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.     

Measuring human immunodeficiency virus type 1 RNA loads in dried blood spot specimens using NucliSENS EasyQ HIV-1 v2.0

BackgroundHIV-1 RNA plasma level is a key parameter for anti-viral treatment monitoring in HIV-1 infected individuals. Plasma stability and accurate measurement of clinical state is at risk when transporting from remote areas. Dried blood spot (DBS) testing can reduce this risk.ObjectivesDetermine the performance of NucliSENS EasyQ HIV-1 v2.0 for DBS.Study design100 HIV-1 negative, and 129 HIV-1 spiked blood specimens (2180 copies/ml) were used for diagnostic specificity and system robustness. Analytical performance was tested in the range 50–85,000,000 copies/ml. Clinical reactivity was measured with specimens obtained from 224 HIV-1 infected individuals. HIV-1 RNA stability was analyzed after applying several different storage conditions.ResultsDiagnostic specificity was 100% and system robustness was demonstrated by 100% detection rate without invalids. Limit of detection (95% detection rate) was 800 copies/ml. Linear results were obtained over the whole range tested. For clinical specimens, percentage positive results were comparable for DBS (57%) and plasma (58%). DBS quantification was on average 0.36 log 10 lower as compared to plasma. Specimen stability was demonstrated for 1 week at 55 °C/60% humidity, 3 weeks at 37 °C/80% humidity, 9 weeks at 37 °C/40% humidity, 3 months at ?20 °C/70% humidity, 3 weeks at 4 °C/100% humidity, 9 months at room temperature (15–30 °C), and 9 weeks shipment simulation.ConclusionResults obtained fully support the use of DBS for the NucliSENS EasyQ HIV-1 v2.0 assay. These findings are especially of importance in cases that plasma stability is currently at risk due to for example, long transport routes from remote areas under less controlled conditions.     

Comparison of HIV-1 RNA measurements obtained by using plasma and dried blood spots in the automated abbott real-time viral load assay

Dried blood spots (DBS) may be a promising alternative specimen type to plasma for measuring the viral load (VL) in HIV-infected individuals in resource-limited settings. However, characterization of assay performance using DBS is incomplete. In this prospective study, the VL was measured in parallel using plasma and DBS specimens collected at the same time from 157 HIV-1-infected individuals. DBS were prepared by dispensing 50 μl of blood onto filter paper cards and were stored desiccated at -20°C. Nucleic acid extraction from plasma and DBS was performed automatically using the Abbott m2000sp instrument, and the VL was measured using the RealTime HIV-1 VL assay, which has a lower limit of detection of 40 HIV RNA copies/ml. The correlation between plasma and DBS results was good (R = 0.91; P < 0.001). The mean difference in the VL (DBS minus plasma) was 0.35 log copies (standard deviation [SD], 0.47 log copies). A total of 40 (26%) paired specimens had a difference of >0.5 log copy, and in 12 (7.8%) it was >1 log copy. the VL from DBS was measurable in 95.7% of specimens with a plasma VL of >2.74 log copies (550 HIV RNA copies/ml). In summary, the VL can reliably be measured using DBS with the Abbott RealTime HIV-1 assay. The estimated lower limit of detection of this automated methodology on DBS is 550 copies/ml, a threshold that may be acceptable for periodic VL monitoring in patients on antiretroviral therapy in resource-limited settings, where early detection of virologic treatment failure is often problematic.     

RNA versus DNA (NucliSENS EasyQ HIV-1 v1.2 versus Amplicor HIV-1 DNA test v1.5) for early diagnosis of HIV-1 infection in infants in Senegal

The objective of this study was to compare the performance of the NucliSENS EasyQ HIV-1 v1.2 platform (bioMérieux, France) to the Amplicor HIV-1 DNA test v1.5 (Roche Molecular Systems, Switzerland) in detecting HIV-1 infection in infants using venipuncture-derived whole blood in tubes and dried blood spots. A total of 149 dried blood spots and 43 EDTA-anticoagulated peripheral blood samples were collected throughout Dakar and other areas in Senegal from infants and children aged 3 weeks to 24 months who were born to HIV-1-infected mothers. Samples were tested using the NucliSENS and Amplicor technologies. The NucliSENS and Amplicor results were 100% concordant using either EDTA-anticoagulated peripheral blood or dried blood spots. Compared to Amplicor, the sensitivity and specificity of the NucliSENS test were 100%. The NucliSENS EasyQ HIV-1 RNA assay performed as well as the Amplicor HIV-1 DNA test in detecting HIV-1 infection in infants. In addition, this platform can give an indication of the viral load baseline. The NucliSENS EasyQ platform is a good alternative for early infant diagnosis of HIV-1 infection.     

A novel RealTime HIV-1 Qualitative assay for the detection of HIV-1 nucleic acids in dried blood spots and plasma

Abbott RealTime HIV-1 Qualitative is an in vitro real-time PCR assay for detecting HIV-1 nucleic acids in human plasma and dried blood spots (DBS). The assay was designed to be used in diagnosis of HIV-1 infections in pediatric and adult patients, with an emphasis on the applicability in resource-limited settings. Use of DBS facilitates specimen collection from remote areas and transportation to testing laboratories. Small sample input requirement facilitates testing of specimens with limited collection volume. The Abbott RealTime HIV-1 Qualitative assay is capable of detecting HIV-1 group M subtypes A-H, group O and group N samples. HIV-1 virus concentrations detected with 95% probability were 80 copies/mL of plasma using the plasma protocol, and 2469 copies/mL of whole blood using the DBS protocol. The assay detected HIV-1 infection in 13 seroconversion panels an average 10.5 days earlier than an HIV-1 antibody test and 4.9 days earlier than a p24 antigen test. For specimens collected from 6 weeks to 18 months old infants born to HIV-1 positive mothers, assay results using both the DBS and plasma protocols agreed well with the Roche Amplicor HIV-1 DNA Test version 1.5 (95.5% agreement for DBS and 97.8% agreement for plasma).     

Detection of human immunodeficiency virus type 1 DNA in dried blood spots by a duplex real-time PCR assay

A dried blood spot (DBS) is a well-accepted means for the collection, transport, and storage of blood samples for various epidemiologic, serologic, and molecular assays for human immunodeficiency virus (HIV) studies. It is particularly important for mother-to-infant-transmission studies of affected individuals living in remote areas. We have developed a real-time PCR method to detect HIV type 1 (HIV-1) DNA in dried blood spots. A cellular gene, RNase P, was coamplified with the HIV-1 DNA in the same tube to monitor the DNA extraction efficiency and the overall assay performance. Our assay is a one-tube, single-step closed-system assay and uses a dUTP/uracil DNA glycosidase anti-PCR contamination control. The HIV-1 primers and probe were derived from a conserved region of the long terminal repeat. The detection of RNase P is attenuated by lowering the forward and reverse primer concentrations so that its amplification will not overwhelm the HIV-1 amplification and yet will provide a semiquantitative measurement of the quality of the isolated DBS DNA. We examined 103 HIV-1-seropositive and 56 seronegative U.S. adults and found that our assay has a sensitivity of 98.1% (95% confidence interval [CI], 95.5% to 100%) and specificity of 100% (95% CI, 99% to 100%). The positive and negative predictive values are 100% and 96.6%, respectively. This duplex PCR assay may be useful in identifying HIV-1-infected persons, particularly infants born to seropositive mothers in remote areas of the world.     

Correlation between HIV-1 viral load quantification in plasma,dried blood spots,and dried plasma spots using the Roche COBAS Taqman assay

BackgroundThe use of simplified methods for viral load determination could greatly increase access to treatment monitoring of HIV patients in resource-limited countries.ObjectiveThe aim of the present study was to optimize and evaluate the performance of the Roche COBAS Taqman assay in HIV-RNA quantification from dried blood spots (DBS) and dried plasma spots (DPS).Study designEDTA blood samples from 108 HIV-infected women were used to prepare 129 DBS and 76 DPS on Whatman 903 card. DBS and DPS were stored at ?20 °C. HIV-1 RNA was extracted from DBS/DPS using the MiniMAG system (bioMerieux). Amplification and detection were performed using the Roche COBAS TaqMan assay. Plasma viral load results were used as standard.ResultsThere was a high correlation between measures of viral load in plasma and in DBS/DPS (r = 0.96 and 0.85 respectively, P < 0.001). Overall, viral load values in DBS and DPS tended to be lower than in plasma with mean (SD) differences of 0.32 log (0.22) for DBS and of 0.35 (0.33) for DPS. Detection rates were 96.4% for DBS and 96.1% for DPS in samples with corresponding plasma values >3.0 log copies/ml. Samples with HIV-RNA below 50 copies/ml were correctly identified in 18/19 DBS and in 7/7 DPS.ConclusionsBoth DBS and DPS provided results highly correlated to the plasma values. High detection rate was obtained with both DBS and DPS when HIV-RNA was >3.0 log copies/ml. Our results support the use of DBS/DPS to detect virologic failure in resource-limited settings.     

Multicenter evaluation of the NucliSens EasyQ HIV-1 v1.1 assay for the quantitative detection of HIV-1 RNA in plasma

The Nuclisens EasyQ HIV-1 v1.1 assay (Biomerieux) is a real-time detection method combined with NASBA technology designed to measure plasma HIV-RNA. Its performance was assessed in 1008 clinical specimens collected from individuals infected with clade B (774) and non-B (234) HIV-1 variants at four European laboratories. The results were compared with those obtained using three other commercial viral load assays: Cobas Amplicor Monitor HIV-1 v1.5 (Roche), Versant HIV-1 RNA assay (Bayer) and Nuclisens HIV-1 QT (Biomerieux). Overall, the linearity, specificity and reproducibility of the EasyQ assay was comparable with that from the other tests. The correlation coefficient (R) between methodologies was 0.85 for Amplicor; 0.87 for Versant; and 0.91 for Nuclisens. The specificity of the assay was 99.4%. Of note, Versant missed 17% of specimens with non-B subtypes which could be detected by EasyQ, while Amplicor provided similar results than EasyQ. HIV-1 group O specimens were only detected by the EasyQ assay. In conclusion, the performance of the EasyQ assay seems to be similar to that of other HIV-1 viral load tests currently on the market, but it is more sensitive than Versant for HIV-1 non-B subtypes and shows a wider dynamic range than Amplicor. Moreover, as it incorporates the advantage of real-time detection procedures, it facilitates high throughput and short turnaround time.     

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.     

Usage of dried blood spots for molecular diagnosis and monitoring HIV-1 infection

The usage of dried blood spots as specimens for diagnosis and monitoring of HIV-1 infection in Thailand was evaluated. EDTA blood samples, which were collected from 100 HIV seronegative and 109 HIV seropositive individuals, were tested on dried blood spots; Whatman, Schleicher and Schuell (S&S) No. 903 and S&S IsoCode filter paper. Nucleic acid was extracted and used as a template for HIV-1 proviral DNA detection by an "in-house" multiplex PCR and a commercial Amplicor HIV-1 PCR test (Roche, version 1.0). HIV-1 RNA qualitative (QL) and quantitative (QT) detection was determined by Nucleic Acid Sequence Based Amplification (NASBA). The average DNA per blood spot recovered from Whatman and S&S IsoCode was not statistically different (p = 0.512) with a range of 218.9+/-46.84 and 225.63+/-88.33 microg, respectively. The concordance of HIV-1 proviral DNA detection by PCR from dried blood spots Whatman and S&S IsoCode was 94% versus 89.4% for sensitivity and 100% versus 100% for specificity. The sensitivity and specificity of HIV-1 RNA QL detection in dried blood spots was 89.7 and 97.5%, respectively. The HIV-1 RNA QT from dried blood spots showed a good correlation in paired dried blood spots and plasma with Pearson correlation, r = 0.817 (R2 = 0.667, P < 0.05). The data showed that dried blood spots could be used for the diagnosis and monitoring of HIV-1 infection.     

Evaluation of the NucliSens EasyQ assay in HIV-1-infected individuals in South Africa

We compared the performance of the NucliSens EasyQ assay (bioMerieux) combined with the manual NucliSens miniMag extraction methodology to the Roche Cobas Ampliprep/Standard Amplicor Monitor methodology (Roche Diagnostics) for HIV-1 RNA quantitation in HIV-1-infected individuals in South Africa. Plasma samples (284) from HIV sero-positive patients at different stages of infection were analyzed. The distribution of results was typical of the clinical samples received at the laboratory where 20% have viral load results <400 copies/ml (2.6 log) and 18% have viral load results >750000 copies/ml (5.8 log) using the Roche Amplicor Monitor standard assay. All statistical analyses were performed using log10-transformed values for all the variables in the analyses, i.e. log10EasyQIU/ml, and log10RNA (log10 copies/ml, Amplicor). Roche values were converted from RNA copies per ml to IU/ml by multiplying the Roche value by 0.51. HIV RNA levels quantitated by the NucliSens EasyQ assay correlated significantly with those of the Roche Cobas Amplicor Monitor assay (r=0.874, p<0.0001). Reproducibility of the NucliSens EasyQ assay in the log6IU range yielded CV variance of 1.3-2.84% for two well-trained technologists. In addition, a retrospective evaluation of the performance of the NucliSens EasyQ assay in 102 runs (2448) samples was conducted in the laboratory over a 4-month interval. Factors considered during this evaluation included time taken to perform the assay, volume requirements, number of required repeats, potential for contamination.     

Evaluation of the ultrasensitive human immunodeficiency virus type 1 (HIV-1) p24 antigen assay performed on dried blood spots for diagnosis of HIV-1 infection in infants

The diagnostic accuracy of the modified p24 antigen assay performed on pediatric dried blood spots was evaluated. Samples analyzed within 6 weeks of collection yielded no false-positive results (specificity, 100%) and few false-negative results (sensitivity, 96.5% to 98.3%). Laboratory services with limited resources should assess this option for routine infant diagnosis.     

Evaluation of the NucliSens EasyQ HIV-1 v1.1 and RealTime HIV-1 kits for quantitation of HIV-1 RNA in plasma

Human immunodeficiency virus type-1 (HIV-1) RNA viral load is an important biomarker to evaluate the therapeutic efficacy of antiretroviral drugs and to monitor disease progression in HIV-infected individuals. We compared HIV-1 RNA quantitation between two different kits, the NucliSens EasyQ^® HIV-1 v1.1 (EasyQ, bioMérieux) and RealTime HIV-1 (RealTime, Abbott), using HIV-1 RNA quality control (QC) materials, cell-cultivated viruses, and the plasma samples of 104 patients with HIV. Correlation between the two kits for HIV RNA-1 quantitation with clinical samples was high (R = 0.91). Based on results obtained with quality control standards, the reproducibility of the RealTime kit was higher than the EasyQ kit: the viral load value and coefficient of variation of each kit was 4.11 ± 0.136 and 3.3% for EasyQ and 3.55 ± 0.042 and 1.2% for RealTime, respectively (P < 0.002).This is the first comparative analysis of the detection limit and reproducibility of two different quantitation kits using clinical plasma samples from Korean HIV-1-infected patients. It will serve a useful reference to determine correction values for each HIV-1 RNA quantitation kits and to choose an appropriate assay kit for each laboratory.     

Evaluation of NucliSens EasyQ HIV-1 assay for quantification of HIV-1 subtypes prevalent in South-east Asia.

BACKGROUND: Monitoring anti-retroviral therapy requires that viral load assays for human immunodeficiency virus type 1 (HIV-1) be applicable to diverse HIV-1 subtypes. OBJECTIVES: To evaluate NucliSens EasyQ HIV-1 assay for quantitation of common HIV-1 subtypes prevalent in South-east Asia. STUDY DESIGN: One hundred and nineteen plasma samples collected in Hong Kong and Cambodia were used to compare the performance of NucliSens EasyQ HIV-1 and COBAS Amplicor HIV-1 Monitor version 1.5 assays. Viral RNA extracted from the NucliSens MiniMAG was also used for HIV-1 subtyping. RESULTS: Performance of NucliSens EasyQ correlated well with COBAS Amplicor (r=0.777, p<0.001) and the small mean difference (0.0462log(10)IU/mL) obtained in the Bland and Altman model indicated good agreement between two assays. The NucliSens EasyQ assay demonstrated a 95% sensitivity at 500IU/mL and 100% specificity. Reproducibility of this assay was within log(10)2-4IU/mL and had a coefficient of variation between 2.3% and 10.4%. Among the 109 specimens included in the analysis, HIV-1 subtyping identified 64 CRF01_AE, 38 subtype B, 3 subtype C, 3 CRF07_BC and 1 subtype G viruses. CONCLUSIONS: Performance of NucliSens EasyQ was comparable to COBAS Amplicor for HIV-1 viral load monitoring. RNA extracts from NucliSens MiniMAG could be used for HIV-1 viral load monitoring, subtyping and drug resistance mutations detection. Our findings highlight the versatility of both NucliSens EasyQ and COBAS Amplicor in monitoring prevalent subtypes and rare circulating recombinant forms (CRFs) in the South-east Asia region.     

Evaluation of the Abbott Real-Time HIV-1 quantitative assay with dried blood spot specimens

The Abbott Real-Time HIV-1 assay was evaluated for its performance in quantification of human immunodeficiency virus type 1 (HIV-1) RNA in dried blood spot (DBS) samples. In total, 169 blood samples with detectable plasma HIV-1 RNA were used to extract RNA from paired DBS and liquid plasma samples, using the automated Abbott m Sample Preparation System (m2000sp). HIV-1 RNA was then quantitated by the m2000rt RealTime analyser. RNA samples suitable for real-time PCR were obtained from all but one (99.4%) of the DBS samples and HIV-1 RNA was detected in 163/168 (97.0%) samples. The correlation between HIV-1 RNA values measured in paired DBS and plasma samples was very high ( r  = 0.882), with 78.5% and 99.4% of cases differing by <0.5 and 1.0 log, respectively. Retesting of DBS replicates following 6 months of storage at 2–8°C showed no loss of HIV-1 RNA in a subset of 89 samples. The feasibility of DBS testing coupled with automated sample processing, and the use of a latest-generation FDA-approved real-time PCR-based system, represents an encouraging first step for viral load measurement in reference centres in developing countries where access to antiretroviral therapy is expanding.     

Evaluation of dried blood spot specimens for HIV-1 drug-resistance testing using the Trugene HIV-1 genotyping assay.

BACKGROUND: Efforts to simplify the collection and shipping of specimens for HIV drug-resistance testing in resource-limited settings are needed as antiretroviral therapy increases worldwide. OBJECTIVE: To evaluate the reliability and practicality of using dried blood spots (DBS) for HIV-1 drug-resistance testing with the Trugene HIV-1 genotyping assay. STUDY DESIGN: Nucleic acids from 33 DBS and counterpart plasma specimens were extracted using the Nuclisens MiniMAG system and genotyped using the Trugene HIV-1 genotyping assay. Results were evaluated for sensitivity, accuracy, and reproducibility. RESULTS: A genotype was obtained for 33 (100%) plasma specimens and 26 (78.8%) DBS specimens, including 19 of 21 (90.5%) DBS specimens with a viral load greater than 6000 copies/mL. The mean nucleotide sequence concordance for the 940-nucleotide region evaluated was 99.3% for 26 DBS and plasma pairs, and 99.2% for 15 replicate DBS pairs. All 58 resistance-associated mutations detected in plasma specimens were detected in the corresponding DBS specimens. CONCLUSIONS: We show that DBS can be reliably and accurately genotyped using standard clinical assay methods, offering a practical alternative to plasma. This method is well suited for pre-treatment resistance testing and has potential for use in monitoring drug resistance in ART-treated individuals.