Clinical evaluation of a fully automated CMV PCR assay

BackgroundThere is a growing need for sensitive high-throughput cytomegalovirus (CMV) PCR tests due to the increasing number of immunocompromised patients requiring monitoring for active CMV infection.ObjectivesTo compare the fully automated COBAS^® AmpliPrep/COBAS^® TaqMan^® (CAP/CTM) CMV test (this test is currently under development and not commercially available) for EDTA–plasma to the reference method COBAS^® AMPLICOR CMV MONITOR.Study designA prospective feasibility study with parallel analysis of 433 EDTA–plasma samples from 277 patients on both systems was carried out after the analytical performance of the new system had been assessed.ResultsThe new system has a wide linear range from 2.0 to 7.3 log₁₀ CMV-DNA copies/ml EDTA–plasma and a detection limit of 46 copies/ml with excellent accuracy and precision. When testing clinical samples, the CAP/CTM CMV test compared extremely well with the COBAS^® AMPLICOR CMV MONITOR (R² = 0.93, p < 0.001) with increased sensitivity and linear range. Discrepant samples all contained low titers of CMV-DNA. In two of the study patients, CMV-DNAemia was detected by the CAP/CTM CMV test up to eight weeks earlier than by COBAS^® AMPLICOR CMV MONITOR.ConclusionAn IVD/CE marked version of the CAP/CTM CMV test will enable laboratories to provide a sensitive, fully automated high-throughput CMV PCR.     

Evaluation of the COBAS® AmpliPrep/COBAS® TaqMan® HCV Test,v2.0 and comparison to assays used in routine clinical practice in an international multicenter clinical trial: The ExPECT study

BackgroundThe COBAS^® AmpliPrep^®/COBAS^® TaqMan^® HCV Test, v2.0 (CAP/CTM2) is used for HCV RNA viral load monitoring.ObjectivesThe performance of the CAP/CTM2 was compared to other widely used tests, including a manual version of the assay (the COBAS^® TaqMan^® HCV Test, v2.0 for use with the High Pure System, HPS/CTM2) predominantly used during phase III clinical trials for the new direct acting antiviral therapies.Study designLow HCV RNA level comparisons were performed across tests (Abbott Realtime HCV Test, ART; COBAS^® AmpliPrep^®/COBAS^® TaqMan^® HCV Test, v1.0, CAP/CTM1; CAP/CTM2; and HPS/CTM2) using dilutions of the 2nd HCV WHO International Standard. Additionally, the clinical performance of the CAP/CTM2 was evaluated with 421 leftover HCV RNA-positive routine clinical samples.ResultsAll quantifiable WHO dilutions were within ±0.3 log₁₀ IU/mL of the expected results across tests and the analytical sensitivity resulted in a limit of detection of 12 IU/mL (95% confidence interval, 10, 15). When clinical samples were tested the results for 87% (367 of 421) of all sample comparisons were within ±0.5 log₁₀ IU/mL. When low viral load results (25–3500 IU/mL) were compared, values obtained by the ART assay were significantly lower (p < 0.0001) than those obtained with the CAP/CTM2.ConclusionsThe new CAP/CTM2 showed good accuracy with comparable sensitivity to comparator assays. The new kit is well-suited for use in the routine diagnostic laboratory, especially for accurate monitoring of patients receiving triple therapy or interferone-free regimens.     

Quantification of cytomegalovirus DNA by a fully automated real-time PCR for early diagnosis and monitoring of active viral infection in solid organ transplant recipients

BackgroundQuantification of cytomegalovirus (CMV) DNA by real-time PCR is currently considered an alternative diagnostic approach for the evaluation of active infection in transplant patients. The pp65 antigenemia assay has been used as reference test for monitoring active CMV infection and guiding preemptive therapy in transplant recipients. However, this assay suffers from some limitations: need for immediate processing of the samples, labour-intensive process, lack of standardization and subjective result interpretation.ObjectivesThe aim of this study was to evaluate the performance of a new commercially available real-time PCR assay coupled with a fully automated DNA extraction system (COBAS Ampliprep/COBAS Taqman CMV Test, Roche Diagnostics) for the detection of CMV-DNA in plasma comparing it with pp65 antigenemia assay for monitoring active CMV infection in solid organ transplant recipients (SOTRs).Study designA total of 266 consecutive samples from 45 SOTRs were monitored with pp65 antigenemia and in parallel with CMV-DNA quantitation by real-time PCR assay.ResultsFifty-eight samples resulted PCR-positive, 163 negative and for 45 samples the CMV-DNA values obtained were below the lower limit of quantification (<150 copies/ml); pp65 antigen was detected in 47 samples and resulted negative in 219 specimens. Concordance between the two evaluations was 76.7%; also a good correlation was observed (r = 0.718). Considering the existing treatment criteria based on pp65 antigenemia evaluation corresponding to pp65 levels ≥ 20 positive cells/200,000, preemptive therapy was administered to four asymptomatically infected patients. The corresponding cut-off value of CMV-DNA load calculated for discrimination between self-clearing infections and those requiring therapy was 2500 copies/ml (or 2275 IU/ml).ConclusionThe fully automated real-time PCR from Roche provided specific and sensitive results and represented a rapid and simple assay for the evaluation and monitoring of CMV infection in SOTRs. Further studies are required to validate the threshold level for the initiation of preemptive therapy.     

A European multicentre study on the comparison of HCV viral loads between VERIS HCV assay and COBAS® TaqMan® HCV Test and RealTime HCV Assay

BackgroundBeckman Coulter has developed the VERIS HCV Assay for use on the new fully automated DxN VERIS Molecular Diagnostic System^¥ for HCV viral load monitoring.ObjectivesEvaluate the clinical performance of the new quantitative VERIS HCV Assay.Study designComparison was performed on 279 plasma specimens from HCV infected patients tested with the VERIS HCV Assay and COBAS^® Ampliprep/COBAS^® Taqman^® HCV Test and 369 specimens tested with the VERIS HCV Assay and RealTime HCV Assay. Patient monitoring sample results from four time points were also compared.ResultsThe average bias between the VERIS HCV Assay and the COBAS^® Ampliprep/COBAS^® Taqman^® HCV Test was 0.04 log₁₀ IU/mL, while between the VERIS HCV Assay and the RealTime HCV Assay average bias was 0.21 log₁₀ IU/mL. Bias, however, was not consistent across the measuring range. Analysis at the lower end of quantification levels 50, 100, and 1000 IU/mL showed a predicted bias for VERIS HCV Assay versus COBAS^® Ampliprep/COBAS^® Taqman^® HCV Test between −0.42 and −0.22 log₁₀ IU/mL and for VERIS HCV Assay versus RealTime HCV Assay between 0.00 and 0.13 log₁₀ IU/mL. Patient monitoring of HCV viral load over time demonstrated similar levels between VERIS HCV Assay results and COBAS^® Ampliprep/COBAS^® Taqman^® HCV Test (52 samples from 13 patients) and RealTime HCV Assay (112 samples from 28 patients).ConclusionsVERIS HCV Assay for use on the DxN VERIS Molecular Diagnostic System represents a reliable new tool for easy sample to result HCV RNA viral load monitoring.     

Performance of a completely automated system for monitoring CMV DNA in plasma

BackgroundCompletely automated systems for monitoring CMV-DNA in plasma samples are now available.ObjectivesEvaluate analytical and clinical performances of the VERIS™/MDx System CMV Assay^®.Study designAnalytical performance was assessed using quantified quality controls. Clinical performance was assessed by comparison with the COBAS^® Ampliprep™/COBAS^® Taqman CMV test using 169 plasma samples that had tested positive with the in-house technique in whole blood.ResultsThe specificity of the VERIS™/MDx System CMV Assay^® was 99% [CI 95%: 97.7–100]. Intra-assay reproducibilities were 0.03, 0.04, 0.05 and 0.04 log₁₀ IU/ml (means 2.78, 3.70, 4.64 and 5.60 log₁₀ IU/ml) for expected values of 2.70, 3.70, 4.70 and 5.70 log₁₀ IU/ml. The inter-assay reproducibilities were 0.12 and 0.08 (means 6.30 and 2.85 log₁₀ IU/ml) for expected values of 6.28 and 2.80 log₁₀ IU/ml. The lower limit of detection was 14.6 IU/ml, and the assay was linear from 2.34 to 5.58 log₁₀ IU/ml. The results for the positive samples were concordant (r = 0.71, p < 0.0001; slope of Deming regression 0.79 [CI 95%: 0.56–1.57] and y-intercept 0.79 [CI 95%: 0.63–0.95]). The VERIS™/MDx System CMV Assay^® detected 18 more positive samples than did the COBAS^® Ampliprep™/COBAS^® Taqman CMV test and the mean virus load were higher (0.41 log₁₀ IU/ml). Patient monitoring on 68 samples collected from 17 immunosuppressed patients showed similar trends between the two assays. As secondary question, virus loads detected by the VERIS™/MDx System CMV Assay^® were compared to those of the in-house procedure on whole blood. The results were similar between the two assays (−0.09 log₁₀ IU/ml) as were the patient monitoring trends.ConclusionThe performances of the VERIS™/MDx System CMV Assay^® facilitated its routine use in monitoring CMV-DNA loads in plasma samples.     

HCV RNA measurement in samples with diverse genotypes using versions 1 and 2 of the Roche COBAS® AmpliPrep/COBAS® TaqMan® HCV test

BackgroundAccurate HCV RNA measurement is required for monitoring treatment. Underquantification has been reported with some genotypes, particularly genotype 4, using version 1 of the Roche COBAS^® AmpliPrep/COBAS^® TaqMan^® HCV Test.ObjectivesCompare the viral loads of clinical specimens representing diverse genotypes from across the United States using versions 1 (V1) and 2 (V2) of the Roche COBAS^® AmpliPrep/COBAS^® TaqMan^® HCV Test. Assess the frequency of nt145 and nt165 variants in the 5′ UTR associated with detection failures and underquantification in a large clinical sample database.Study designThree hundred archived clinical samples were measured using V1 and V2. Bland–Altman analysis was performed on log-transformed results and compared by genotype. The frequencies of nt145 and nt165 variants from 15,817 sequences were calculated.ResultsOn average, V2 results were 0.16 log IU/mL lower than V1 results. The average genotype 4 sample was 0.08 log IU/mL higher in V1 than V2. The largest individual sample differences were −2.10 (genotype 2b) and 1.57 (genotype 2a) log IU/mL. For genotype 4 samples, the greatest underquantification by V1 was 1.46 log IU/mL. There were 13 (0.082%) variants at nt145 and 24 variants at nt165 (0.152%), including one sequence with variants at both positions (0.0063%).ConclusionsGenotype 4 samples from the U.S. are rarely underquantified and not disproportionately so compared to other genotypes using the COBAS^® AmpliPrep/COBAS^® TaqMan^® HCV Tests. Variants at the nt145 and nt165 positions are uncommon in the U.S. and double variants are exceedingly rare. Underquantification of HCV samples with the V1 assay is likely a very rare occurrence in U.S. patients.     

Viral load assay sensitivity and low level viremia in HAART treated HIV patients

BackgroundThe recent introduction of highly sensitive viral load assays resulted in a significant increase in number of treated HIV-infected patients with a detectable viral load. The significance of a viral load between 20 and 50 copies/mL remains unclear.ObjectivesTo compare the performance of three viral load assays, with special attention for specificity and sensitivity at the lowest level of quantification.Study designSamples (n = 181) were selected from 62 HIV-positive individuals that experience viral blips or episodes of low but detectable viremia under antiretroviral treatment, and from 216 HIV-negative individuals. Each sample was tested in at least two of three assays: the Cobas Amplicor HIV-1 Monitor (CAP/CA), the Cobas Ampliprep/Cobas TaqMan HIV-1 version 1 (CAP/CTM1) and the Cobas Ampliprep/Cobas TaqMan HIV-1 version 2 (CAP/CTM2).ResultsNo false positive results were recorded. Kappa statistics revealed fair to moderate agreement between the results of the three assays, but important differences in sensitivity were observed, with the highest sensitivity reported for CAP/CTM2 followed by CAP/CTM1 and CAP/CA. The differences in sensitivity remained after equalization of the detection limit for all assays at 50 copies/mL. Analysis of samples collected over time showed that patients with single blips in CAP/CA present with recurrent blips in CAP/CTM1 and persistent detectable viremia in CAP/CTM2.ConclusionsViral load results between 20 and 50 copies/mL in either CAP/CTM1 or CAP/CTM2, indicate true viremia. The availability of highly sensitive assays force reconsideration of the terms ‘undetectable’ viral load and ‘virological success’ of antiretroviral treatment.     

A European multicientre study on the comparison of HIV-1 viral loads between VERIS HIV-1 Assay and Roche COBAS® TAQMAN® HIV-1 test,Abbott RealTime HIV-1 Assay,and Siemens VERSANT HIV-1 Assay

BackgroundViral load monitoring is essential for patients under treatment for HIV. Beckman Coulter has developed the VERIS HIV-1 Assay for use on the novel, automated DxN VERIS Molecular Diagnostics System.^¥ObjectivesEvaluation of the clinical performance of the new quantitative VERIS HIV-1 Assay at multiple EU laboratories.Study designMethod comparison with the VERIS HIV-1 Assay was performed with 415 specimens at 5 sites tested with COBAS^® AmpliPrep/COBAS^® TaqMan^® HIV-1 Test, v2.0, 169 specimens at 3 sites tested with RealTime HIV-1 Assay, and 202 specimens from 2 sites tested with VERSANT HIV-1 Assay. Patient monitoring sample results from 4 sites were also compared.ResultsBland-Altman analysis showed the average bias between VERIS HIV-1 Assay and COBAS HIV-1 Test, RealTime HIV-1 Assay, and VERSANT HIV-1 Assay to be 0.28, 0.39, and 0.61 log₁₀ cp/mL, respectively. Bias at low end levels below 1000 cp/mL showed predicted bias to be <0.3 log₁₀ cp/mL for VERIS HIV-1 Assay versus COBAS HIV-1 Test and RealTime HIV-1 Assay, and <0.5 log₁₀ cp/mL versus VERSANT HIV-1 Assay. Analysis on 174 specimens tested with the 0.175 mL volume VERIS HIV-1 Assay and COBAS HIV-1 Test showed average bias of 0.39 log₁₀ cp/mL. Patient monitoring results using VERIS HIV-1 Assay demonstrated similar viral load trends over time to all comparators.ConclusionsThe VERIS HIV-1 Assay for use on the DxN VERIS System demonstrated comparable clinical performance to COBAS^® HIV-1 Test, RealTime HIV-1 Assay, and VERSANT HIV-1 Assay.     

Comparison of three quantitative HCV RNA assays in samples from HCV genotype 1- or 4-infected patients treated with the NS3/4A protease inhibitor simeprevir

BackgroundMonitoring HCV RNA levels during treatment is an important tool for managing protease-inhibitor-based regimens, and different assays used in clinical practice can impact treatment decisions.ObjectivesThe concordance of three HCV RNA assays was determined, and their impact on treatment decisions assessed using samples from HCV genotype (GT) 1- and GT4-infected patients treated with the NS3/4A inhibitor simeprevir in combination with pegylated interferon-α/ribavirin.Study designPlasma samples collected during the simeprevir Phase III studies QUEST-1 and QUEST-2 (GT1), and RESTORE (GT4) were analyzed with the Roche High-Pure-System COBAS^® TaqMan^® HCV v2.0 (HPS), the Roche AmpliPrep COBAS^® TaqMan^® HCV v2.0 (CAP), and the Abbott RealTime HCV (ART) assay.ResultsIn GT1, of the 440 samples, 81% were undetectable (rapid virological response; RVR) by HPS at Week 4, 76% by CAP and 44% by ART. In GT4 (103 samples), RVR rates were 67% by HPS and 24% by ART. HCV RNA <25 IU/mL at Week 4 was observed for 95–96% and 92% GT1 samples and 86% and 74% GT4 samples by HPS/CAP and ART, respectively. At Week 12, assay concordance for undetectability was high in GT1 and GT4, (95–98% and 93%, respectively).ConclusionsWhile different HCV RNA assays can lead to substantially different RVR rates, a good concordance was observed with a cut-off of 25 IU/mL. Sustained virologic response rates among GT1 patients achieving RVR or <25 IU/mL at Week 4 were high and similar between assays used. At later time points, when viremia is low, assay concordance was high.     

Clinical performances of two real-time PCR assays and bDNA/TMA to early monitor treatment outcome in patients with chronic hepatitis C

BackgroundEarly viral monitoring is essential for the management of treatment outcome in patients with chronic hepatitis C. A variety of commercially available assays are now available to quantify HCV-RNA in routine clinical practice.ObjectivesCompare the clinical results of 3 commercially available assays to evaluate the positive predictive value (PPV) and the negative predictive value (NPV) of rapid virological response (RVR) at week 4 and early virological response (EVR) at week 12.Study design287 patients treated with standard care regimen combination therapy were studied. HCV-RNA values measured at baseline, week 4, week 12 with VERSANT^® HCV 3.0 Assay (bDNA), and VERSANT^® HCV-RNA Qualitative Assay (TMA) (bDNA/TMA); COBAS Ampliprep/COBAS/TaqMan (CAP/CTM) and Abbott m2000sp extraction/m2000rt amplification system (ART). RVR was defined as undetectable serum HCV-RNA and EVR as a ≥2 log decline in baseline viral load (BLV).ResultsMedian (range) BVLs were: 5.585(2.585–6.816), 5.189(2.792–7.747) and 4.804(2.380–6.580) log₁₀ IU/ml, with bDNA/TMA, CAP/CTM and ART, respectively (p < 0.01); RVR was observed in 22%, 30% and 27% of the patients and PPVs were 97%, 91% and 94% with bDNA/TMA, CAP/CTM and ART, respectively (p = 0.317). EVR was observed in 76%, 73% and 67% of the patients and NPVs were 93%, 83% and 79% with bDNA/TMA, CAP/CTM and ART, respectively (p = 0.09).ConclusionsTreatment monitoring should include both detection of serum HCV-RNA at week 4 to predict SVR and at week 12 to predict non-SVR. The value of all 3 assays was similar for evaluating RVR or EVR. Because of viral load discrepancies the same assay should be used throughout patient treatment follow-up.     

Quantitative analysis of cytomegalovirus (CMV) viremia using the pp65 antigenemia assay and the COBAS AMPLICOR CMV MONITOR PCR test after blood and marrow allogeneic transplantation

The performance of a commercially available qualitative PCR test for plasma (AMPLICOR CMV Test; Roche Diagnostics) and a quantitative PCR test for plasma and leukocytes (COBAS AMPLICOR CMV MONITOR Test; Roche Diagnostics) was evaluated with samples from 50 blood or marrow allogeneic transplant recipients who received short courses of sequential ganciclovir therapy (2 weeks intravenously followed by 2 weeks orally) based on a positive cytomegalovirus (CMV) pp65 antigenemia (AG) assay. The number of persons with a positive CMV test was significantly higher for leukocyte-based assays (AG, 67.5%; PCR, 62.5%) compared to both quantitative and qualitative PCR tests of plasma (42.5 and 35%, respectively). One person developed CMV disease during the study despite a negative AG assay; in this particular case, all PCR assays were found to be positive 10 days before his death. There was a trend for earlier positivity after transplantation and more rapid negativity after initiation of ganciclovir for the tests performed on leukocytes. The mean number of CMV copies as assessed by PCR was significantly higher in leukocytes than in plasma (P = 0.02). Overall, excellent agreement (kappa coefficient, >0.75) was found only between the two PCR assays (qualitative and quantitative) based on plasma. These results suggest that either the pp65 AG assay or the COBAS AMPLICOR CMV MONITOR Test using leukocytes could be used to safely monitor CMV viremia in related allogeneic blood or marrow transplant recipients. Such a strategy will result in preemptive treatment for about two-thirds of the persons with a relatively low rate (<33%) of secondary viremic episodes following short courses of ganciclovir therapy.     

Comparative performance of the new Aptima HIV-1 Quant Dx assay with three commercial PCR-based HIV-1 RNA quantitation assays

BackgroundQuantitative measurement of HIV-1 RNA levels in plasma (‘viral load’) plays a central role in clinical management. The choice of assay platform can influence results and treatment decisions.ObjectiveTo compare the analytical performance of the new TMA-based Hologic Aptima^® HIV-1 Quant Dx assay with that of three PCR-based assays: Abbott RealTime HIV-1, Qiagen Artus^® HI Virus-1 QS-RGQ, and Roche CAP/CTM HIV-1 Test v2.Study designAssay performance was evaluated using Acrometrix HIV-1 RNA Standard panels; the 3rd WHO HIV-1 RNA International Standard (12–500 copies/ml; 6 dilutions; 9 replicates); and plasma samples from 191 HIV-positive patients.ResultsAptima showed high (>0.99) precision, accuracy and concordance with the Acrometrix Standards across a wide dynamic range (2.0–6.7 log₁₀ copies/ml). Variance caused up to 2.1 (Aptima), 1.7 (RealTime), 7.5 (Artus), and 1.9 (CAP/CTM) fold changes in the International Standard quantifications at 50–500 copies/ml. HIV-1 RNA detection rates in plasma samples were 141/191 (74%), 119/191 (62%), 108/191 (57%), and 145/191 (76%) for Aptima, RealTime, Artus and CAP/CTM, respectively. For categorising samples either side of 50 copies/ml, Aptima had excellent agreement with RealTime (kappa 0.92; 95% CI 0.87–0.98); lowest agreement was with Artus (kappa 0.79; 95%CI 0.70–0.88). Aptima quantifications were mean 0.12 and 0.06 log₁₀ copies/ml higher compared with RealTime and CAP/CTM, respectively, and 0.05 log₁₀ copies/ml lower compared with Artus. Limits of agreement were narrowest when comparing Aptima to RealTime.ConclusionsThe new Aptima HIV assay is sensitive, precise, and accurate. HIV assays exhibit discordance at low HIV-1 RNA copy numbers.     

Comparison of quantitative cytomegalovirus (CMV) PCR in plasma and CMV antigenemia assay: clinical utility of the prototype AMPLICOR CMV MONITOR test in transplant recipients

The correlation between the prototype AMPLICOR CMV MONITOR test (Roche Molecular Systems), a quantitative PCR assay, and the cytomegalovirus (CMV) pp65 antigenemia assay was evaluated in transplant recipients. Sequential blood specimens were collected on 29 patients (491 specimens), the leukocyte fraction was tested by CMV antigenemia, and quantitative PCR was performed on plasma specimens. None of the 15 patients (242 specimens) who were antigenemia negative were positive for CMV DNA by PCR, and none of these patients developed active CMV disease. There were 14 antigenemia-positive patients, 8 of whom developed active CMV disease. In all patients, there was a good association between the antigenemia and PCR assays. Ganciclovir-resistant virus was isolated from three patients with active CMV disease. These three patients had persistently elevated levels of antigenemia and CMV DNA by PCR when resistance to ganciclovir developed. This standardized, quantitative CMV PCR assay on plasma has clinical utility for the diagnosis of active disease and in monitoring the response to antiviral therapy in transplant recipients.     

Cytomegalovirus load at treatment initiation is predictive of time to resolution of viremia and duration of therapy in hematopoietic cell transplant recipients

BackgroundPreemptive antiviral therapy relies on viral load measurements and is the mainstay of cytomegalovirus (CMV) prevention in hematopoietic cell transplant (HCT) recipients. However, optimal CMV levels for the initiation of preemptive therapy have not been defined.ObjectivesThe objectives of our work were to evaluate the relationship between plasma CMV DNA levels at initiation of preemptive therapy with time to resolution of viremia and duration of treatment.Study designRetrospective analysis of HCT recipients undergoing serial CMV PCR testing between June 2011 and June 2014 was performed.Results221 HCT recipients underwent preemptive therapy for 305 episodes of CMV viremia. Median time to resolution was shorter when treatment was initiated at lower CMV levels (15 days at 135–440 international units (IU)/mL, 18 days at 441–1000 IU/mL, and 21 days at >1000 IU/mL, P < .001). Prolonged viremia lasting >30 days occurred less frequently when treatment was initiated at 135–440 IU/mL compared to 441–1000 IU/mL and >1000 IU/mL (1%, 15%, 24%, P < .001). Median treatment duration was also shorter in the lower viral load groups (28, 34, 37 days, P < .001).ConclusionInitiation of preemptive therapy at low CMV levels was associated with shorter episodes of viremia and courses of antiviral therapy. These data support the utility of initiating preemptive CMV therapy at viral loads as low as 135 IU/mL in HCT recipients.     

Persistent rhinovirus infection in pediatric hematopoietic stem cell transplant recipients with impaired cellular immunity

BackgroundHRV infections are generally self-limiting in healthy subjects, whereas in immunocompromised hosts HRV infections can lead to severe complications and persistent infections. The persistence of HRV shedding could be due to the inefficient immunological control of a single infectious episode.ObjectivesTo investigate the clinical, virologic and immunologic characteristics of pediatric HSCT recipients with HRV-PI infection.Study designDuring the period 2006–2012, eight hematopoietic stem cell transplant (HSCT) recipients presented with persistent rhinovirus infection (HRV-PI, ≥30 days). Viral load and T-CD4⁺, T-CD8⁺, B and NK lymphocyte counts at the onset of infection were compared with those of fourteen HSCT recipients with acute HRV infection (HRV-AI, ≤15 days).ResultsThe median duration of HRV positivity in patients with HRV-PI was 61 days (range 30–174 days) and phylogenetic analysis showed the persistence of a single HRV type in all patients (100%). In HSCT recipients with HRV-PI, T-CD4⁺, T-CD8⁺ and NK cell counts at the onset of infection were significantly lower than those observed in recipients with HRV-AI (p < 0.01), while B cell counts were similar in the two groups (p =  0.25). A decrease in HRV load was associated with a significant increase in T-CD4⁺, T-CD8⁺and NK lymphocyte counts in HRV-PI patients (p < 0.01).ConclusionsThis study suggests a role for cellular immunity in HRV clearance and highlights the importance of its recovery for the control of HRV infection in HSCT recipients.     

Diagnostic usefulness of dynamic changes of CMV-specific T-cell responses in predicting CMV infections in HCT recipients

BackgroundCMV-specific cell mediated immune responses before and after hematopoietic stem cell transplantation (HCT) can categorize patients as at high or low risk of CMV development.ObjectivesWe evaluated the usefulness of the CMV-specific T-cell ELISPOT assay for predicting the development of CMV infections after HCT in recipients with donor-positive and recipient-positive CMV serology (D+/R+ ).Study designCMV pp65 and IE1-specific ELISPOT assays were performed before HCT (D0), and at 30 (D30) and 90 (D90) days after HCT.ResultsOf the 84 HCT recipients with D+/R+, 42 (50%) developed ≥ 1 episode of CMV infection. Thirty-nine (64%) of 61 patients with Δ(D30-D0) pp65 < 42 developed CMV infections compared with 3 (14%) of 21 patients with Δ(D30-D0) pp65 ≥ 42 (P < 0.001). Twenty-three (74%) of 31 patients with Δ(D30-D0) IE1 < −4 developed CMV infections compared with 19 (37%) of 51 patients with Δ(D30-D0) IE1 ≥ −4 (P = 0.001). pp65 Δ(D30-D0) ≥ 42 had 93% sensitivity for ruling out subsequent CMV infection, and pp65 Δ(D30-D0) < 42 followed by Δ(D30-D0) IE1 < −4 had 100% specificity for ruling in the subsequent CMV infection. In addition, 10 (53%) of 19 patients with Δ(D90-D30) pp65 < 23 had relapsing CMV infections, compared with 3 (15%) of 20 patients with Δ(D90-D30) pp65 ≥ 23 (P = 0.02). The sensitivity and specificity of Δ(D90-D30) pp65 were 77% (95% CI 50–92) and 65% (95% CI, 46–81).ConclusionDynamic change in the CMV-specific ELISPOT assay before versus after HCT appears to predict the subsequent development of CMV infection and relapsing CMV infection.     

A European multicientre study on the comparison of HBV viral loads between VERIS HBV assay and Roche COBAS® TAQMAN® HBV test,Abbott RealTime HBV assay,Siemens VERSANT HBV assay,and Qiagen artus HBV RG kit

BackgroundHepatitis B viral load testing is essential to treatment and monitoring decisions in patients with chronic Hepatitis B. Beckman Coulter has developed the VERIS HBV Assay (Veris) for use on the fully automated DxN VERIS Molecular Diagnostics System.¹ObjectivesTo evaluate the clinical performance of the Veris HBV Assay at multiple EU laboratoriesStudy designMethod comparison was performed with a total of 344 plasma specimens from HBV infected patients tested with Veris and COBAS^® TaqMan^® HBV Test (Cobas), 207 specimens tested with Veris and RealTime HBV Assay (RealTime), 86 specimens tested with Veris and VERSANT^® HBV Assay (Versant), and 74 specimens tested with Veris and artus^® HBV RG PCR kit (artus).ResultsBland-Altman analysis showed average bias of −0.46 log₁₀ IU/mL between Veris and Cobas, −0.46 log₁₀ IU/mL between Veris and RealTime, −0.36 log₁₀ IU/mL between Veris and Versant, and −0.12 log₁₀  IU/mL between Veris and artus. Bias was consistent across the assay range. Patient monitoring results using Veris demonstrated similar viral load trends over time to Cobas, RealTime, and artus.ConclusionsThe VERIS HBV Assay demonstrated comparable clinical performance, with varying degrees of negative bias, compared to other currently marketed assays for HBV DNA monitoring. This negative bias should be taken into consideration if switching monitoring methods to Veris.     

Comprehensive comparison of the VERSANT® HIV-1 RNA 3.0 (bDNA) and COBAS AMPLICOR HIV-1 MONITOR® 1.5 assays on 1000 clinical specimens

BackgroundPlasma human immunodeficiency virus type 1 (HIV-1) RNA level is an important parameter for patient management, yet viral load assays from different manufacturers are not standardized.Objectives and study designIn this study, we evaluated the concordance between test results obtained for 1000 plasma specimens collected from HIV-1-infected individuals measured with the VERSANT^® HIV-1 RNA 3.0 assay (bDNA) and the COBAS AMPLICOR HIV-1 MONITOR^® 1.5 test (PCR). We compared viral load values obtained by each of these assays throughout their dynamic ranges, with particular focus on samples with low viral load (i.e. 50–250 copies/mL), and calculated the estimated distribution of distinct plasma viral load levels for the entire study population modeled from the data observed in the study.ResultsWe found that these two assays show excellent agreement, with a correlation (R²) of 0.957 and a slope of 1.004. The mean difference in viral load values between the two assays was less than 0.10-log₁₀ throughout the dynamic range and 98.2% of all samples had bDNA and PCR results within 0.5-log₁₀ of each other, a difference that is within the range considered to be a minimal change in plasma viremia. Moreover, the two assays show very similar results across all assay ranges tested. The estimated prevalence of samples with results <50 copies/mL, 50–250 copies/mL, and 250–500,000 copies/mL were 41.6%, 7.7%, and 49.7%, respectively, by the bDNA assay, and 42.4%, 6.9%, and 50.7%, respectively, by the PCR assay.ConclusionBased on our findings from 1000 clinical specimens, we do not see the need to re-establish a baseline value or apply a conversion factor when switching from one assay to the other. Since the majority of our patient population likely is infected with subtype B virus, it is unclear if our findings will apply to other patient populations with a greater incidence of infection with non-B subtypes.     

Effect of artemether-lumefantrine (Coartem) on cytomegalovirus urine viral load during and following treatment for malaria in children

BackgroundArtemisinins, commonly used to treat malaria, have shown activity against cytomegalovirus (CMV) in vitro, in an animal model, and in case reports; however, the in vivo anti-CMV activity has not been well investigated.ObjectivesTo evaluate whether artemisinins affect CMV shedding among subjects co-infected with CMV and malaria.Study designA prospective observational study of children in Mali (6 month–10 year) presenting with fever. Urine samples were collected at day 0, 3, and 14 from children treated with artemether-lumefantrine (Coartem^®) for malaria and those who had other illnesses not treated with Coartem. CMV DNA was quantified using a real-time PCR. Resulting urine viral loads were compared between the groups at three time points.Results164 malaria cases and 143 non-malaria comparisons were enrolled. Eighty-one (49%) cases and 88 (62%) comparisons shed CMV at day 0. Day 0 and day 3 viral loads were similar, but at day 14 the median viral load of cases was lower than that of comparisons (360 vs 720 copies/mL or 2.56 vs 2.86 log₁₀), p = 0.059. A stratified analysis of day 0 high viral shedders (defined as >1000 copies/mL) showed significantly lower median viral load at day 14 among cases (490 copies/mL, 2.69 log₁₀) vs comparisons (1200 copies/mL, 3.08 log₁₀), p = 0.045.ConclusionA high rate of urinary CMV shedding was found in a malaria-endemic area. Among high virus shedders artemether-lumefantrine decreased urine viral load, but the effect was not observed when analysis of both high and low shedders was performed. These results support additional studies of artemisinin dosing and duration in CMV infection.