Automated Multireplicate Quantification of Persistent HIV-1 Viremia in Individuals on Antiretroviral Therapy.

Clearance of low-level viremia that persists in most HIV-1-positive individuals on antiretroviral therapy (ART) is an important milestone for efforts to cure HIV-1 infection. The level of persistent viremia on ART is generally below the lower limit of quantification (LOQ) of current FDA-cleared plasma HIV-1 RNA assays (20 to 40 copies/ml) but can be quantified by reverse transcriptase PCR (RT-PCR) assays with single-copy sensitivity. Such assays require multistep manual methods, and their low throughput limits the capacity to monitor the effects of interventions on persistent viremia. Recently, S. Bakkour, X. Deng, P. Bacchetti, E. Grebe, et al. (J Clin Microbiol 58:e01400-20, 2020, https://doi.org/10.1128/JCM.01400-20), reported the use of multiple replicates and Poisson statistics to infer HIV-1 RNA concentrations below the commercial LOQ of an automated platform (Hologic Panther Aptima). Here, we evaluate the detection and quantitation of low-level viremia using the following two adaptions of the automated platform: a multireplicate strategy (9×) and a concentrated single-replicate strategy in which 5 ml of plasma is concentrated by centrifugation (1×, concentrated). We compare these new methods to a recently reported manual integrase-targeting single-copy assay version 2 (iSCA v2). Using laboratory-generated HIV-1 RNA plasma samples at known concentrations, all three methods had similar sensitivity for HIV-1 RNA detection, although iSCA v2 was most sensitive (95% LOD, 2.3 copies/ml), 9× was marginally less sensitive (95% LOD, 3.0 copies/ml), and 1×, concentrated was least sensitive (95% LOD, 3.9 copies/ml). In contrast, for clinical plasma samples, 9× had greater sensitivity than iSCA v2 (82% of samples were quantifiable compared with 62% of samples by iSCA v2). These results support 9× as an acceptable high-throughput alternative to iSCA v2 for quantifying low-level viremia in individuals on ART.

Replicate Aptima Assay for Quantifying Residual Plasma Viremia in Individuals on Antiretroviral Therapy.

Detection of residual plasma viremia in antiretroviral therapy (ART)-suppressed HIV-infected individuals is critical for characterizing the latent reservoir and evaluating the impact of cure interventions. Ultracentrifugation-based single-copy assays are sensitive but labor intensive. Fully automated replicate testing using a standard clinical viral load assay was evaluated as a high-throughput alternative for the quantification of low-level viremia. Four plasma samples from blood donors with acute HIV-1 infection and one viral culture supernatant were serially diluted into 25-ml samples to nominal viral loads ranging from 39 to <0.5 copies (cp)/ml. Each dilution was tested with 45 replicates (reps) using 0.5 ml/rep with the Aptima HIV-1 Quant assay. The nominal and estimated viral loads based on the single-hit Poisson model were compared, and a hybrid Poisson digital model for calibrated viral load estimation was derived. Testing performed using 45 reps on longitudinal plasma samples from 50 ART-suppressed individuals in the Reservoir Assay Validation and Evaluation Network (RAVEN) study cohort (range of 1 to 19 years of continuous ART suppression) showed a median viral load of 0.54 cp/ml (interquartile range [IQR], 0.22 to 1.46 cp/ml) and a 14% (95% confidence interval [CI], 9% to 19%) decline in viral load for each additional year in duration suppressed. Within the RAVEN cohort, the expected false-negative rate for detection at lower rep numbers using 9 and 18 reps was 26% and 14%, respectively. Residual plasma viremia levels positively correlated with cell-associated HIV RNA and DNA. The performance characteristics of the replicate Aptima assay support its use for quantifying residual plasma viremia to study the latent HIV reservoir and cure interventions.

Performance Characteristics of a High-Throughput Automated Transcription-Mediated Amplification Test for SARS-CoV-2 Detection.

The COVID-19 pandemic caused by the new SARS-CoV-2 coronavirus has imposed severe challenges on laboratories in their effort to achieve sufficient diagnostic testing capability for identifying infected individuals. In this study, we report the analytical and clinical performance characteristics of a new, high-throughput, fully automated nucleic acid amplification test system for the detection of SARS-CoV-2. The assay utilizes target capture, transcription-mediated amplification, and acridinium ester-labeled probe chemistry on the automated Panther system to directly amplify and detect two separate target sequences in the open reading frame 1ab (ORF1ab) region of the SARS-CoV-2 RNA genome. The probit 95% limit of detection of the assay was determined to be 0.004 50% tissue culture infective dose (TCID50)/ml using inactivated virus and 25 copies/ml (c/ml) using synthetic in vitro transcript RNA targets. Analytical sensitivity (100% detection) was confirmed to be 83 to 194 c/ml using three commercially available SARS-CoV-2 nucleic acid controls. No cross-reactivity or interference was observed with testing of six related human coronaviruses, as well as 24 other viral, fungal, and bacterial pathogens, at high titers. Clinical nasopharyngeal swab specimen testing (n = 140) showed 100%, 98.7%, and 99.3% positive, negative, and overall agreement, respectively, with a validated reverse transcription-PCR nucleic acid amplification test (NAAT) for SARS-CoV-2 RNA. These results provide validation evidence for a sensitive and specific method for pandemic-scale automated molecular diagnostic testing for SARS-CoV-2.

Evaluation of the Aptima HIV-1 Quant Dx Assay for HIV-1 RNA Quantitation in Different Biological Specimen Types.

The search for a cure for HIV infection has highlighted the need for increasingly sensitive and precise assays to measure viral burden in various tissues and body fluids. We describe the application of a standardized assay for HIV-1 RNA in multiple specimen types. The fully automated Aptima HIV-1 Quant Dx assay (Aptima assay) is FDA cleared for blood plasma HIV-1 RNA quantitation. In this study, the Aptima assay was applied for the quantitation of HIV RNA in peripheral blood mononuclear cells (PBMCs; n = 72), seminal plasma (n = 20), cerebrospinal fluid (CSF; n = 36), dried blood spots (DBS; n = 104), and dried plasma spots (DPS; n = 104). The Aptima assay was equivalent to or better than commercial assays or validated in-house assays for the quantitation of HIV RNA in CSF and seminal plasma. For PBMC specimens, the sensitivity of the Aptima assay in the detection of HIV RNA decayed as background uninfected PBMC counts increased; proteinase K treatment demonstrated some benefit in restoring signal at higher levels of background PBMCs. Finally, the Aptima assay yielded 100% detection rates of DBS in participants with plasma HIV RNA levels of ≥35 copies/ml and 100% detection rates of DPS in participants with plasma HIV RNA levels of ≥394 copies/ml. The Aptima assay can be applied to a variety of specimens from HIV-infected subjects to measure HIV RNA for studies of viral persistence and cure strategies. It can also detect HIV in dried blood and plasma specimens, which may be of benefit in resource-limited settings.

Evaluation of Hologic Aptima HIV-1 Quant Dx Assay on the Panther System on HIV Subtypes.

Quantitation of the HIV-1 viral load in plasma is the current standard of care for clinical monitoring of HIV-infected individuals undergoing antiretroviral therapy. This study evaluated the analytical and clinical performances of the Aptima HIV-1 Quant Dx assay (Hologic, San Diego, CA) for monitoring viral load by using 277 well-characterized subtype samples, including 171 cultured virus isolates and 106 plasma samples from 35 countries, representing all major HIV subtypes, recombinants, and circulating recombinant forms (CRFs) currently in circulation worldwide. Linearity of the Aptima assay was tested on each of 6 major HIV-1 subtypes (A, B, C, D, CRF01_AE, and CRF02_AG) and demonstrated an R(2) value of ≥0.996. The performance of the Aptima assay was also compared to those of the Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 v.2 (CAP/CTM) and Abbott m2000 RealTime HIV-1 (RealTime) assays on all subtype samples. The Aptima assay values averaged 0.21 log higher than the CAP/CTM values and 0.30 log higher than the RealTime values, and the values were >0.4 log higher than CAP/CTM values for subtypes F and G and than RealTime values for subtypes C, F, and G and CRF02_AG. Two samples demonstrated results with >1-log differences from RealTime results. When the data were adjusted by the average difference, 94.9% and 87.0% of Aptima results fell within 0.5 log of the CAP/CTM and RealTime results, respectively. The linearity and accuracy of the Aptima assay in correctly quantitating all major HIV-1 subtypes, coupled with the completely automated format and high throughput of the Panther system, make this system well suited for reliable measurement of viral load in the clinical laboratory.

Analytical characteristics and comparative evaluation of Aptima HIV-1 Quant Dx assay with Ampliprep/COBAS TaqMan HIV-1 test v2.0.

BACKGROUND: Quantitation of HIV-RNA is critically important for diagnosis, prognosis, treatment, monitoring and assessment of infectivity in HIV-1 infection. The objective of this study was to assess performance characteristics of the Aptima HIV-1 Quant Dx assay (Aptima), a new transcription mediated amplification (TMA), fully integrated and automated assay from Hologic Inc., San Diego, CA, USA. The analytical sensitivity, analytical specificity, precision and detection of HIV-1 subtypes were tested based on commercially available international standards or panels. A selected group of 244 anti-HIV-1 (+) plasma samples was used for comparison with Roche COBAS Ampliprep/COBAS TaqMan HIV- 1 test v2.0 (Roche CAP/CTM), (Roche Molecular Systems, Pleasanton, CA). RESULTS: The 50 and 95 % limit of detection were estimated at 4.9 (95 % CI 3.9-5.7) and 17.6 (15.2-21.2) IU/mL respectively. The specificity was found 99.83 (99.06-99.97) %. The standard deviations and coefficient of variations for panels with 50 and 100 copies/mL (1.7 and 2 log copies/mL) were 0.14 log copies/mL (8.67 %CV) and 0.18 log copies/mL (9.91 %CV) respectively. The detection rate for Aptima and Roche assays was 220/244 (90.2 %) and 217/244 (88.9 %) respectively. CONCLUSION: The Aptima assay is a sensitive, specific, precise and accurate test for measuring HIV-1 viral loads and for the detection of HIV-1 infections.

Prospective evaluation of accuracy of HIV viral load monitoring using the Aptima HIV Quant Dx assay with fingerstick and venous dried blood spots prepared under field conditions in Kenya.

Quantification of HIV-1 RNA is essential for clinical management of HIV patients. The limited throughput and significant hands-on time required by most HIV Viral load (VL) tests makes it challenging for laboratories with high test volume, to turn around patient results quickly. The Hologic Aptima HIV-1 Quant Dx Assay (Aptima), has the potential to alleviate this burden as it is high throughput and fully automated. This assay is validated for both plasma and dried blood spots (DBS), which are commonly used in resource limited settings. The objective of this study was to compare the performance of Aptima to Abbott RealTime HIV-1 Assay (Abbott RT), which was used as reference. This was a cross-sectional prospective study where HIV VL in finger stick (FS) DBS, venous blood (VB) DBS and plasma, collected from 258 consenting adults visiting 5 medical facilities in Kenya, Africa were tested in Aptima. The results were compared to plasma VL in Abbott RT at the medical decision point (MDP) of 1000 copies/mL and across Aptima assay range. The total agreement at MDP between plasma HIV VL in Abbott RT and plasma, FS and VB DBS tested in Aptima were 97.7%, 92.2% and 95.3% respectively with kappa statistic of 0.95, 0.84 and 0.90. The positive and negative agreement for all 3 sample types were >92%. Regression analysis between VL in Abbott RT plasma and various sample types tested in Aptima had a Pearson's correlation coefficient ≥0.91 with systematic bias of < 0.20 log copies/mL on Bland-Altman analysis. The high level of agreement in Aptima HIV VL results for all 3 sample types with Abbott RT plasma VL along with the high throughput, complete automation, and ease of use of the Panther platform makes Aptima a good option for HIV VL monitoring for busy laboratories with high volume of testing.

Performance evaluation of the Aptima HIV-1 Quant Dx assay for detection of HIV in infants in Kenya.

BACKGROUND: Early infant diagnosis (EID) of HIV-1 exposed infants enables timely initiation of antiretroviral therapy (ART), thereby allowing early diagnosis and treatment to slow disease progression and reduce mortality. Turn-around time to results, partially caused by low to medium throughput technology, remains a hindrance to early treatment. A major solution to this challenge is to incorporate high throughput and accurate technologies in the testing process. The Hologic Aptima Quant Dx Assay (Aptima) is a CE marked Real-Time TMA assay running on the high throughput Panther system. OBJECTIVES: The objective of this study was to evaluate the performance of Aptima for EID using dried blood spots. STUDY DESIGN: This was a cross-sectional prospective study of 2,048 infants seeking HIV services from health facilities in Western Kenya, Africa. Capillary Dried Blood Spot samples DBS were collected from infants with the consent of their mothers. The qualitative performance of Aptima was compared with the Roche COBAS Ampliprep/ COBAS Taqman HIV-1 Qualitative Test v2.0 (CAP/CTM), using these DBS. Demographic information of the participants was also collected. RESULTS: A total of 1,975 successful comparisons between the two platforms were included in the analysis. The overall agreement between the assays was 99.65 %. The sensitivity and specificity of Aptima was 95.24 % (95 % CI 88.40-98.19 %) and 99.84 % (95 % CI 99.49-99.92 %) respectively. CONCLUSIONS: Aptima assay has performance characteristics that are comparable to those of the Roche CAP/CTM for qualitative testing on DBS taken from infants. The two assays can therefore be used interchangeably for Early Infant Diagnosis of HIV.

Aptima HIV-1 Quant Dx--A fully automated assay for both diagnosis and quantification of HIV-1.

BACKGROUND: Separate assays are available for diagnosis and viral load (VL) monitoring of HIV-1. Studies have shown that using a single test for both confirmatory diagnosis and VL increases linkage to care. OBJECTIVE: To validate a single assay for both diagnosis and VL monitoring of HIV-1 on the fully automated Panther platform. STUDY DESIGN: Validate the assay by assessing specificity, sensitivity, subtype detection, seroconversion, reproducibility and linearity. Also assess diagnostic agreement with the Procleix(®) Ultrio Elite™ discriminatory assay (Procleix), and agreement of VL results (method comparison) with Ampliprep/COBAS TaqMan HIV-1 version 2.0 (CAP/CTM), using clinical samples. RESULTS: The assay was specific (100%) and sensitive with a 95% limit of detection of 12 copies/mL with the 3rd WHO standards. Aptima detected HIV in seroconversion panels 6 and 11 days before p24 antigen and antibody tests, respectively. Diagnostic agreement with Procleix, was 100%. Regression analysis showed good agreement of VL results between Aptima and CAP/CTM with a slope of 1.02, intercept of 0.07, and correlation coefficient (R(2)) of 0.97. Aptima was more sensitive than CAP/CTM. Equivalent quantification was seen on testing clinical samples and isolates belonging to HIV group M, N, O and P and commercially available subtype panels. Assay results were linear (R(2) 0.9994) with standard deviation of <0.17 log copies across assay range. CONCLUSIONS: The good specificity, sensitivity, precision, subtype performance and clinical agreement with other assays demonstrated by Aptima combined with the complete automation provided by the Panther platform makes Aptima a good candidate for both VL monitoring and diagnosis of HIV-1.

Systemic Cytokine Levels Do Not Predict CD4(+) T-Cell Recovery After Suppressive Combination Antiretroviral Therapy in Chronic Human Immunodeficiency Virus Infection.

Background. Subjects on suppressive combination antiretroviral therapy (cART) who do not achieve robust reconstitution of CD4(+) T cells face higher risk of complications and death. We studied participants in the Women's Interagency HIV Study with good (immunological responder [IR]) or poor (immunological nonresponder [INR]) CD4(+) T-cell recovery after suppressive cART (n = 50 per group) to determine whether cytokine levels or low-level viral load correlated with INR status. Methods. A baseline sample prior to viral control and 2 subsequent samples 1 and 2 years after viral control were tested. Serum levels of 30 cytokines were measured at each time point, and low-level human immunodeficiency virus (HIV) viral load and anti-HIV antibody levels were measured 2 years after viral suppression. Results. There were minimal differences in cytokine levels between IR and INR subjects. At baseline, macrophage inflammatory protein-3ß levels were higher in IR subjects; after 1 year of suppressive cART, soluble vascular endothelial growth factor-R3 levels were higher in IR subjects; and after 2 years of suppressive cART, interferon gamma-induced protein 10 levels were higher in INR subjects. Very low-level HIV viral load and anti-HIV antibody levels did not differ between IR and INR subjects. Conclusions. These results imply that targeting residual viral replication might not be the optimum therapeutic approach for INR subjects.

Whole genome amplification of DNA from laser capture-microdissected tissue for high-throughput single nucleotide polymorphism and short tandem repeat genotyping.

Genome-wide screening of genetic alterations between normal and cancer cells, as well as among subgroups of tumors, is important for establishing molecular mechanism and classification of cancer. Gene silencing through loss of heterozygosity is widely observed in cancer cells and detectable by analyzing allelic loss of single nucleotide polymorphism and/or short tandem repeat markers. To use minute quantities of DNA that are available through laser capture microdissection (LCM) of cancer cells, a whole genome amplification method that maintains locus and allele balance is essential. We have successfully used a ø29 polymerase-based isothermal whole genome amplification method to amplify LCM DNA using a proteinase K lysis procedure coupled with a pooling strategy. Through single nucleotide polymorphism and short tandem repeat genotype analysis we demonstrate that using pooled DNA from two or three separate amplification reactions significantly reduces any allele bias introduced during amplification. This strategy is especially effective when using small quantities of source DNA. Although a convenient alkaline lysis DNA extraction procedure provided satisfactory results from using 1500 to 3000 LCM cells, proteinase K digestion was superior for lower cell numbers. Accurate genotyping is achieved with as few as 100 cells when both proteinase K extraction and pooling are applied.