Laboratory Evaluation of the Liat HIV Quant (IQuum) Whole-Blood and Plasma HIV-1 Viral Load Assays for Point-of-Care Testing in South Africa

Point-of-care (POC) HIV viral load (VL) testing offers the potential to reduce turnaround times for antiretroviral therapy monitoring, offer near-patient acute HIV diagnosis in adults, extend existing centralized VL services, screen women in labor, and prompt pediatrics to early treatment. The Liat HIV Quant plasma and whole-blood assays, prerelease version, were evaluated in South Africa. The precision, accuracy, linearity, and agreement of the Liat HIV Quant whole-blood and plasma assays were compared to those of reference technologies (Roche CAP CTMv2.0 and Abbott RealTime HIV-1) on an HIV verification plasma panel (n = 42) and HIV clinical specimens (n = 163). HIV Quant plasma assay showed good performance, with a 2.7% similarity coefficient of variation (CV) compared to the Abbott assay and a 1.8% similarity CV compared to the Roche test on the verification panel, and 100% specificity. HIV Quant plasma had substantial agreement (p_c [concordance correlation] = 0.96) with Roche on clinical specimens and increased variability (p_c = 0.73) in the range of <3.0 log copies/ml range with the HIV Quant whole-blood assay. HIV Quant plasma assay had good linearity (2.0 to 5.0 log copies/ml; R² = 0.99). Clinical sensitivity at a viral load of 1,000 copies/ml of the HIV Quant plasma and whole-blood assays compared to that of the Roche assay (n = 94) was 100% (confidence interval [CI], 95.3% to 100%). The specificity of HIV Quant plasma was 88.2% (CI, 63.6% to 98.5%), and that for whole blood was 41.2% (CI, 18.4% to 67.1%). No virological failure (downward misclassification) was missed. Liat HIV Quant plasma assay can be interchanged with existing VL technology in South Africa. Liat HIV Quant whole-blood assay would be advantageous for POC early infant diagnosis at birth and adult adherence monitoring and needs to be evaluated further in this clinical context. LIAT cartridges currently require cold storage, but the technology is user-friendly and robust. Clinical cost and implementation modeling is required.     

Comparison of a Transplant Multiplex Viral Panel on the ICEPlex System with Real-Time PCR for Detection of Cytomegalovirus,Epstein-Barr Virus,and BK Virus in Clinical Specimens

We compared a multiplex viral transplant panel on the ICEPlex system to real-time PCR for the detection of cytomegalovirus (CMV), Epstein-Barr virus (EBV), and BK virus (BKV). The sensitivities of the ICEPlex were 83.3%, 95.5%, and 65.5% for the detection of CMV, EBV, and BKV, respectively. Interestingly, the multiplex assay detected dual infections in 16/280 (5.7%) samples tested.     

Monitoring of Cytomegalovirus Viral Loads by Two Molecular Assays in Whole-Blood and Plasma Samples from Hematopoietic Stem Cell Transplant Recipients

Cytomegalovirus (CMV) viral loads in hematopoietic stem cell transplant (HSCT) recipients are typically monitored using quantitative molecular assays. The Roche Cobas AmpliPrep/Cobas TaqMan CMV test (Cobas CMV) has recently been cleared by the FDA for the monitoring of CMV viral loads in plasma samples from transplant patients. In this study, we compare and correlate the viral loads obtained by a laboratory-developed test (LC CMV) (using Roche analyte-specific reagents [ASR] on the LightCycler 2.0) on whole-blood specimens with those obtained on corresponding plasma and whole-blood specimens by the Cobas CMV assay. Testing was performed on 773 archived patient specimens. The strength of the agreement was good for the two assays performed on whole blood (κ = 0.6; 95% confidence interval [CI], 0.51 to 0.7) and moderate when the tests were performed on different sample types (κ = 0.54; 95% CI, 0.47 to 0.62 for the LC CMV whole blood [WB] assay versus Cobas plasma [PL], and κ = 0.57; 95% CI, 0.5 to 0.65 for the Cobas WB assay versus Cobas PL), although the difference was not statistically significant. Using a combination gold standard (i.e., a true positive was a specimen that was positive by two or more methods), the sensitivity and specificity of the assays were 78.8% and 99.3% for the LC CMV assay, 85.2% and 98.1% for the Cobas CMV WB assay, and 100% and 90.5% for Cobas CMV PL assay, respectively. A comparison of the CMV viral load trends in both plasma and whole blood from a few patients with multiple positive successive samples showed similar slopes, with differences in the slope ranging from 0.01 to 0.22. However, the absolute value for individual viral load differed markedly with whole-blood viral loads, being on average 0.5- to 1.22-log higher than those in plasma. The Cobas CMV assay provides a valid option for the monitoring of viral loads in transplant patients. Due to its increased sensitivity, the detection of CMV DNA in patients with low viral loads (i.e., those below limit of quantification [LOQ]) is increased with the Cobas CMV assay in plasma specimens. Longitudinal prospective studies will be needed to examine the clinical significance of these low-level viral loads.     

Field Evaluation of the InBios Chagas Detect Plus Rapid Test in Serum and Whole-Blood Specimens in Bolivia

Trypanosoma cruzi causes Chagas disease, which affects an estimated 7 million to 8 million people. Chagas disease is endemic throughout Latin America, with the highest prevalence in Bolivia. Conventional diagnosis requires a well-equipped laboratory with experienced personnel. We evaluated the Chagas Detect Plus (CDP) (InBios, Seattle, WA), a rapid immunochromatographic assay for IgG antibodies to T. cruzi. CDP performance was compared to infection status based on results obtained by indirect hemagglutination assay, immunofluorescent-antibody test, and enzyme-linked immunosorbent assay. Confirmed infection required positive results by at least 2 conventional assays. We used specimens from adults of both sexes in a general hospital in the city of Santa Cruz and from pregnant women in a hospital and children in villages in the Bolivian Chaco, an area of hyperendemicity. CDP was performed in paired whole-blood and serum specimens from 385 individuals in the two hospital studies and in 200 serum specimens from the community study. CDP showed sensitivities/specificities of 96.2% (95% confidence interval, 92.7 to 98.4)/98.8% (95.9 to 99.9) in whole blood and 99.3% (97.5 to 99.9)/96.9% (94.2 to 98.6) in serum, with no differences by sex, age group, or study site. CDP showed excellent sensitivity and specificity in our study population, comparable to those of conventional serology. The test is reliable for field surveys, requires no laboratory equipment, and performed well in serum and whole blood. The CDP could also be used for accurate maternal screening to identify neonates at risk of congenital transmission. CDP performance data in diverse geographic areas are needed to strengthen the evidence base for its use.     

Comparative Evaluation of a Commercially Available Automated System for Extraction of Viral DNA from Whole Blood: Application to Monitoring of Epstein-Barr Virus and Cytomegalovirus Load

The NucliSENS easyMAG automated system was compared to the column-based Qiagen method for Epstein-Barr virus (EBV) or cytomegalovirus (CMV) DNA extraction from whole blood before viral load determination using the corresponding R-gene amplification kits. Both extraction techniques exhibited a total agreement of 81.3% for EBV and 87.2% for CMV.Epstein-Barr virus (EBV) and cytomegalovirus (CMV) infections represent a significant clinical threat for immunocompromised patients. The frequent determination of EBV and CMV viral load permits the early diagnosis of infection, start of preemptive or curative therapy, and monitoring of treatment efficiency (5, 17, 20). By comparison to serum, plasma, or white-blood-cell fractions, whole-blood samples are now recognized as the most suitable sample for the determination of viral loads for EBV and CMV (3, 4, 7, 9, 10, 12, 13, 18, 19).Although the methods relying on silica columns are time-consuming and need trained experimenters, these methods are considered the gold standard for the extraction of nucleic acids from whole-blood samples. Due to the large amount of genetic material in such samples, new extraction methods must be carefully evaluated, including those relying on automated devices (1, 7, 10, 14, 15). The fully automated NucliSENS easyMAG instrument (bioMérieux) using magnetic silica particles (2) allows the simultaneous process of up to 24 extractions. The use of magnetic particles eliminates the several centrifugation steps that could be a source of cross-contamination, and manual steps are limited to the loading of samples, reagents, and disposables. The performance of this method in the extraction of DNA from whole-blood samples prior to viral quantification has not been yet evaluated. The present study was undertaken to answer this question in the clinical context of EBV or CMV infection.The whole-blood specimens selected for this study included 80 samples for EBV analysis and 94 samples for CMV analysis, taken from patients hospitalized at the University Hospital of Saint-Etienne, Saint-Etienne, France, from December 2007 to September 2008. The samples were kept frozen at −20°C. After the samples were thawed, whole-blood aliquots were tested and kept at 4°C for up to 24 h for potential retest. Two hundred microliters of each selected sample was extracted by two different technicians either by the reference manual method, i.e., QIAamp column DNA blood extraction kit according to the manufacturer''s recommendations (Qiagen), or by the new specific B protocol on the NucliSENS easyMAG instrument. The latter protocol consists of the treatment of 200 μl of whole blood in 2 ml of lysis buffer and the capture of nucleic acids by 140 μl of magnetic silica. After incubation and washing procedures, nucleic acids were recovered in 50 μl of elution buffer. EBV and CMV loads were quantified by using the respective R-gene amplification kit (Argene Biosoft) according to the manufacturer''s recommendations. Both amplification kits have been previously validated for quantification of EBV and CMV load in whole blood (8, 11). DNA extracts from both methods were amplified in the same run using an ABI 7500 instrument (Applied Biosystems).     

Monitoring the Quality of HIV-1 Viral Load Testing through a Proficiency Testing Program Using Dried Tube Specimens in Resource-Limited Settings

HIV-1 viral load (VL) levels are used for monitoring disease progression and antiretroviral therapy outcomes in HIV-infected patients. To assess the performance of laboratories conducting HIV-1 VL testing in resource-limited settings, the U.S. Centers for Disease Control and Prevention implemented a voluntary, free-of-charge, external quality assurance program using dried tube specimens (DTSs). Between 2010 and 2012, DTS proficiency testing (PT) panels consisting of 5 specimens were distributed at ambient temperature to participants. The results from the participants (n ≥ 6) using the same assay were grouped, analyzed, and graded as acceptable within a group mean ± 3 standard deviations. Mean proficiency scores were calculated by dividing the combined PT scores by the number of testing cycles using a linear regression model. Between 2010 and 2012, the number of participants enrolled increased from 32 in 16 countries to 114 in 44 countries. A total of 78.2% of the participants reported results using 10 different VL assays. The rates of reporting of acceptable results by the participants were 96.6% for the Abbott assay, 96.3% for the Roche Cobas assay, 94.5% for the Roche Amplicor assay, 93.0% for the Biocentric assay, and 89.3% for the NucliSens assay. The overall mean proficiency scores improved over time (P = 0.024). DTSs are a good alternative specimen type to plasma specimens for VL PT programs, as they do not require cold chain transportation and can be used on PCR-based assays. Our data suggest that the CDC HIV-1 VL PT program using DTSs positively impacts the testing performance of the participants, which might translate into better and more accurate VL testing services for patients.     

Evaluation of Effect of Specimen-Handling Parameters for Plasma Preparation Tubes on Viral Load Measurements Obtained by Using the Abbott RealTime HIV-1 Load Assay

HIV-1 viral load testing is essential to the management of HIV-1-infected patients, and proper specimen handling ensures accurate viral load (VL) results. This study was performed to (i) evaluate the effect of freezing plasma in situ in BD Vacutainer plasma preparation tubes (PPT) on the accuracy of HIV-1 viral load results using the Abbott RealTime HIV-1 assay and (ii) evaluate the effect of whole-blood storage in the PPT for 6 h at room temperature prior to centrifugation (PPT6H) rather than 2 h as specified in the PPT product insert. Of the 64 HIV-positive subjects evaluated, 29 had average viral load counts of >40 copies/ml in at least one of the tubes tested and 35 subjects had a result of either “undetected target” or “below the limit of quantification” (LOQ) for all or some of the tubes regardless of handling condition. For the 29 subjects with VLs that were >LOQ, the mean biases between plasma from Vacutainer K₂EDTA tubes and plasma frozen in situ in PPT and between K₂EDTA tube plasma and plasma from PPT6H (log₁₀ copies/ml) were 0.005 and −0.001, respectively, and r² was >0.92 for all correlations. We conclude that VLs determined from plasma frozen in situ in PPT are equivalent to VLs in K₂EDTA tube plasma and can be used for accurate quantification of HIV-1 RNA in the Abbott RealTime HIV-1 assay. Furthermore specimens collected in PPT can be stored for 6 h at room temperature with no effect on viral load results as measured by the Abbott RealTime HIV-1 assay.Accurate quantification of human immunodeficiency virus type 1 (HIV-1), also referred to as HIV-1 viral load (VL) testing, is essential for effective management of HIV-1-infected patients. The BD Vacutainer plasma preparation tube (PPT) (BD Preanalytical Systems, Franklin Lakes, NJ) was developed to facilitate the handling of plasma specimens used for HIV-1 viral load testing. The PPT contains a K₂EDTA additive and a polyester gel which, upon centrifugation, forms a barrier that separates blood cells from plasma, allowing storage, freezing, and shipment of the plasma specimen in situ in PPT. Use of PPT also results in a decrease in the amount of labor required to process specimens, elimination of a potential source of error in specimen labeling, and reduction in the risk of HIV exposure associated with the transfer of separated plasma to secondary tubes for shipping.Earlier studies using PPT for specimen collection demonstrated compatibility of this tube with HIV-1 VL tests (1, 4, 5). Over the past few years, however, investigators have reported that freezing plasma in situ in a PPT produced higher HIV-1 viral load results compared to plasma from K₂EDTA tubes when tested in the Cobas Amplicor HIV-1 monitor system (Roche Molecular Diagnostics, Pleasanton, CA) (2, 3, 11). Elevated levels of HIV-1 VL from PPT frozen in situ after centrifugation were first reported by Squires et al., who showed that PPT yielded higher HIV RNA levels than K₂EDTA tubes. The disparity in quantification, seen in both standard and ultrasensitive assays, was more apparent in specimens with VLs between the limit of quantification (LOQ), 50 copies/ml, and 1,000 copies/ml and more pronounced in specimens close to or below 50 copies/ml (3, 10, 11). In fact, studies that compared plasma aspirated from K₂EDTA tubes with plasma frozen in situ in PPT show that VLs that are clearly below the LOQ in the former are quantifiable in the latter (3, 8, 11). Such discrepant results for plasma collected in different tubes from the same subject could be interpreted as virological failure, and these results, therefore, have therapeutic implications.Additional studies reported that specimens which were aspirated and transferred to another tube after centrifugation did not show patterns of artificially increased viral load (8, 10). Similarly, recentrifugation of specimens transported in PPT at 4°C eliminated the inaccurate quantification of HIV seen in plasmas frozen in situ in PPT (6, 9).Such observations led investigators to associate elevated viral loads in plasma in PPT with the presence of cell-associated nucleic acids released from cells trapped within or adhered to the surface of the gel barrier in PPT (6). Depletion of cellular material by recentrifugation of separated, unfrozen plasma from PPT resulted in lower VLs, indicating that cell-associated nucleic acid contributed to the elevation in VL. This finding was compatible with an earlier study which had shown that the HIV proviral DNA associated with cellular (genomic) DNA was in part responsible for the increase in viral load (13).The aim of this study was to evaluate the performance of PPT with the new automated RealTime HIV-1 assay (Abbott Molecular, Des Plaines, IL) for the quantification of HIV-1 viral load using the Abbott m2000sp and m2000rt systems for sample processing and amplification/detection, respectively. Specifically, we were interested in determining the effect of freezing plasma in situ in PPT on HIV-1 viral load analyzed using the Abbott RealTime HIV-1 assay. In addition, the study evaluated the effect on viral load of whole-blood storage in PPT for 6 h before centrifugation rather than 2 h, as specified in the PPT product insert.     

Sequence Conservation of the Region Targeted by the Abbott RealTime HBV Viral Load Assay in Clinical Specimens

The Abbott RealTime HBV assay targets the N-terminal region of the S gene. Here we analyzed the sequence variability of the assay target region from >2,100 clinical specimens. Thermodynamic modeling of the percentage of bound primer/probe at the assay annealing temperature was performed to assess the potential effect of sequence variability.     

Evaluation of Quantification of HIV-1 RNA Viral Load in Plasma and Dried Blood Spots by Use of the Semiautomated Cobas Amplicor Assay and the Fully Automated Cobas Ampliprep/TaqMan Assay,Version 2.0, in Kisumu,Kenya

In Kenya, HIV-1 viral load monitoring is commonly performed with the Cobas Amplicor using plasma specimens. Interest is growing in transitioning to real-time PCR (RT-PCR), such as the Cobas Ampliprep/Cobas TaqMan (CAP/CTM), using dried blood spots (DBS). Before implementation, direct evaluation of the two assays using DBS field specimens is required. This study compares the sensitivity, specificity, negative and positive predictive values (NPV and PPV, respectively), concordance, and agreement between HIV-1 viral load measurements using plasma and DBS specimens obtained from 512 HIV-1-infected pregnant females enrolled in the Kisumu Breastfeeding Study and tested with the Cobas Amplicor and CAP/CTM assays. The sensitivity and NPV of viral load detection in DBS specimens were higher with CAP/CTM (sensitivity, 100%; 95% confidence interval [CI], 99.1 to 100.0%; NPV, 100%; 95% CI, 59.0 to 100.0%) than the Cobas Amplicor (sensitivity, 96.6%; 95% CI, 94.3 to 98.1%; NPV, 58.8%; 95% CI, 40.7 to 75.4%). The PPVs were comparable between both assays when using DBS. The specificity of viral load detection in DBS specimens was lower with CAP/CTM (77.8%; 95% CI, 40.0 to 97.2%) than that of the Cobas Amplicor (95.2%; 95% CI, 76.2 to 99.9%). Good concordance and agreement were observed when paired plasma and DBS specimens were tested with both assays. Lower specificity with the CAP/CTM is likely due to proviral HIV-1 DNA amplification and lower detection limits with RT-PCR. However, the CAP/CTM has better sensitivity and higher throughput than the Cobas Amplicor. These findings suggest that DBS may be a suitable alternative to plasma when using RT-PCR, which could increase access to viral load monitoring in resource-limited settings.     

Evaluation of the Qiagen artus C. difficile QS-RGQ Kit for Detection of Clostridium difficile Toxins A and B in Clinical Stool Specimens

We compared the Qiagen artus C. difficile QS-RGQ kit, a new nucleic acid amplification test for the detection of Clostridium difficile toxins in stool specimens, with the Cepheid Xpert C. difficile test. The sensitivity, specificity, positive predictive value, and negative predictive value for the Qiagen artus C. difficile QS-RGQ test were 100%, 89.5%, 60.9%, and 100%, and those for the Cepheid Xpert C. difficile test were 100%, 90%, 62.2%, and 100%, respectively.     

Sequence Variation in Amplification Target Genes and Standards Influences Interlaboratory Comparison of BK Virus DNA Load Measurement

International guidelines define a BK virus (BKV) load of ≥4 log₁₀ copies/ml as presumptive of BKV-associated nephropathy (BKVN) and a cutoff for therapeutic intervention. To investigate whether BKV DNA loads (BKVL) are comparable between laboratories, 2 panels of 15 and 8 clinical specimens (urine, whole blood, and plasma) harboring different BKV genotypes were distributed to 20 and 27 French hospital centers in 2013 and 2014, respectively. Although 68% of the reported results fell within the acceptable range of the expected result ±0.5 log₁₀, the interlaboratory variation ranged from 1.32 to 5.55 log₁₀. Polymorphisms specific to BKV genotypes II and IV, namely, the number and position of mutations in amplification target genes and/or deletion in standards, arose as major sources of interlaboratory disagreements. The diversity of DNA purification methods also contributed to the interlaboratory variability, in particular for urine samples. Our data strongly suggest that (i) commercial external quality controls for BKVL assessment should include all major BKV genotypes to allow a correct evaluation of BKV assays, and (ii) the BKV sequence of commercial standards should be provided to users to verify the absence of mismatches with the primers and probes of their BKV assays. Finally, the optimization of primer and probe design and standardization of DNA extraction methods may substantially decrease interlaboratory variability and allow interinstitutional studies to define a universal cutoff for presumptive BKVN and, ultimately, ensure adequate patient care.