Fully Automated Quantification of Cytomegalovirus (CMV) in Whole Blood with the New Sensitive Abbott RealTime CMV Assay in the Era of the CMV International Standard

Fully standardized reproducible and sensitive quantification assays for cytomegalovirus (CMV) are needed to better define thresholds for antiviral therapy initiation and interruption. We evaluated the newly released Abbott RealTime CMV assay for CMV quantification in whole blood (WB) that includes automated extraction and amplification (m2000 RealTime system). Sensitivity, accuracy, linearity, and intra- and interassay variability were validated in a WB matrix using Quality Control for Molecular Diagnostics (QCMD) panels and the WHO international standard (IS). The intra- and interassay coefficients of variation were 1.37% and 2.09% at 5 log₁₀ copies/ml and 2.41% and 3.80% at 3 log₁₀ copies/ml, respectively. According to expected values for the QCMD and Abbott RealTime CMV methods, the lower limits of quantification were 104 and <50 copies/ml, respectively. The conversion factor between international units and copies (2.18), determined from serial dilutions of the WHO IS in WB, was significantly different from the factor provided by the manufacturer (1.56) (P = 0.001). Results from 302 clinical samples were compared with those from the Qiagen artus CMV assay on the same m2000 RealTime system. The two assays provided highly concordant results (concordance correlation coefficient, 0.92), but the Abbott RealTime CMV assay detected and quantified, respectively, 20.6% and 47.8% more samples than the Qiagen/artus CMV assay. The sensitivity and reproducibility of the results, along with the automation, fulfilled the quality requirements for implementation of the Abbott RealTime CMV assay in clinical settings. Our results highlight the need for careful validation of conversion factors provided by the manufacturers for the WHO IS in WB to allow future comparison of results obtained with different assays.     

Quantitation of Cytomegalovirus DNA Load in Dried Blood Spots Correlates Well with Plasma Viral Load

An assay to accurately quantitate cytomegalovirus (CMV) load in finger-stick-collected dried blood spots (DBS) could potentially be useful for field studies or for analyzing patient self-collected specimens. We therefore assessed CMV DNA load in paired venipuncture-collected plasma samples and finger-stick DBS, using a previously validated quantitative PCR assay. Assay variability, sensitivity, and changes in viral load during antiviral therapy in finger-stick DBS were compared to the reference plasma quantitative PCR assay, using 106 prospectively collected pairs of finger-stick DBS and plasma samples from 35 solid-organ transplant (SOT) patients. The DBS assay showed good agreement with the reference plasma viral load assay on the log₁₀ scale (Pearson correlation coefficient, 0.92; P < 0.001). The 95% limit of detection of the DBS assay was estimated at 2,700 plasma copies/ml (675 plasma IU/ml). In 94% (76/81) of paired DBS and plasma samples above the limit of detection, the difference in CMV load was <1 log₁₀. CMV viral load changes during antiviral treatment were comparable in plasma and DBS. We conclude that finger-stick DBS provides a convenient sample type for quantitation of CMV load that correlates well with plasma levels. Future studies to optimize and evaluate this methodology for patient self-collected samples are warranted.     

Intra- and Interlaboratory Variabilities of Results Obtained with the Quantiplex Human Immunodeficiency Virus Type 1 RNA bDNA Assay, Version 3.0

Normal assay variation associated with bDNA tests for human immunodeficiency virus type 1 (HIV-1) RNA performed at two laboratories with different levels of test experience was investigated. Two 5-ml aliquots of blood in EDTA tubes were collected from each patient for whom the HIV-1 bDNA test was ordered. Blood was stored for no more than 4 h at room temperature prior to plasma separation. Plasma was stored at −70°C until transported to the Central Pennsylvania Alliance Laboratory (CPAL; York, Pa.) and to the Hershey Medical Center (Hershey, Pa.) on dry ice. Samples were stored at ≤−70°C at both laboratories prior to testing. Pools of negative (donor), low-HIV-1-RNA-positive, and high-HIV-1-RNA-positive plasma samples were also repeatedly tested at CPAL to determine both intra- and interrun variation. From 11 August 1999 until 14 September 2000, 448 patient specimens were analyzed in parallel at CPAL and Hershey. From 206 samples with results of ≥1,000 copies/ml at CPAL, 148 (72%) of the results varied by ≤0.20 log₁₀ when tested at Hershey and none varied by >0.50 log₁₀. However, of 242 specimens with results of <1,000 copies/ml at CPAL, 11 (5%) of the results varied by >0.50 log₁₀ when tested at Hershey. Of 38 aliquots of HIV-1 RNA pool negative samples included in 13 CPAL bDNA runs, 37 (97%) gave results of <50 copies/ml and 1 (3%) gave a result of 114 copies/ml. Low-positive HIV-1 RNA pool intrarun variation ranged from 0.06 to 0.26 log₁₀ while the maximum interrun variation was 0.52 log₁₀. High-positive HIV-1 RNA pool intrarun variation ranged from 0.04 to 0.32 log₁₀, while the maximum interrun variation was 0.55 log₁₀. In our patient population, a change in bDNA HIV-1 RNA results of ≤0.50 log₁₀ over time most likely represents normal laboratory test variation. However, a change of >0.50 log₁₀, especially if the results are >1,000 copies/ml, is likely to be significant.     

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

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

Comparative effectiveness of dried plasma HIV-1 viral load testing in Brazil using ViveST for sample collection

BackgroundUtilization of dried plasma for HIV-1 viral load testing would significantly decrease sample shipping costs.ObjectivesTo describe the precision and reproducibility of ViveST^® (ST) as a transportation method for shipping specimens for HIV-1 viral load (VL) testing.Study designThirty clinical plasma samples were used to generate replicate samples with HIV VL values of 4 log₁₀, 3 log₁₀ and 2 log₁₀ copies/mL for reproducibility testing and an additional 299 samples with HIV VL <50 copies/mL (99); 1.7 log₁₀ to 3.99 log₁₀ (100); and 4 log₁₀ to 5.99 log₁₀/mL (100) were used to compare ViveST to frozen plasma samples using the VERSANT^® HIV-1 RNA 3.0 Assay. Results were compared using Student t-test, Pearson correlation and Bland–Altman analyses.ResultsMean intra-assay variance among frozen and dried plasma triplicates was 0.15 log₁₀ and 0.09 log₁₀ copies/mL respectively (n = 10, P = NS). Compared to frozen plasma, there was a mean reduction of 0.3 log₁₀, 0.27 log₁₀, and 0.35 log₁₀ copies/mL at the 4 log₁₀, 3 log₁₀, and 2 log₁₀ copy/mL samples respectively (n = 30, all comparisons, P < 0.01). Overall correlation between 299 frozen and ViveST samples was r = 0.97, where 12 of 99 undetectable frozen VL were positive with ST, and 12 of 200 frozen detectable VL were undetectable with ViveST (mean VL 2.1, 1.9 log₁₀ copies/mL respectively).ConclusionsHIV-1 viral load results using ViveST were reproducible, correlated well with frozen plasma, though yielding minimally lower values. Our data suggest that dried plasma for HIV-1 VL testing using ViveST has promise for use in HIV clinical practice.     

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

Dried blood spots (DBS) collected onto filter paper have eased the difficulty of blood collection in resource-limited settings. Currently, Whatman 903 (W-903) filter paper is the only filter paper that has been used for HIV load and HIV drug resistance (HIVDR) testing. We therefore evaluated two additional commercially available filter papers, Ahlstrom grade 226 (A-226) and Munktell TFN (M-TFN), for viral load (VL) testing and HIVDR genotyping using W-903 filter paper as a comparison group. DBS specimens were generated from 344 adult patients on antiretroviral therapy (ART) in Botswana. The VL was measured with NucliSENS EasyQ HIV-1 v2.0, and genotyping was performed for those specimens with a detectable VL (≥2.90 log₁₀ copies/ml) using an in-house method. Bland-Altman analysis revealed a strong concordance in quantitative VL analysis between W-903 and A-226 (bias = −0.034 ± 0.246 log₁₀ copies/ml [mean difference ± standard deviation]) and W-903 and M-TFN (bias = −0.028 ± 0.186 log₁₀ copies/ml) filter papers, while qualitative VL analysis for virological failure determination, defined as a VL of ≥3.00 log₁₀ copies/ml, showed low sensitivities for A-266 (71.54%) and M-TFN (65.71%) filter papers compared to W-903 filter paper. DBS collected on M-TFN filter paper had the highest genotyping efficiency (100%) compared to W-903 and A-226 filter papers (91.7%) and appeared more sensitive in detecting major HIVDR mutations. DBS collected on A-226 and M-TFN filter papers performed similarly to DBS collected on W-903 filter paper for quantitative VL analysis and HIVDR detection. Together, the encouraging genotyping results and the variability observed in determining virological failure from this small pilot study warrant further investigation of A-226 and M-TFN filter papers as specimen collection devices for HIVDR monitoring surveys.     

Concurrent genotyping and quantitation of cytomegalovirus gB genotypes in solid-organ-transplant recipients by use of a real-time PCR assay

We have developed a real-time genotyping and quantitative PCR (RT-GQ-PCR) assay to genotype cytomegalovirus (CMV) and quantify viral loads simultaneously in solid organ transplant (SOT) recipients. Special minor-groove DNA-binding probes were designed based on sequence polymorphism in the gB gene to increase genotyping specificity for gB1 to gB4. For validation, 28 samples with known genotypes determined by restriction fragment analysis (RFA) and 121 with unknown genotypes were tested. All samples were from SOT patients with CMV viremia. A 100% concordance for genotyping was achieved by using the RT-GQ-PCR with known genotypes determined by RFA. The RT-GQ-PCR identified more cases of CMV infections with mixed genotypes than RFA did. No cross-reaction between genotypes was observed. All four gB genotypes were detected in the 121 samples of unknown genotype. gB1 was the predominant single genotype (n = 61, 50.4%), followed by gB2 (n = 26, 21.0%), gB3, (n = 11, 9.1%), and gB4 (n = 3, 2.5%). Mixed-genotype infections were detected in 17% (20/121) of the samples. Patients with mixed-genotype infections had significantly higher CMV viral loads than those with single-genotype infections (P = 0.019). The RT-GQ-PCR assay was found to be highly sensitive and specific, with a wide dynamic range (2.7 to 10.7 log₁₀ copies/ml) and very good precision (coefficient of variation, ~1.78%). With the prominent feature of concurrent CMV gB genotyping and quantitation in a single reaction, the new assay provides a rapid and cost-effective method for monitoring CMV infection in SOT recipients.     

Neutralization Profiles of Sera from Human Immunodeficiency Virus (HIV)-Infected Individuals: Relationship to HIV Viral Load and CD4 Cell Count

The relationship of the neutralizing activity (NA) profile of sera from human immunodeficiency virus (HIV)-infected individuals to the HIV viral load and the absolute CD4 count was examined. The NA of 24 serum samples against autologous isolates (AI) and HIV type 1 strain MN was examined. Three NA patterns were recognized. Nine sera neutralized both AI and MN (+/+), six sera neutralized MN but not AI (−/+), and nine sera failed to neutralize both AI and MN (−/−). The identification of the three neutralization patterns (+/+, −/+, and −/−) indicated that resistance to neutralization was progressive. A reciprocal relationship between the viral burden of the patients and the NA profiles was observed. The nine subjects with a −/− NA profile had a plasma viral load of ≥5 × 10⁴ copies/ml and a cellular viral burden of ≥1,122 infectious units per million viable cells, which were significantly different from those of the other groups (P < 0.02). These patterns were independent of the phenotypic characteristics of the virus. Longitudinally, subjects with a −/− profile at baseline gained their HIV-specific NA by 24 weeks of antiretroviral therapy when this was associated with a ≥1-log₁₀ decline in the plasma HIV viral load. The sera from week 24 from some patients were able to neutralize both the 24-week and the baseline dominant virus isolates. A change in CD4 cell count of 50 or more in either direction predicted a −/− or +/+ profile. The verification of the autologous NA profile might be important in selecting patients who may benefit from immune-based therapies involving neutralizing monoclonal antibodies.     

Comparison of NucliSens and Amplicor Monitor Assays for Quantification of Human Immunodeficiency Virus Type 1 (HIV-1) RNA in Plasma of Persons with HIV-1 Subtype A Infection in Abidjan, Côte d’Ivoire

We compared the sensitivity and accuracy of the NucliSens assay and those of both the standard and modified (addition of a new primer set, primer mix 1, supplied by Roche) Amplicor HIV Monitor assays to quantify human immunodeficiency virus type 1 (HIV-1) RNA in persons infected with HIV-1 subtype A in Abidjan, Côte d’Ivoire. Seventy-one plasma samples from HIV-1-seropositive persons at different stages of HIV infection and 15 samples from HIV antibody-negative persons were analyzed. The HIV-1 genetic subtype was determined either by DNA sequencing or by a restriction fragment length polymorphism assay. Of the 71 samples, 70 (98%) were subtype A and 1 was subtype G. Of the 70 subtype A samples, the proportion of RNA-positive plasma samples and mean HIV-1 RNA levels were significantly higher by the modified HIV Monitor assay (n = 67 [96%]; mean RNA levels, 5.2 log₁₀ HIV-1 RNA copies/ml) than the NucliSens assay (n = 56 [80%]; 4.3 log₁₀ HIV-1 RNA copies/ml) or the standard HIV Monitor assay (n = 44 [63%]; mean RNA levels, 3.8 log₁₀ HIV-1 RNA copies/ml) (all P values were <0.05). The HIV-1 RNA levels by the modified HIV Monitor assay correlated significantly with those by the NucliSens assay (r = 0.76; P < 0.001) and the standard HIV Monitor assay (r = 0.57; P < 0.001), as did the RNA levels by the NucliSens and the standard HIV Monitor assays (r = 0.60; P < 0.001). Lower CD4 cell counts were significantly correlated with higher HIV-1 RNA levels by all three assays (r = −0.47 for the NucliSens assay, −0.45 for the standard HIV Monitor assay, and −0.62 for the modified HIV Monitor assay). These results indicate that the modified HIV Monitor assay has the highest sensitivity and efficiency at quantifying the levels of RNA in persons infected with HIV-1 subtype A and thus constitutes a valuable tool for the monitoring of RNA levels in areas of Africa were HIV-1 subtype A is predominant.     

Toward Standardization of BK Virus Monitoring: Evaluation of the BK Virus R-gene Kit for Quantification of BK Viral Load in Urine,Whole-Blood,and Plasma Specimens

Screening of BK virus (BKV) replication is recommended to identify patients at increased risk of BKV-associated diseases. However, the heterogeneity of molecular techniques hinders the establishment of universal guidelines for BKV monitoring. Here we aimed to compare the performance of the CE-marked BK virus R-gene kit (R-gene) to the performance of our in-house assay for quantification of BKV DNA loads (BKVL). A 12-specimen panel from the Quality Control for Molecular Diagnostics (QCMD) organization, 163 urine samples, and 88 paired specimens of plasma and whole blood (WB) from transplant recipients were tested. Both the R-gene and in-house assays showed a good correlation within the QCMD panel (r = 0.995 and r = 0.989, respectively). BKVL were highly correlated between assays, although positive biases were observed with the in-house assay in analysis of urine (0.72 ± 0.83 log₁₀ copies/ml), plasma (1.17 ± 0.63 log₁₀ copies/ml), and WB (1.28 ± 0.37 log₁₀ copies/ml). Recalibration with a common calibrator significantly reduced the bias in comparisons between assays. In contrast, BKVL was underestimated with the in-house PCR in eight samples containing BKV genotype II, presenting point mutations at primer-annealing sites. Using the R-gene assay, plasma and WB specimens were found to be equally suitable for quantification of BKVL, as indicated by the high correlation coefficient (r = 0.965, P < 0.0001). In conclusion, the R-gene assay demonstrated reliable performance and higher accuracy than the in-house assay for quantification of BKVL in urine and blood specimens. Screening of BKV replication by a well-validated commercial kit may enable clinical laboratories to assess viral loads with greater reproducibility and precision.     

Efficiencies of Four Versions of the AMPLICOR HIV-1 MONITOR Test for Quantification of Different Subtypes of Human Immunodeficiency Virus Type 1

Three versions of a commercial human immunodeficiency virus (HIV) type 1 (HIV-1) load test (the AMPLICOR HIV-1 MONITOR Test versions 1.0, 1.0+, and 1.5; Roche Diagnostics, Branchburg, N.J.) were evaluated for their ability to detect and quantify HIV-1 RNA of different genetic subtypes. Plasma samples from 96 patients infected with various subtypes of HIV-1 (55 patients infected with subtype A, 9 with subtype B, 21 with subtype C, 2 with subtype D, 7 with subtype E, and 2 with subtype G) and cultured virus from 29 HIV-1 reference strains (3 of subtype A, 6 of subtype B, 5 of subtype C, 3 of subtype D, 8 of subtype E, 3 of subtype F, and 1 of subtype G) were tested. Detection of subtypes A and E was significantly improved with versions 1.0+ and 1.5 compared to that with version 1.0, whereas detection of subtypes B, C, D, and G was equivalent with the three versions. Versions 1.0, 1.0+, and 1.5 detected 65, 98, and 100% of the subtype A-infected samples from patients, respectively, and 71, 100, and 100% of the subtype E-infected samples from patients, respectively. Version 1.5 yielded a significant increase in viral load for samples infected with subtypes A and E (greater than 1 log₁₀ HIV RNA copies/ml). For samples infected with subtype B, C, and D and tested with version 1.5, only a slight increase in viral load was observed (<0.5 log₁₀). We also evaluated a prototype automated version of the test that uses the same PCR primers as version 1.5. The results with the prototype automated test were highly correlated with those of the version 1.5 test for all subtypes, but were lower overall. The AMPLICOR HIV-1 MONITOR Test, version 1.5, yielded accurate measurement of the HIV load for all HIV-1 subtypes tested, which should allow the test to be used to assess disease prognosis and response to antiretroviral treatment in patients infected with a group M HIV-1 subtype.     

HIV Load Testing with Small Samples of Whole Blood

Access to human immunodeficiency virus (HIV) viral load (VL) testing is of paramount importance for the success of antiretroviral therapy treatment campaigns throughout the world. In many countries, limited laboratory infrastructure and transport capacities preclude a substantial number of people infected with HIV from accessing the necessary testing. Point-of-care diagnostic testing methods for those with HIV infection provide a compelling solution to addressing this challenge. To facilitate ease of use in such tests, finger-stick whole blood (WB) would constitute an ideal sample type if test performance equivalent to laboratory testing could be ensured. To determine the diagnostic sensitivity of a VL assay based on small volumes of WB, we analyzed 1,094 sample pairs of 1 ml of plasma and 10 μl of WB from donors confirmed to be HIV positive. The probability of detecting HIV nucleic acids in 10 μl of blood was 59.3% (95% confidence interval, 54.9 to 63.6%), 85.1% (80.0 to 90.2%), 91.5% (88.1 to 95%), and 100% when the corresponding plasma samples had an undetectable VL, a detectable VL less than 40 viral copies/ml (cp/ml), a VL between 40 and 4,000 cp/ml, and a VL greater than 4,000 cp/ml, respectively. Capillary blood and venous blood yielded comparable diagnostic sensitivities. Furthermore, our data indicate that WB could be used to monitor VL changes after highly active antiretroviral therapy (HAART) started. Thus, we have demonstrated the feasibility of small volumes of venous and finger-stick WB as valid samples for VL testing. This approach should facilitate the development of robust point-of-care HIV VL tests.Universal access to highly active antiretroviral therapy (HAART) is crucial in the fight against human immunodeficiency virus (HIV) and AIDS throughout the world. Through international efforts, more than 4 million patients were placed on HAART in December 2008 (43). In the same period, more than 5 million people were in need of HAART but had no access to treatment, according to the World Health Organization (WHO) (43). Increased access to therapy calls for careful monitoring to detect therapy failure and to ensure adherence (17, 20). Maintenance of a low viral load (VL) under HAART will help to substantially decrease the spread of the epidemic (10). Moreover, models suggest that universal access to therapy could eventually lead to epidemiological eradication of the disease (16).The plasma HIV RNA level is well established as a prognostic marker for the HIV-1 infection (29, 30) for monitoring the response to antiretroviral therapy (33) and therapy adherence (6, 17, 23, 37). For high-income countries, monitoring treatment response by measuring the plasma VL every 3 or 4 months is recommended by the International AIDS Society (19). Numerous HIV VL tests have been developed and commercialized using EDTA plasma as the sample of choice, but in settings with limited infrastructure the transport of fresh samples and generation of plasma is difficult and sometimes impossible. Dried blood and plasma spots have been evaluated as an alternative sample material to obtain VL data (1, 12, 25). However, as summarized in a systematic review, these methods are less sensitive, with a lower detection limit between 2.9 and 3.6 log₁₀ copies/ml (cp/ml), depending on the spot volume (18). In addition, commercially available tests target RNA from viral particles present in the plasma. When using dried blood spots with such tests, a substantial portion of proviral DNA integrated into the host genome may also be amplified and not excluded from the analysis, thus making a comparison of data difficult with measurements on EDTA plasma (32, 41).There is an urgent need for a simple, rapid, and affordable point-of-care VL assay. Such an assay will require small volumes of whole blood (WB) instead of large volumes of plasma and, therefore, would be particularly useful for infant diagnostics, where large samples volumes are difficult if not impossible to obtain. A “whole-blood approach” is supported by study data wherein the Procleix Discriminatory HIV-1 assay was used to qualitatively analyze 63 WB samples in comparison to corresponding plasma samples. It was found that of 11 plasma samples below the level of detection, 8 contained detectable amounts of HIV-1 RNA (W. Andrews, P. Yan, C. Harrington, B. Phelps, T. Elbeik, E. Fiebig, and V. Ng, poster presented at the annual meeting of the American Association of Blood Banks [AABB], 2003). In an earlier publication, one frozen WB sample was successfully analyzed by using the Procleix Discriminatory HIV-1 assay to prove an infection with HIV (39). However, no comprehensive study has been undertaken thus far to demonstrate utility of small-volume WB samples for VL monitoring of HIV-1. Therefore, in our study we measured the VL in 1 ml of plasma and in 10 μl of venous WB to determine the diagnostic sensitivities (36) of both assays. Furthermore, we compared the diagnostic sensitivities obtained with capillary and venous WB. To demonstrate that WB VL can be used for HAART monitoring, we performed a longitudinal analysis of plasma and blood samples from patients starting HAART. We show the feasibility of VL monitoring using only 10 μl of WB.     

Immunological responses and long-term treatment interruption after human immunodeficiency virus type 1 (HIV-1) lipopeptide immunization of HIV-1-infected patients: the LIPTHERA study

We studied the time course of immunological and virological markers after highly active antiretroviral therapy (HAART) interruption in chronically human immunodeficiency virus type 1 (HIV-1)-infected patients immunized with an HIV lipopeptide preparation. In a prospective open pilot study, 24 HIV-1-infected HAART-treated patients with undetectable plasma viral loads (pVLs) and CD4⁺ T-cell counts above 350/mm³ were immunized at weeks 0, 3, and 6 with a candidate vaccine consisting of six HIV lipopeptides. At week 24, patients with pVLs of <1.7 log₁₀ copies/ml were invited to stop taking HAART. Antiretroviral therapy was resumed if the pVL rose above 4.47 log₁₀ copies/ml and/or if the CD4⁺ cell count fell below 250/mm³. Immunological and virologic parameters were studied before and after HAART interruption. The median baseline and nadir CD4⁺ cell counts were 482 (interquartile range [IQR], 195 to 826) and 313 (IQR, 1 to 481)/mm³, respectively. New specific CD8⁺ cell responses to HIV-1 epitopes were detected after immunization in 13 (57%) of 23 assessable patients. Twenty-one patients were evaluated 96 weeks after HAART interruption. The median time to pVL rebound was 4 weeks (IQR, 2 to 6), and the median peak pVL was 4.26 (IQR, 3 to 5) log₁₀ copies/ml. Thirteen of these 21 patients resumed HAART a median of 60 weeks after immunization (IQR, 9.2 to 68.4 weeks), when the median pVL was 4.8 (IQR, 2.9 to 5.7) log₁₀ copies/ml and the median CD4⁺ cell count was 551 (IQR, 156 to 778)/mm³. Eight patients were still off therapy at 96 weeks, with a median pVL of 4 (IQR, 1.7 to 4.6) log₁₀ copies/ml and a median CD4⁺ cell count of 412 (IQR, 299 to 832)/mm³. No clinical disease progression had occurred. Despite the lack of a control arm, these findings warrant a randomized study of therapeutic vaccination with HIV lipopeptides followed by long-term HAART interruption in AIDS-free chronically infected 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.     

Nation-wide measure of variability in HCMV,EBV and BKV DNA quantification among centers involved in monitoring transplanted patients

BackgroundInter-laboratory variability in quantifying pathogens involved in viral disease following transplantation may have a great impact on patient care, especially when pre-emptive strategies are used for prevention.ObjectivesThe aim of this study was to analyze the variability in quantifying CMV, EBV and BKV DNA from 15 virology laboratories of the Italian Infections in Transplant Working Group (GLaIT) involved in monitoring transplanted patients.Study designPanels from international Quality Control programs for Molecular Diagnostics (QCMD, year 2012), specific for the detection of CMV in plasma, CMV in whole blood (WB), EBV and BKV were used. Intra- and inter-laboratory variability, as well as, deviations from QCMD consensus values were measured.Results100% specificity was obtained with all panels. A sensitivity of 100% was achieved for EBV and BKV evaluations. Three CMV samples, with concentrations below 3 log₁₀ copies/ml, were not detected by a few centers. Mean intra-laboratory variability (% CV) was 1.6 for CMV plasma and 3.0 for CMV WB. Mean inter-laboratory variability (% CV) was below 15% for all of the tested panels. Inter-laboratory variability was higher for CMV in WB with respect to the CMV plasma panel (3.0 vs 1.6% CV). The percentiles 87.7%, 58.6%, 89.6% and 74.7% fell within ± 0.5 log₁₀ difference of the consensus values for CMV plasma, CMV WB, EBV and BKV panels, respectively.ConclusionsAn acceptable intra- and inter-laboratory variability, in comparison with international standards was observed in this study. However, further harmonization in viral genome quantification is a reasonable goal for the future.     

Quantitative PCR in the diagnosis of CMV infection and in the monitoring of viral load during the antiviral treatment in renal transplant patients

Cytomegalovirus (CMV) infection is a significant problem in transplantation. In this study, a quantitative PCR test was compared with the CMVpp65 antigenemia assay not only in the diagnosis CMV infections but especially in the monitoring of viral loads during ganciclovir treatment of CMV disease in individual renal transplant patients. Altogether 342 blood specimens were obtained from 116 patients. Blood specimens were used for Cobas Amplicor Monitor plasma PCR and for the pp65 assay. Also shell vial culture was performed. The patients with a positive pp65 finding were monitored for CMV weekly during ganciclovir treatment and/or until the antigenemia subsided. CMV was detected in 31/116 (27%) patients, of whom 14 (12%) developed CMV disease and were treated with ganciclovir. CMV was found by shell vial culture in 13/14 cases, but by PCR and pp65 test in all 14 patients. CMV was detected in 156 (45%) samples; by PCR in 121/156 (range 344-103,000 copies/ml) and by pp65 test in 138/156 (range 1-1,000 positive cells/50,000 leukocytes) and by culture in 59/156 (38%) only. The peak viral loads were significantly (P<0.0001) higher in CMV disease than in untreated infections (19,650 vs. 379 copies/ml, and 100 vs. 5pp65 positive cells). In the monitoring of individual patients, the time-related CMV-DNAemia and pp65 antigenemia correlated well during the treatment of CMV disease. In conclusion, Cobas Amplicor Monitor plasma PCR and CMVpp65 antigen assays can be equally used in the diagnosis CMV infection and in the monitoring of viral load during antiviral treatment.     

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.     

Immunity to Human Immunodeficiency Virus (HIV) in Children with Chronic HIV Infection Receiving Highly Active Antiretroviral Therapy

Our objective was to describe the CD4-mediated human immunodeficiency virus (HIV)-specific cell-mediated immunity (CMI) and its virologic and immunologic correlates in children with chronic HIV infection on highly active antiretroviral therapy (HAART). Twelve HIV-infected children on stable antiretroviral therapy with a median level of CD4⁺ lymphocytes (CD4%) of 25.5% and a median viral load (VL) of 786 HIV RNA copies/ml were enrolled in this study. Nine of these children were also cytomegalovirus (CMV) seropositive. Blood mononuclear cells, stimulated with HIV and CMV antigens, were used to measure lymphocyte proliferation and to enumerate gamma interferon (IFN-γ)-producing CD4⁺ cells. HIV CMI and CMV CMI were detected in similar proportions of patients and correlated with each other, although the HIV responses were less robust. HIV lymphocyte proliferation significantly increased with lower HIV VL and showed a trend to increase with higher CD4% and longer time on HAART. The in vitro IFN-γ response to HIV or CMV was not affected by CD4%, VL, or HAART. Pediatric patients with established HIV infection on HAART frequently exhibit HIV CMI despite undetectable HIV replication. We concluded that the association between HIV CMI and CMV CMI indicates that the same factors govern responsiveness to either antigen.     

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.