regression slope and a Bland-Altman slope indicated a linear relationship between the two assays. TaqMan quantitations were on average 2.6 times higher than those of HIV 3.0. A linear relationship was observed between serial dilutions of spiked cell-free HIV-1 and both the VERSANT HIV 3.0 and the TaqMan RNA assays. CONCLUSION: The two methods correlated well although the VERSANT HIV 3.0 research protocol quantified HIV-1 RNA slightly lower than TaqMan.     

Undetectable plasma HIV-1 RNA with strong gag-pol specific interferon-gamma ELISPOT response in an HIV-1 clade A/E-infected child untreated with antiretroviral therapy

We report a 7-year HIV-1 clade A/E-infected child untreated with antiretroviral therapy who had positive HIV antibody testing but undetectable plasma HIV-1 RNA by Roche Amplicor version 1.5 and bDNA version 3.0. DNA PCR was positive by methods using gag/pol primers but not env/pol primers. The patient had strong HIV-1-specific cytotoxic T lymphocyte responses, which likely contributed to her low viral burden and undetectable plasma HIV-1 RNA.     

Quantitation of HIV-1 RNA in dried blood and plasma spots

Dried blood spots (DBSs) and dried plasma spots (DPSs) are an attractive method for serological and molecular diagnosis of HIV infection. Recently, Youngpairoj et al. [Youngpairoj, A.S., Masciotra, S., Garrido, C., Zahonero, N., de Mendoza, C., Garcia-Lerma, J.G., 2008. HIV-1 drug resistance genotyping from dried blood spots stored for 1 year at 4 degrees C. J. Antimicrob. Chemother. 61, 1217–1220] showed that HIV-1 can be genotyped efficiently from DBS specimens stored at 4 °C for 1 year. The viral load obtained from DBS and DPS samples was compared with that obtained from plasma samples. A total of 86 samples was prepared from people infected with HIV subtype B or non-B and spotted on 903 filter papers stored with desiccant at 4 °C. RNA was extracted using the QIAamp Viral RNA mini kit (Qiagen, Courtaboeuf, France). RNA from DBS or DPS samples was quantified in accordance with the Agence Nationale de Recherche sur le SIDA (ANRS, AC11, Paris, France) assay for HIV-1 quantitation. When the mean viral load of plasma samples and DPS samples or plasma samples and DBS samples were compared, there were no significant differences. The overall data showed that although the sensitivity threshold of the assays was different, there was a correlation between the three specimen types and that DBS and DPS samples can be routinely used for viral load quantification particularly in resource-limited settings.     

Use of Sno Strip filter-paper wicks for collection of genital-tract samples allows reproducible determination of human immunodeficiency virus type 1 (HIV-1) RNA viral load with a commercial HIV-1 viral load assay

To assess the reproducibility of measurements of cervical and vaginal human immunodeficiency virus (HIV) viral load, 92 duplicate cervical and 88 duplicate vaginal samples were collected from 13 HIV-infected women using Sno Strip filter-paper wicks. RNA was eluted from the strips, extracted, and assayed using a modified protocol for the Roche Cobas Amplicor HIV-1 Monitor assay. Pearson's correlation coefficient (R), coefficient of determination (D), and Bland-Altman plots (BA) were used to compare paired log10-transformed viral loads. Analysis of duplicate same-site samples showed good reproducibility (cervix: R = 0.72, D = 52%, BA = 89% within range; vagina: R = 0.72, D = 51%, BA = 87% within range); paired cervix/vagina measurements showed moderate correlation only (R = 0.56; D = 31.3%). Standardized sample collection and simple modification of the Roche Cobas Amplicor HIV-1 Monitor assay allows reproducible measurement of genital viral load.