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.     

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.     

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.     

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.     

Evaluation of a new random-access HBV DNA molecular assay: The VERIS HBV assay

BackgroundDetection and quantification of HBV DNA are essential to diagnose chronic HBV infection, monitor the virological response to treatment and the possible selection of resistant viruses in order to tailor therapy. The VERIS/MDx System HBV Assay is a random-access system that quantifies HBV DNA in clinical samples using unique single sample and reagent access during the workflow process without the need to reload other tests and delivers results within 1.2 h following sampling.Objective and study designThe goal of this study was to evaluate the analytical performance of the VERIS HBV assay for HBV DNA detection and quantification in clinical samples from a series of patients chronically infected with different HBV genotypes.ResultsThe specificity of the VERIS HBV assay was estimated to be over 99.5%. The limit of detection (LOD) was estimated to be 4.1 IU/mL (95%CI: 3.20–5.90 IU/mL). Using an HBV linearity panel and controls (Seracare LifeScience), intra-assay and inter-assay coefficients of variation ranged from 0.12% to 3.64% and from 1.05% to 7.35%, respectively. The influence of the HBV genotype was evaluated from 120 clinical specimens containing HBV genotypes A to G tested in parallel with the VERIS HBV assay and the COBAS AmpliPrep/COBAS TaqMan HBV v2.0 assay. A linear relationship between the HBV DNA levels measured with both assays was found. A modest bias of HBV DNA levels was observed in the VERIS assay as compared to CAP/CTM HBV v2.0 in most of the samples tested (mean VERIS minus CAP/CTM difference: −0.395 log IU/mL). Overall, the VERIS HBV assay is well suited to monitoring clinical HBV DNA levels in infected patients according to current clinical practice guidelines.     

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.     

Hepatitis B viral load in dried blood spots: A validation study in Zambia

BackgroundAccess to hepatitis B viral load (VL) testing is poor in sub-Saharan Africa (SSA) due to economic and logistical reasons.ObjectivesTo demonstrate the feasibility of testing dried blood spots (DBS) for hepatitis B virus (HBV) VL in a laboratory in Lusaka, Zambia, and to compare HBV VLs between DBS and plasma samples.Study designPaired plasma and DBS samples from HIV-HBV co-infected Zambian adults were analyzed for HBV VL using the COBAS AmpliPrep/COBAS TaqMan HBV test (Version 2.0) and for HBV genotype by direct sequencing. We used Bland-Altman analysis to compare VLs between sample types and by genotype. Logistic regression analysis was conducted to assess the probability of an undetectable DBS result by plasma VL.ResultsAmong 68 participants, median age was 34 years, 61.8% were men, and median plasma HBV VL was 3.98 log IU/ml (interquartile range, 2.04–5.95). Among sequenced viruses, 28 were genotype A1 and 27 were genotype E. Bland–Altman plots suggested strong agreement between DBS and plasma VLs. DBS VLs were on average 1.59 log IU/ml lower than plasma with 95% limits of agreement of −2.40 to −0.83 log IU/ml. At a plasma VL ≥2,000 IU/ml, the probability of an undetectable DBS result was 1.8% (95% CI: 0.5–6.6). At plasma VL ≥20,000 IU/ml this probability reduced to 0.2% (95% CI: 0.03–1.7).ConclusionsIn a Zambian laboratory, we observed strong agreement between DBS and plasma VLs and high sensitivity in DBS at plasma VL ≥2,000 IU/ml. As HBV treatment expands, DBS could increase access to HBV VL testing and care in SSA settings.     

Peginterferon alfa-2a and peginterferon alfa-2b combined with ribavirin in patients with genotype 1 chronic hepatitis C: Results of a prospective single-centre study

PurposeThis prospective, randomized, single-centre study compared peginterferons alfa-2a and alfa-2b, combined with ribavirin, in treating patients infected with hepatitis C virus (HCV) genotype 1.Material/methodsHundred-and-one patients received 48 weeks of open-label treatment with peginterferon alfa-2a (180 μg/week) and 111 patients received peginterferon alfa-2b (1.5 μg/kg/week). All patients received the same dose of ribavirin 1000/1200 mg/day, depending on weight. The primary efficacy endpoint was sustained virologic response (SVR), defined as undetectable HCV RNA (<50 IU/mL) 24 weeks after the end of treatment.ResultsEarly virologic response (EVR), defined as at least 2 log₁₀ IU/mL reduction of viral load at 12 weeks, was more common in patients treated with peginterferon alfa-2a (88% vs. 74.8%; p = 0.04). However, the difference in SVR was not statistically significant (49.5% vs. 44.1%; p = 0.43).ConclusionsPeginterferon alfa-2a treated patients were also more likely to be HCV RNA negative at the end of treatment (67.3% vs. 57.7%), but this difference did not reach statistical significance. Multivariate logistic regression analysis found that SVR was associated with low fibrosis stage (F1–2 by Scheuer; p = 0.001) and low serum HCV RNA level (<400,000 IU/L; p = 0.023). While both forms of peginterferon showed similar efficacy as measured by SVR, use of peginterferon alfa-2b could lower the number of patients receiving unnecessary treatment beyond 12 weeks.     

Point -of -care testing (POCT) in molecular diagnostics: Performance evaluation of GeneXpert HCV RNA test in diagnosing and monitoring of HCV infection

BackgroundMolecular testing at the point-of-care may turn out to be game changer for HCV diagnosis and treatment monitoring, through increased sensitivity, reduced turnaround time, and ease of performance. One such assay GeneXpert^® has recently been released.ObjectivesComparative analysis between performances of GeneXpert^® and Abbott HCV-RNA was done.Study design174 HCV infected patients were recruited and, one time plasma samples from 154 patients and repeated samples from 20 patients, obtained at specific treatment time-points (0, 4, 12 and 24) weeks were serially re-tested on Xpert^®.ResultsGenotype 3 was the commonest, seen in 80 (66%) of the cases, genotype 1 in 34 (28.3%), genotype 4 in 4 (3.3%) and genotypes 2 and 5 in 1 (0.8%) each. Median HCV RNA load was 4.69 log₁₀ (range: 0–6.98 log₁₀) IU/ml. Overall a very good correlation was seen between the two assays (R² = 0.985), concordance of the results between the assays was seen in 138 samples (89.6%). High and low positive standards were tested ten times on Xpert^® to evaluate the precision and the coefficient of variation was 0.01 for HPC and 0.07 for the LPC. Monitoring of patients on two different regimes of treatment, pegylated interferon plus ribavirin and sofosbuvir plus ribavirin was done by both the systems at baseline, 4, 12 and 24 weeks. Perfect correlation between the assays in the course of therapy at different treatment time- point in genotypes 3 and 1 was seen.ConclusionThe study demonstrates excellent performance of the Xpert^® HCV assay in viral load assessment and in treatment course monitoring consistency.     

Profound week 4 interferon responsiveness is mandatory for hepatitis C genotype 1 patients with unfavorable IL-28B genotype

BackgroundViral kinetics and host interleukin 28B (IL-28B) genotype determine treatment outcome in hepatitis C virus genotype 1 (HCV-1) infection.ObjectivesWe aimed to explore the interplay between interferon responsiveness at treatment week 4 and IL28B genotype in the achievement of a sustained virological response (SVR; undetectable HCV RNA 24-weeks after end-of-treatment).Study designsRs8099917 genotypes were determined in 528 HCV-1 patients with peginterferon/ribavirin. Interferon responsiveness were evaluated by the degree of week 4 viral reduction: <1 log₁₀ IU/mL, 1–2 logs₁₀ IU/mL, 2–3 logs₁₀ IU/mL, 3–4 logs₁₀ IU/mL and ≥4 logs₁₀ IU/mL reduction and/or undetectable HCV RNA, respectively.ResultsThe SVR rate was significantly higher in patients with great interferon responsiveness at week 4. A great interferon responsiveness was associated with younger age (P < 0.0001), lower body mass index (P = 0.0056), lower aspartate aminotransferase levels (P = 0.0009), higher hemogloblin concentration (P = 0.0033), higher platelet counts (P < 0.0001), male gender (P < 0.0001) and rs809997 TT-genotype (P < 0.0001). Comparing to non-TT genotype patients, TT genotype patients had a significantly higher SVR rate with moderate viral reduction (1–3 logs₁₀ IU/mL) at week 4 (58.9% vs. 18.2%, P < 0.001), and the SVR rate did not differ between TT/non-TT patients on the extreme ends (<1 or >3 log₁₀ IU/mL reduction) of week 4 interferon responsiveness. For non-TT genotype carriers who were with <3 logs₁₀ reduction, none (0/15) could have a complete early virological response and only 10.9% (7/64) of the patients had an SVR.ConclusionsMore profound interferon responsiveness is mandatory for HCV-1 patients with unfavorable IL-28B genotype.     

Development and persistence of DAA resistance associated mutations in patients failing HCV treatment

BackgroundDirect-acting antiviral agents (DAAs) combined with pegylated-interferon (PegIFN) and ribavirin (RBV) are still a standard treatment in patients with genotype 1HCV infection. However, virologic response could be impaired by baseline or early selection of resistant HCV strains.ObjectivesThe aim of this study was to determine the onset and persistence of resistance-associated mutations (RAMs) in the NS3 and NS5B genes of DAA-naïve patients failing treatment.Study designDirect sequencing of HCV NS3 was performed in 49 DAA-naïve patients with HCV genotype 1 infection.ResultsEight out of 23 patients (34.7%) failed PegIFN/RBV/telaprevir during the 12-weeks of therapy. Treatment failure was associated with the development of RAMs at amino-acids 36,54,80 and 155 of the HCV protease in 6/8 patients (75%). Among patients treated with PegIFN/RBV/boceprevir treatment, 4/18 (22.2%) failed therapy. Of these, 2 (50%) carried virus strains which developed a RAM at amino-acids 54 and 155. Among HCV strains with RAMs, 7 belonged to genotype 1a and 1 to 1b. Finally, in 6/10 (60%) patients, drug-resistant variants could still be detected for up to 3–7 months after stopping therapy.ConclusionsA higher rate (p = 0.49) of treatment failure was observed in patients receiving telaprevir- compared to the boceprevir-based combination. In addition, compared with genotype 1b, genotype 1a was associated with higher rates (p = 0.01) of treatment failure due to virus resistant strains.Resistance testing at baseline and during DAA treatment should be taken into consideration when treating patients with new HCV combination therapies.     

TLR3 polymorphisms are associated with virologic response to hepatitis C virus (HCV) treatment in HIV/HCV coinfected patients

BackgroundToll-like receptor-3 (TLR3) is a cellular receptor that may recognize double-stranded RNA (dsRNA) from viruses, resulting in production of proinflammatory cytokines and interferons, which are important for the adaptive immune response.ObjectivesTo analyze the association between Toll-like receptor-3 (TLR3) polymorphisms (rs3775291 and rs13126816) and virologic response to pegylated interferon-alpha plus ribavirin (pegIFNα/RBV) therapy in HIV/HCV coinfected patients.Study designWe performed a retrospective study in 321 naïve patients treated with pegIFNα/RBV. Genotyping was performed by using the GoldenGate^® assay with VeraCode^®. The outcome variables were early virologic response (EVR) and sustained virologic response (SVR).ResultsIn a multivariate analysis, rs3775291 A allele decreased the likelihood of achieving EVR (aOR = 0.20; p = 0.018) and SVR (aOR = 0.38; p = 0.024). Regarding rs13126816, the percentage of EVR decreased with each minor A allele (p = 0.034) in HCV-GT2/3 patients, although no significant association was obtained in the multivariate analysis (p = 0.076). Regarding TLR3 haplotypes (comprised of rs3775291 and rs13126816), GT2/3 patients with AA haplotype had decreased odds of achieving EVR (p = 0.030), whereas GG haplotype increased the likelihood (p = 0.018). Regarding SVR, GG haplotype carriers had increased odds of achieving SVR (p = 0.019, p = 0.043 and p = 0.070 for all, GT2/3 and GT1/4 patients, respectively). Besides, GT1/4 patients with GA haplotype had lower odds of achieving SVR (p = 0.039).ConclusionsOur study shows the first evidence that two TLR3 polymorphisms (rs3775291 and rs13126816) seem to be related to the HCV therapy response in HCV/HIV coinfected patients.     

CMV antigenemia and quantitative viral load assessments in hematopoietic stem cell transplant recipients

BackgroundSensitive and reliable diagnostic tests are essential for the prevention of cytomegalovirus (CMV) disease after hematopoietic stem cell transplantation (HSCT). pp65 antigenemia and polymerase chain reaction (PCR) assays are commonly used to monitor CMV in HSCT recipients. However, there is considerable intra- and inter-laboratory variability in the results, which impact comparability and clinical practice.Objectives/study designUsing 380 samples from 135 HSCT recipients, we compared the new FDA approved quantitative PCR assay, COBAS^® AmpliPrep/COBAS^® TaqMan^® CMV test (CAP/CTM CMV test) developed and standardized using the 1st WHO International Standard for CMV with pp65 antigenemia and COBAS^® AMPLICOR MONITOR CMV tests.ResultsThe median time between transplantation and testing samples was 57 days (range, 0–207 days). The median CMV load (log₁₀) was 3.17 IU/mL (3.21 copies/mL). Among samples with detectable CMV load, 52% were negative by pp65 antigenemia. CMV loads were higher in pp65 antigenemia-positive than in negative samples. One pp65-antigenemia-positive cell per 100,000 leukocytes corresponded to a median CMV load of 1200 IU/mL. CMV loads determined by the CAP/CTM CMV test were slightly lower than the ones by the AMPLICOR MONITOR CMV test (?0.15 [95% CI, ?0.18 to ?0.13] copies/mL), but slope differences indicated only limited co-linearity.ConclusionsThe CAP/CTM CMV test is more sensitive than pp65 antigenemia and the AMPLICOR MONITOR CMV test in HSCT recipients. The lower limit of quantification and co-linearity with the international WHO standard renders the CAP/CTM CMV test suitable for future clinical trials defining viral load thresholds of CMV therapy.     

Comparative evaluation of the Geenius HCV supplemental assay and Inno-LIA HCV score assay in detecting anti-HCV antibodies

ObjectiveThis study compared two assays aimed at confirming the presence of anti-HCV antibodies (Ab) after a positive screening: Geenius HCV supplemental assay (Bio-Rad, Marne la Coquette, France) and the Inno-LIA HCV score assay (Fujirebio, Les Ulis, France).Material and methodsA total of 180 archived samples were investigated including 119 samples collected at different stages of HCV infection in 25 hemodialyzed patients who underwent HCV seroconversion, 14 samples from 4 commercial seroconversion panels, 47 Ab positive/HCV-RNA positive blood donations of which 7 showing an single reactivity in confirmatory assays. Samples were investigated and results were interpreted with the two assays according to the manufacturers’ instructions.ResultsOverall, Geenius and Inno-LIA were concordant for 84% (151/180) samples: 38 negative, 17 indeterminate and 96 positive. Of the 29 discrepant results, 26 were overclassified with Inno-LIA. HCV seroconversion was detected with Inno-LIA 4 and 7 days prior to Geenius in two panels. The high positive rate observed with Inno-LIA (64%) compared to Geenius (54%) was mainly due to low reactivities considered positive according to interpretation criteria, which could affect specificity.ConclusionAlthough HCV supplemental assays are not recommended for the diagnostic of HCV infection, which is primarily based on HCV-RNA testing, both assays are suitable as second line anti-HCV tests when Ab screening is positive and RNA testing cannot be performed. Moreover, Geenius system provides an objective result in less than 30 minutes, which is compatible when a rapid diagnostic is required.     

Evolving strategy for HCV testing in an Italian tertiary care hospital

BackgroundDiagnostic tests for hepatitis C virus (HCV) infection should be adapted according to the clinical status of the patient.ObjectivesWe exploited the application of different HCV diagnostic algorithms in a tertiary care hospital practice.Study designThe laboratory clinical reports to the medical orders for HCV testing during three years were clustered by different combinations of assays for anti-HCV antibodies (HCV Ab) (screening and confirmatory), HCV nucleic acid (HCV-RNA), HCV core antigen (HCV Ag). The latter was the first-line assay in acute HCV infections requiring a rapid assessment of the infectious state.ResultsThe majority (91.9%) of the 2726 subjects whose samples were analyzed were inpatients. Most of the patients/subjects were tested for clinical suspicion of viral hepatitis (49.2%), or occupational accident to health care professionals (20.0%). On 66% of samples HCV Ag test alone was performed and resulted positive in 116 cases (6%), while it was detected in 50.3% of anti-HCV positive samples. The agreement between HCV Ag and HCV-RNA was very high (k = 0.97); HCV Ag positivity rates increased according to the signal of the HCV Ab screening test.ConclusionsThe use of different testing strategies according to the patients’ history and clinical status allowed a significant reduction of the number of tests performed and the time needed to provide a diagnostic response useful for patients’ management without compromising the overall diagnostic accuracy for HCV infection.     

Rate and predictors of serum HCV-RNA >6 million IU/mL in patients with chronic hepatitis C

BackgroundBaseline serum HCV-RNA predicts sustained virological response in chronic hepatitis C patients treated with antiviral therapy. A threshold at 6 million IU/mL has been proposed to best discriminate treatment outcomes on sofosbuvir-based regimens. In comparison with the general population, immunosuppressed individuals exhibit greater viral load values.ObjectivesTo estimate the rate and predictors of serum HCV-RNA above 6 million IU/mL in chronic hepatitis C patients on care outside clinical trials.Study designSerum HCV-RNA values recorded from all chronic hepatitis C patients consecutively attended at our clinic during the last decade were analyzed. Testing had been performed using the COBAS TaqMan HCV test v2.0.ResultsA total of 816 individuals with detectable serum HCV-RNA were identified. The main characteristics of this population were as follows: mean age 48.6 years-old; 73.4% males; mean ALT 82.6 IU/L; mean HCV-RNA 6.02 log IU/mL; 80.6% HCV genotypes 1 or 4; 34.9% advanced liver fibrosis; 35.4% IL28B-CC alleles. HIV coinfection in 78.7%, of whom 91% were on antiretroviral therapy.Overall, 127 (15.6%) had serum HCV-RNA values >6 million IU/mL. This high viremia was found in 18.2% of HIV-positive versus 5.7% of HIV-negative subjects (p < 0.001). In multivariate analysis, serum HCV-RNA >6 million IU/mL was only significantly associated with HIV coinfection (OR: 4.03; 95% CI: 1.98–8.19, p < 0.01) and HCV genotypes 1 or 4 (OR: 1.88; 95% CI: 1.05–3.37, p = 0.03).ConclusionsSerum HCV-RNA >6 million IU/mL is roughly seen in 6% of chronic hepatitis C monoinfected patients, and increases up to 18% in HIV coinfection.