NucliSENS EasyQ HIV-1 Research Articles

Performance of patient-collected dried blood spot specimens for HIV-1 viral load testing: evidence from the DO ART Study in South Africa.

Evaluate the clinical utility of patient-collected dried blood spots (DBS) in measuring HIV-1 viral load (VL) for monitoring antiretroviral therapy (ART) compared to provider-collected DBS and blood plasma. In a randomized trial of community-based delivery of ART in South Africa, we assessed performance of: (1) DBS specimens compared to plasma, and (2) participant-collected versus staff-collected DBS specimens, to measure HIV-1 VL. The bioMérieux NucliSENS EasyQ HIV-1 v2.0 assay was used for VL measurement. From October 2017 to November 2019, we collected 996 pairs of plasma/DBS specimens from 760 participants and 315 pairs of staff-/participant-collected DBS cards from 261 participants. We assessed DBS test sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) using the WHO failure threshold of 1000 copies/mL. Log-transformed VL was compared using concordance correlation coefficients (CCC) and mean differences from linear mixed models. In a population with 13% detectable VL, DBS VL compared with plasma VL had 91% (95% CI: 86-95) sensitivity, 99% (98-100) specificity, 94% (90-98) PPV, and 99% (98-99) NPV. We observed high agreement between staff-collected DBS VL and plasma VL (CCC: 0.94), and between participant-collected DBS VL and plasma VL (CCC: 0.92). Correlation between participant- and staff-collected DBS was very high (CCC: 0.97; mean difference for those with a detectable result: -0.10 log 10 copies/mL [-0.21-0.02]). VL results from participant-collected DBS are clinically comparable with those collected by clinical staff and using blood plasma. Self-collected DBS has potential for use for ART monitoring outside the clinic.

Analysis of antibodies involved in cases of discordant and indeterminate HIV serology in Togo from 2016 to 2018

Introduction: The national algorithm for the diagnosis of HIV infection in Togo leads sometimes to discordant or indeterminate results. The objective of this study was to explore the characteristics of discordant and indeterminate HIV serologies at the National Reference Center for HIV testing (CNR-VIH) in Togo. Material and methods: Through a cross-sectional study, we analyzed the cases of discordant and indeterminate serologies from 2016 to 2018 at the CNR-VIH. Three kind of tests were used. The first one is a screening test also called “test 1” is very sensitive while the confirmatory test also called “test 2” is very specific and used when the sample is reactive with test 1. The third test is an immunoblotting test which is used in case of discordance between test 1 and test 2 results. We used Vironostika HIV Uni-Form II Ag/Ab (BioMerieux, Geneva, Switzerland), Determine Alere HIV-1/2 Serum/Plasma (Chiba, Japan) and Murex HIV Ag/Ab (Dartford, UK) as test 1 while Tri-Dot HIV-I/II (J. Mitra & Pvt Ltd. New Delhi-110-India Co.) and Inno-Lia HIV-I/II Score (Fujirebio, Ghent, Belgium) were used respectively as test 2 and test 3. The test 3 allowed us to determine the antibodies involved in the occurrence of indeterminate serologies. A pool of indeterminate samples was tested for qualitative detection of HIV-1 RNA by RT-PCR using the NucliSENS EasyQ HIV-1 platform from BioMérieux. Results: A total of 555 discordant serologies corresponding to 4.3% of all serologies performed over the 3 years have been analyzed. The average age of the subjects tested was 36.9 ± 17.4 years and men were slightly more represented with a sex ratio of 1.03. Of the 555 samples, 81 (14.7%) were reactive with 53 (9.6%) cases of HIV-1 and 28 (5.1%) cases of HIV-2. Two hundred and thirty-one (41.6%) samples were negative while the remaining 243 (43.8%) were indeterminate. The frequencies of antibodies (Ab) against gp120, gp41, p24, p31, p17, gp105 and gp36 were respectively 10.3%, 46.3%, 15.5 %, 11.2%, 8.8%, 5.4% and 5.0%. Among the 257 cases for which gp41 Ab had been detected, 202 (78.6%) were cases of indeterminate serology with the only presence of gp41 Ab. Qualitative HIV-1 RNA testing on 71 (29.2%) indeterminate samples was negative. Conclusion: This study showed that discordant serologies are frequently found in our common practice in Togo. The use of immunoblotting tests has the advantage of making it possible to elucidate more than half of these discordant serologies. The contribution of molecular biology techniques is uncertain. The large majority of these indeterminate serologies are due to cross-reactions particularly with gp41. These indeterminate serologies require not only further studies for their understanding but also an update of HIV diagnosis algorithm in Togo.

Sensitivity and specificity of dried blood spots for HIV-1 viral load quantification

The use of dried blood spots (DBS) instead of plasma as a specimen type for HIV-1 viral load (VL) testing facilitates the decentralization of specimen collection and can increase access to VL testing in resource-limited settings. The performance of DBS for VL testing is lower, however, when compared to the gold standard sample type plasma. In this diagnostic accuracy study, we evaluated 3 VL assays with DBS.Participants were recruited between August 2012 and April 2015. Both plasma and DBS specimens were prepared and tested for HIV-1 VL with the Roche CAP/CTM HIV-1 test v2.0, the Abbott RealTime HIV-1, and the bioMérieux NucliSENS EasyQ HIV-1 v2.0. Sensitivity and specificity to detect treatment failure at a threshold of 1000 cps/mL with DBS were determined.A total of 272 HIV-positive patients and 51 HIV-negative people were recruited in the study. The mean difference or bias between plasma and DBS VL was <0.5 log cps/mL with all 3 assays but >25% of the specimens differed by >0.5 log cps/mL.All 3 assays had comparable sensitivities around 80% and specificities around 90%. Upward misclassification rates were around 10%, but downward misclassification rates ranged from 20.3% to 23.6%. Differences in between assays were not statistically significant (P > 0.1).The 3 VL assays evaluated had suboptimal performance with DBS but still performed better than immunological or clinical monitoring. Even after the introduction of the much-anticipated point-of-care VL devices, it is expected that DBS will remain important as a complementary option for supporting access to VL monitoring, particularly in rural, resource-limited settings. Manufacturers should accelerate efforts to develop more reliable, sensitive and specific methods to test VL on DBS specimens.

English

The use of plasma is an obstacle to realize HIV-1 viral load in sub-Saharan Africa. In this context, the dried blood spot (DBS) is an interesting tool for sample collections. This approach was tested using a DBS hole-punch device (PUNCHER). Plasma and DBS samples were obtained from 102 patients, comprising 17 HIV-1 negative patients and 85 HIV-1 infected patients. The PUNCHER’s performance used to cut DBS was evaluated with the following criteria: ease of use, time savings and safety. VL was measured in parallel on plasma and DBS samples using NucliSENS EasyQ HIV-1. The correlation between plasma and DBS results was strong (R = 0.91; P < 0.001). The mean difference (± standard deviation) was -0.59 ± 0.52 log10 copies/ml. The sensitivity and specificity of DBS were 91.3% (n= 74) for the 81 VL detectable samples and 100% for the 21 VL undetectable samples, respectively. On a scale of 10, the PUNCHER’s performance scored 9.3 for ease of use, 8.6 for time savings and 10 for safety. PUNCHER is highly efficient at cutting DBS, and the VL resulting from DBS correlated well with those obtained from plasma. Key words: Puncher, dried blood spot (DBS), viral load, performance.

Finger Prick Dried Blood Spots for HIV Viral Load Measurement in Field Conditions in Zimbabwe.

BackgroundIn the context of a community-randomized trial of antiretrovirals for HIV prevention and treatment among sex workers in Zimbabwe (the SAPPH-IRe trial), we will measure the proportion of women with HIV viral load (VL) above 1000 copies/mL (“VL>1000”) as our primary endpoint. We sought to characterize VL assay performance by comparing results from finger prick dried blood spots (DBS) collected in the field with plasma samples, to determine whether finger prick DBS is an acceptable sample for VL quantification in the setting.MethodsWe collected whole blood from a finger prick onto filter paper and plasma samples using venipuncture from women in two communities. VL quantification was run on samples in parallel using NucliSENS EasyQ HIV-1 v2.0. Our trial outcome is the proportion of women with VL>1000, consistent with WHO guidelines relating to regimen switching. We therefore focused on this cut-off level for assessing sensitivity and specificity. Results were log transformed and the mean difference and standard deviation calculated, and correlation between VL quantification across sample types was evaluated.ResultsA total of 149 HIV-positive women provided DBS and plasma samples; 56 (63%) reported being on antiretroviral therapy. VL ranged from undetectable-6.08 log10 using DBS and undetectable-6.40 log10 using plasma. The mean difference in VL (plasma-DBS) was 0.077 log10 (95%CI = 0.025–0.18 log10; standard deviation = 0.63 log10,). 78 (52%) DBS and 87 (58%) plasma samples had a VL>1000. Based on plasma ‘gold-standard’, DBS sensitivity for detection of VL>1000 was 87.4%, and specificity was 96.8%.ConclusionThere was generally good agreement between DBS and plasma VL for detection of VL>1000. Overall, finger prick DBS appeared to be an acceptable sample for classifying VL as above or below 1000 copies/mL using the NucliSENS assay.

Prospective Evaluation of Diagnostic Accuracy of Dried Blood Spots from Finger Prick Samples for Determination of HIV-1 Load with the NucliSENS Easy-Q HIV-1 Version 2.0 Assay in Malawi

HIV-1 viral load (VL) testing is not widely available in resource-limited settings. The use of finger prick dried blood spot (FP-DBS) samples could remove barriers related to sample collection and transport. Measurement of VL using DBS from EDTA venous blood (VB-DBS) in place of plasma has previously been validated using the NucliSENS Easy-Q HIV-1 v2.0 assay, but information on the accuracy of FP-DBS samples for measuring VL is limited. This prospective study, conducted at Thyolo District Hospital in southern Malawi, compared VL levels measured on FP-DBS samples and plasma using the NucliSENS Easy-Q HIV-1 v2.0 assay. Comparability was assessed by means of agreement and correlation (131 patients with VLs of ≥100 copies/ml), sensitivity, and specificity (612 patients on antiretroviral treatment [ART]). Samples of EDTA venous blood and FP-DBS from 1,009 HIV-infected individuals were collected and prepared in the laboratory. Bland-Altman analysis found good agreement between plasma and FP-DBS VL levels, with a mean difference of -0.35 log10, and 95% limits of agreement from -1.26 to 0.55 log10. FP-DBS had a sensitivity of 88.7% (95% confidence interval [CI], 81.1 to 94.4%) and a specificity of 97.8% (95% CI, 96.1 to 98.9%) using a 1,000-copies/ml cut point and a sensitivity of 83.0% (95% CI, 73.4 to 90.1%) and a specificity of 100% (95% CI, 99.3 to 100%) using a 5,000-copies/ml cut point. This study shows that FP-DBS is an acceptable alternative to plasma for measuring VL using the NucliSENS Easy-Q HIV-1 v2.0. We are conducting a second study to assess the proficiency of health workers at preparing FP-DBS in primary health care clinics.

Antiretroviral treatment outcome in HIV-1-infected patients routinely followed up in capital cities and remote areas of Senegal, Mali and Guinea-Conakry.

IntroductionAccess to antiretroviral treatment (ART) becomes more and more effective in resource-limited settings (RLS). However, this global effort would be even more profitable if the access to laboratory services especially in decentralized settings was strengthened. We report the virological outcome and HIV-1 drug resistance in three West African countries using dried blood spots (DBS) samples. MethodsWe included HIV-1-infected adults on ART ≥6 months and followed up in capital cities and decentralized sites in Senegal, Mali and Guinea-Conakry. Patients were consecutively enrolled and DBS were collected in field conditions and kept at ambient temperature before transfer to the reference laboratory. Viral load (VL) was quantified using the NucliSENS EasyQ HIV-1 v1.2. Genotyping of HIV-1 pol gene was performed using in-house protocol.ResultsOf the 407 participants, 119, 152 and 136 were from Senegal, Mali and Guinea-Conakry, respectively. The median treatment duration was 36 months [IQR: 6–136]. Virological failure (VF) (VL≥3log10 copies/mL) was observed in 26% (95% confidence interval (CI), 18–35; n=31), 11% (95% CI, 6–17; n=16) and 24% (95% CI, 17–32; n=33) of patients in Senegal, Mali and Guinea-Conakry, respectively (p=0.001). Of samples presenting VL≥3log10 copies/mL (n=80), 70 were successfully genotyped. At least one drug resistance mutation (DRM) was detected in the following proportions: 70% (95% CI, 50–86; n=19), 93% (95% CI, 68–100; n=14) and 68% (95% CI, 48–84; n=19) in Senegal, Mali and Guinea-Conakry, respectively (p=0.22). Twenty-six per cent (26%; 95% CI, 16–38; n=18) of patients in VF harboured wild-type viruses, which is likely indicative of weak adherence. Phylogenetic analysis showed the predominance of CRF02_AG subtype (73%; 95% CI, 61–83; n=51).ConclusionsWe describe the ART outcome in capital and rural settings of Senegal, Mali and Guinea-Conakry. Our results in all of the three countries highlight the need to reinforce the ART adherence in order to minimize the occurrence of drug resistance. In addition, these findings provide additional evidence that the use of DBS as a sampling support could assist virological monitoring of patients on ART in remote areas.

Higher Specificity of Nucleic Acid Sequence-Based Amplification Isothermal Technology than of Real-Time PCR for Quantification of HIV-1 RNA on Dried Blood Spots

Dried blood spots (DBS) are widely proposed as a plasma surrogate for monitoring antiretroviral treatment efficacy based on the HIV-1 RNA level (viral load [VL]) in resource-limited settings. Interfering coamplification of cell-associated HIV-1 DNA during reverse transcription (RT)-PCR can be avoided by using nucleic acid sequence-based amplification (NASBA) technology, which is based on an RNA template and isothermic conditions. We analyzed VL values obtained with DBS and plasma samples by comparing isothermic NASBA (NucliSENS EasyQ HIV-1 V2.0; bioMérieux) with real-time RT-PCR (Cobas TaqMan HIV-1 V2.0; Roche). Samples from 197 HIV-1-infected patients were tested (non-B subtypes in 51% of the cases). Nucleic acid extractions were performed by use of NucliSENS EasyMAG (bioMérieux) and Cobas AmpliPrep (Roche) before the NASBA and RT-PCR quantifications, respectively. Both quantification assays have lower limits of detection of 20 (1.3) and 800 (2.9) log10 copies/ml (log) in plasma and DBS, respectively. The mean (DBS minus plasma) differences were -0.39 and -0.46 log, respectively, for RT-PCR and NASBA. RT-PCR on DBS identified virological failure in 122 of 126 patients (sensitivity, 97%) and viral suppression in 58 of 70 patients (specificity, 83%), yielding 12 false-positive results (median, 3.2 log). NASBA on DBS identified virological failure in 85 of 96 patients (sensitivity, 89%) and viral suppression in 95 of 97 patients (specificity, 98%) and yielded 2 false-positive results (3.0 log for both). Both technologies detected HIV-1 RNA in DBS at a threshold of 800 copies/ml. This higher specificity of NASBA technology could avoid overestimation of poor compliance or the emergence of resistance when monitoring antiretroviral efficacy with the DBS method.

Comparison of the NucliSENS EasyQ HIV-1 v2.0 with Abbott m2000rt RealTime HIV-1 assay for plasma RNA quantitation in different HIV-1 subtypes

Quantitation of HIV-1 RNA levels in plasma has significant prognostic value since high viral load concentrations in plasma are associated with a faster disease progression. Viral load testing became one the most important tools for monitoring HIV patients. New real time methodologies to quantify HIV viral load had arisen in the last decade. HIV is a virus with a high genetic variability, with the potential to negatively affect the performance of the viral load assays. Consequently, any new assay should be challenged against, at least, the most prevalent HIV-1 genetic variants. In the present study, the new version of NucliSENS EasyQ® HIV-1 (Version 2.0) quantitative assay was compared with another ultra-sensitive test – Abbott RealTime HIV-1 – using 175 plasma samples from patients infected with several HIV-1 subtypes and recombinant forms: subtype B (41, 23%), subtype C (19, 11%), subtype G (76, 44%), and CRF02_AG (39, 22%). Overall, there was agreement between the assays in 95.43% of the samples. Both assays have a very good dynamic range [1.4–6.9] and [1.60–7.0] log10 copies/mL and excellent correlation in samples with various subtypes. Based on the fact that no clinically significant differences were observed in the viral load measurements by these two assays, HIV-1 subtypes are quantified equally by both assays. However due to HIV diversity, mainly in regions were non B subtypes are predominant more evaluations are needed, so we do not recommend to switch platform during longitudinal viral load monitoring.

Comparison of Ahlstrom Grade 226, Munktell TFN, and Whatman 903 Filter Papers for Dried Blood Spot Specimen Collection and Subsequent HIV-1 Load and Drug Resistance Genotyping Analysis

Dried blood spots (DBS) collected onto filter paper have eased the difficulty of blood collection in resource-limited settings. Currently, Whatman 903 (W-903) filter paper is the only filter paper that has been used for HIV load and HIV drug resistance (HIVDR) testing. We therefore evaluated two additional commercially available filter papers, Ahlstrom grade 226 (A-226) and Munktell TFN (M-TFN), for viral load (VL) testing and HIVDR genotyping using W-903 filter paper as a comparison group. DBS specimens were generated from 344 adult patients on antiretroviral therapy (ART) in Botswana. The VL was measured with NucliSENS EasyQ HIV-1 v2.0, and genotyping was performed for those specimens with a detectable VL (≥ 2.90 log(10) copies/ml) using an in-house method. Bland-Altman analysis revealed a strong concordance in quantitative VL analysis between W-903 and A-226 (bias = -0.034 ± 0.246 log(10) copies/ml [mean difference ± standard deviation]) and W-903 and M-TFN (bias = -0.028 ± 0.186 log(10) copies/ml) filter papers, while qualitative VL analysis for virological failure determination, defined as a VL of ≥ 3.00 log(10) copies/ml, showed low sensitivities for A-266 (71.54%) and M-TFN (65.71%) filter papers compared to W-903 filter paper. DBS collected on M-TFN filter paper had the highest genotyping efficiency (100%) compared to W-903 and A-226 filter papers (91.7%) and appeared more sensitive in detecting major HIVDR mutations. DBS collected on A-226 and M-TFN filter papers performed similarly to DBS collected on W-903 filter paper for quantitative VL analysis and HIVDR detection. Together, the encouraging genotyping results and the variability observed in determining virological failure from this small pilot study warrant further investigation of A-226 and M-TFN filter papers as specimen collection devices for HIVDR monitoring surveys.

Comparison between the automated Roche Cobas AmpliPrep/Cobas TaqMan HIV-1 test version 2.0 assay and its version 1 and Nuclisens HIV-1 EasyQ version 2.0 assays when measuring diverse HIV-1 genotypes in China

BackgroundSeveral commercially available HIV-1 viral load assays based on real-time detection technology and automated platforms are available. It is not clear how the diversity of HIV-1 genotypes impacts the ability to consistently detect HIV-1 viral loads. ObjectivesTo examine whether the diversity of HIV-1 genotypes impacts the ability of the Cobas AmpliPrep/Cobas TaqMan HIV-1 test version 2.0 (CAP/CTM v2.0), its version 1.0 (CAP/CTM v1.0) and the NucliSens EasyQ HIV-1 version 2.0 (EasyQ v2.0) assays to consistently determine the viral loads. Study designThe three assays were used to measure the viral load in 178 plasma samples with diverse genotypes from treatment-naive patients. ResultsCAP/CTM v2.0 showed significant correlation and high agreement with CAP/CTM v1.0 and EasyQ v2.0. CAP/CTM v2.0 showed excellent detection of clade B samples compared with CAP/CTM v1.0 and EasyQ v2.0. However, significant differences were observed when using CAP/CTM v2.0 to test clade BC and AE samples. The HIV-1 load measured by CAP/CTM v2.0 differed by >0.5logIU/ml in 59.52% and 72.62% of clade BC samples, and in 57.14% and 85.71% of clade AE samples, compared with CAP/CTM v1.0 and EasyQ v2.0, respectively. CAP/CTM v2.0 was more precise (13.18%) than EasyQ v2.0 (29.21%), and both assays showed good linearity (R≥0.9926). ConclusionsThe three assays may not deliver consistent results for samples belonging to clades BC and AE. It is strongly suggested that the version of the HIV-1 viral load assay used initially is also used at follow-up.

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.

Current challenges to viral load testing in the context of emerging genetic diversity of HIV-1

One of the major characteristics of HIV-1 is its extreme genetic diversity. A key factor in assessing the sensitivity of a molecular-based assay measuring HIV-1 RNA viral load (VL) in plasma is its ability to detect/quantify all (or most of) relevant HIV-1 genetic subtype/recombinant forms accurately. This review provides an overview of the current commercially available quantitative real-time assays (the Abbott RealTime HIV-1, Roche TaqMan HIV-1 versions 1.0 and 2.0, BioMérieux Nuclisens EasyQ HIV-1, Siemens VERSANT HIV-1 RNA 1.0 kinetic PCR, and Biocentric Generic HIV Viral Load assays). For each assay, studies from 2005 to 2010 assessing the impact of HIV-1 genetic diversity on the reliability of HIV-1 RNA quantification are described. In light of HIV-1 genetic diversity, a general recommendation to favor one test over the other cannot categorically be made. Larger field evaluations of HIV-1 RNA assays should be conducted in areas where HIV-1 genetic diversity is the highest. The large-scale implementation of HIV-1 VL testing is urgently required in the developing world to change HIV infection from a likely death sentence into a manageable chronic infection, as done in Northern countries.

High prevalence of HIV‐1 drug resistance among patients on first‐line antiretroviral treatment in Lomé, Togo

BackgroundWith widespread use of antiretroviral (ARV) drugs in Africa, one of the major potential challenges is the risk of emergence of ARV drug-resistant HIV strains. Our objective is to evaluate the virological failure and genotypic drug-resistance mutations in patients receiving first-line highly active antiretroviral therapy (HAART) in routine clinics that use the World Health Organization public health approach to monitor antiretroviral treatment (ART) in Togo.MethodsPatients on HAART for one year (10-14 months) were enrolled between April and October 2008 at three sites in Lomé, the capital city of Togo. Plasma viral load was measured with the NucliSENS EasyQ HIV-1 assay (Biomérieux, Lyon, France) and/or a Generic viral load assay (Biocentric, Bandol, France). Genotypic drug-resistance testing was performed with an inhouse assay on plasma samples from patients with viral loads of more than 1000 copies/ml. CD4 cell counts and demographic data were also obtained from medical records.ResultsA total of 188 patients receiving first-line antiretroviral treatment were enrolled, and 58 (30.8%) of them experienced virologic failure. Drug-resistance mutations were present in 46 patients, corresponding to 24.5% of all patients enrolled in the study. All 46 patients were resistant to non-nucleoside reverse-transcriptase inhibitors (NNRTIs): of these, 12 were resistant only to NNRTIs, 25 to NNRTIs and lamivudine/emtricitabine, and eight to all three drugs of their ARV regimes. Importantly, eight patients were already predicted to be resistant to etravirine, the new NNRTI, and three patients harboured the K65R mutation, inducing major resistance to tenofovir.ConclusionsIn Togo, efforts to provide access to ARV therapy for infected persons have increased since 2003, and scaling up of ART started in 2007. The high number of resistant strains observed in Togo shows clearly that the emergence of HIV drug resistance is of increasing concern in countries where ART is now widely used, and can compromise the long-term success of first- and second-line ART.

HIV-1 Load Comparison Using Four Commercial Real-Time Assays

The HIV-1 RNA viral load is commonly used for the monitoring of disease progression and antiretroviral treatment of HIV-1-infected patients. Since the misestimating of values could lead to inappropriate therapeutical management, the comparative performances, especially the ability to span the genetic diversity of HIV-1, of available automated real-time assays need to be evaluated. We conducted a prospective study with 74 consenting patients enrolled between March 2007 and November 2008. A blood sample was obtained at the time of diagnosis of HIV seropositivity and blindly tested for HIV-1 RNA by at least 4 commercial tests: the Abbott m2000 RealTime HIV-1, bioMérieux NucliSens EasyQ HIV-1, version 1.2 (v1.2), and Cobas AmpliPrep/Cobas TaqMan (CAP/CTM) v1.0 and v2.0 assays. The means of difference were null between CAP/CTM v2.0 and Abbott for CRF02_AG subtypes but positive in favor of CAP/CTM v2.0 for genotype B and negative in favor of NucliSens for all genotypes. The standard deviation (SD) of difference ranged from 0.3 to 0.59, depending on the considered couples of assays. Reliabilities of these four tests, appreciated by the standard deviation of difference between the measurement and the estimated "true" viral load and by the coefficient of reliability, were significantly different (P < 10(-4)) among each other. Significant differences were also observed within each group of HIV-1 genotype. The global disparity was higher for CRF02_AG than for B subtypes. This study indicates a risk of viral load misestimating or discrepancies between techniques, depending on the HIV-1 subtype, and speaks in favor of using the same assay for the monitoring of HIV-1-infected patients.

Performance of NucliSens HIV-1 EasyQ Version 2.0 Compared with Six Commercially Available Quantitative Nucleic Acid Assays for Detection of HIV-1 in China

Six HIV-1 viral load assays have been widely used in China. These include the Cobas Amplicor HIV-1 Monitor Version 1.5 ('Amplicor'), Cobas AmpliPrep/Cobas TaqMan HIV-1 test Version 1.0 ('CAP/CTM'), Versant HIV-1 RNA Version 3.0 (branched DNA [bDNA]-based assay; 'Versant bDNA'), Abbott RealTime HIV-1 assay ('Abbott RealTime'), NucliSens HIV-1 QT (nucleic acid sequence-based amplification assay; 'NucliSens NASBA'), and NucliSens EasyQ HIV-1 Version 1.1 ('EasyQ V1.1'). Recently, an updated version of EasyQ V1.1, NucliSens EasyQ HIV-1 Version 2.0 ('EasyQ V2.0') was introduced into China. It is important to evaluate the impact of HIV-1 genotypes on the updated assay compared with the other commercial available assays in China. A total of 175 plasma samples with different HIV-1 clades prevalent in China were collected from treatment-naïve patients. The viral loads of those samples were determined with the seven HIV-1 viral load assays, and the quantitative differences between them were evaluated. Overall, EasyQ V2.0 exhibited a significant correlation (R = 0.769-0.850, p ≤ 0.001) and high agreement (94.77-97.13%, using the Bland-Altman model) with the other six assays. Although no significant differences between EasyQ V2.0 and the other six assays were observed when quantifying clade B' samples, there were statistically significant differences between EasyQ V2.0 and the Amplicor, Versant bDNA, and Abbott RealTime assays when quantifying clade BC samples, and between EasyQ V2.0 and the Versant bDNA and Abbott RealTime assays when quantifying clade AE samples. For clade BC samples, the quantitative differences between EasyQ V2.0 and the Amplicor, Versant bDNA, and Abbott RealTime assays exceeded 0.5 log(10) IU/mL in approximately 50% of samples and exceeded 1 log(10) IU/mL in approximately 15% of samples. For clade AE samples, the quantitative differences between EasyQ V2.0 and the CAP/CTM, Versant bDNA, and Abbott RealTime assays exceeded 0.5 log(10) IU/mL in approximately 50% of samples, and the differences between EasyQ V2.0 and CAP/CTM exceeded 1 log(10) IU/mL in approximately 15% of samples. Genotypes may affect the quantification of HIV-1 RNA, especially in clade BC samples with respect to EasyQ V2.0 and the Amplicor, Versant bDNA, or Abbott RealTime assays, and in clade AE samples with respect to EasyQ V2.0 and the Versant bDNA or Abbott RealTime assays. It is therefore strongly suggested that, where possible, the HIV-1 viral load in infected patients be quantified at follow-up by the same version of the same assay that was used initially.

Quantification of human immunodeficiency Virus -1 Viral load using nucleic acid sequence-based amplification (NASBA) in North Central Nigeria

Viral load (VL) quantification is considered an integral part of the standard care in human immunodeficiency virus (HIV) infected individuals but in Nigeria as in most of sub-Saharan Africa, this has not reached the majority ofpatients. We report the first field application of the NucliSens EasyQ HIV-1 platform for the real time quantification of HIV-1 VL combining NASBA amplification and real time detection with molecular beacons among HIV-1 infected individuals in north central Nigeria where the predominant HIV-1 subtypes are CRF02_AG and G. CD4+ counts were enumerated using a fluorescence-activated cell sorter system. Of one hundred and forty nine (n=149) plasma sample from patients with mean age of 32 years and made up of 77 males and 72 females, fifty {n=50 (37.9%); 28 males and 22 females} had VLs below the lower detection limit (LDL=25 IU/ml) set by the assay while eighty-two {n=82 (62.1%); 39 males and 43 females} had VL levels above the LDL. Furthermore, 13 of 82 (15.9%) patients with viral loads above the LDL had VLs between 26-1000 IU/ml while 69 (84.1%) had VLs of 1001-2,400,000 IU/ml. 17 (11.4%) of the samples could not be analyzed due to poor viral amplification. Among individuals with both CD4+ and VL results (n=56), those with CD4+ of 1-418 cell/microl presented with higher VL usually above 45,000 IU/ml when compared with those with CD4+ of over 500 cell/microl. Our findings highlight the pattern, usefulness and feasibility ofVL quantification by NucliSens EasyQ in monitoring HIV-1 patients in Nigeria.

Reply to ‘Quantification of HIV-1 RNA on Dried Blood Spots’ AIDS 24:475–6

The authors thank Dr Johannessen for the thoughtful comments on our manuscript [1]. In light of growing evidence of the substantial shortcomings of clinical and immunologic monitoring of patients receiving antiretroviral therapy (ART) for informing decisions on switching regimens [2], we agree that practical and reliable approaches to earlier identification of virologic failure in resource-limited settings are urgently needed. A primary purpose of our study was to extend the promising results seen in laboratory-based studies of a dried blood spot method [3] using an assay that was already in service in our setting for liquid plasma-based HIV-1 RNA testing [4] to a field situation similar to that expected under program conditions in Tanzania. As pointed out by Dr Johannessen and described in our paper, at plasma HIV-1 RNA levels below approximately 3 log copies/ml, dried blood spot samples yielded higher HIV-1 RNA levels presumably related to amplification of pro-viral DNA. Accepting this limitation, we explored the performance of dried blood spot samples against liquid plasma samples at two threshold definitions for virologic failure: a conventional threshold of 400 copies/ml or more and at a threshold of 5000 copies/ml or more, a definition for virologic failure used by some national care and treatment programs [5]. Not surprisingly and as shown in Table 2 of our paper, although sensitivity for detection of virologic failure was similar at both thresholds, specificity was higher at a threshold of 5000 copies/ml or more (0.97) than at a threshold of 400 copies/ml or more (0.87). However, we would dispute the characterization of the dried blood spot HIV-1 RNA resulting in ‘a high number of false-positive results’ at either threshold when viewed in light of the shortcomings of clinical and immunologic monitoring [6] and we disagree that there are any substantial differences is the classification of virologic failure between dried blood spot HIV-1 RNA and plasma HIV-1 RNA at a threshold of 5000 copies/ml or more. Furthermore, it is notable that Dr Johannessen's own study from Tanzania that tested dried blood spots by nucleic acid sequenced-based amplification using the NucliSENS EasyQ HIV-1 assay (BioMérieux Inc., Durham, North Carolina, USA) showed a similar specificity for virologic failure at a plasma HIV-1 RNA threshold of 5000 copies/ml or more as the Abbott m2000 system (Abbott Laboratories, Abbott Park, Illinois, USA) did in our study [7]. Nonetheless, we applaud efforts to improve the performance of dried blood spot approaches to HIV-1 nucleic acid amplification for ART monitoring at lower HIV-1 RNA levels by refinement of existing assays and by evaluation of alternative assays. Acknowledgement This study was funded by Abbott Laboratories (Abbott Park, Illinois, USA). None of the authors was employed by Abbott Laboratories. S.M.L. and J.A.C. have received speaking honoraria from Abbott Laboratories and J.A.B. has received speaking honoraria and has served as a consultant for Abbott Laboratories.

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

Background HIV-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. Objectives Determine the performance of NucliSENS EasyQ HIV-1 v2.0 for DBS. Study design 100 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. Results Diagnostic 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. Conclusion Results 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.

Dried Blood Spots Perform Well in Viral Load Monitoring of Patients Who Receive Antiretroviral Treatment in Rural Tanzania

Monitoring of antiretroviral treatment (ART) with human immunodeficiency virus (HIV) viral loads, as recommended in industrialized countries, is rarely available in resource-limited settings because of the high costs and stringent requirements for storage and transport of plasma. Dried blood spots (DBS) can be an alternative to plasma, but the use of DBS has not been assessed under field conditions in rural Africa. The present study investigates the performance of DBS in HIV viral load monitoring of patients who received ART in rural Tanzania. From November 2007 through June 2008, parallel plasma and DBS specimens were obtained from patients who received ART at Haydom Lutheran Hospital in rural Tanzania. DBS specimens were stored at tropical room temperature for 3 weeks before testing with the NucliSENS EasyQ HIV-1 v1.2 assay. Results obtained with DBS were compared with results obtained with use of a gold-standard plasma assay. Ninety-eight plasma-DBS pairs were compared, and plasma viral loads ranged from <40 to >1,000,000 copies/mL. The correlation between plasma and DBS viral load was strong (R(2) = 0.75). The mean difference (+/- standard deviation) was 0.04 +/ 0.57 log(10) copies/mL, and only 8 samples showed >1 log(10) copies/mL difference. HIV type 1 RNA was detected in 7%, 60%, and 100% of DBS specimens with corresponding plasma viral loads of 40-999, 1000-2999, and 3000 copies/mL, respectively. DBS, in combination with the NucliSENS EasyQ HIV-1 v1.2 asay, performed well in monitoring HIV viral loads in patients who received ART in rural Tanzania, although the sensitivity was reduced when viral burden was low. The use of DBS can simplify virological monitoring in resource-limited settings.