Accuracy of quantitative viral secondary standards: a re-examination.

Interlaboratory agreement of viral load assays depends on the accuracy and uniformity of quantitative calibrators. Previous work demonstrated poor agreement of secondary cytomegalovirus (CMV) standards with nominal values. This study re-evaluated this issue among commercially produced secondary standards for both BK virus (BKV) and CMV, using digital polymerase chain reaction (dPCR) to compare the materials from three different manufacturers. Overall, standards showed an improved agreement compared to prior work, against nominal values in both log10 copies/mL and log10 international unit (IU)/mL, with bias from manufacturer-assigned nominal values of 0.0-0.9 log10 units (either copies or IU)/mL. Standards normalized to IU and those values assigned by dPCR rather than by real-time PCR (qPCR) showed better agreement with nominal values. The latter reinforces prior conclusions regarding the utility of using such methods for quantitative value assignment in reference materials. Quantitative standards have improved over the last several years, and the remaining bias from nominal values might be further reduced by universal implementation of dPCR methods for value assignment, normalized to IU. IMPORTANCE: Interlaboratory agreement of viral load assays depends on accuracy and uniformity of quantitative calibrators. Previous work, published in JCM several years ago, demonstrated poor agreement of secondary cytomegalovirus (CMV) standards with nominal values. This study re-evaluated this issue among commercially produced secondary standards for both BK virus (BKV) and CMV, using digital polymerase chain reaction (dPCR) to compare the materials from three different manufacturers. Overall, standards showed an improved agreement compared to prior work, against nominal values, indicating a substantial improvement in the production of accurate secondary viral standards, while supporting the need for further work in this area and for the broad adaption of international unit (IU) as a reporting standard for quantitative viral load results.

Matrix Matters: Assessment of Commutability among BK Virus Assays and Standards.

Quantitative testing of BK virus (BKPyV) nucleic acid has become the standard of care in transplant patients. While the relationship between interassay harmonization and commutability has been well characterized for other transplant-related viruses, it has been less well studied for BKPyV, particularly regarding differences in commutability between matrices. Here, interassay agreement was evaluated among six real-time nucleic acid amplification tests (NAATs) and one digital PCR (dPCR) BKPyV assay. Differences in the commutability of three quantitative standards was examined across all assays using a variety of statistical approaches. Panels, including 40 samples each of plasma and urine samples previously positive for BKPyV, together with one previously negative plasma sample and four previously negative urine samples, were tested using all assays, with each real-time NAAT utilizing its usual quantitative calibrators. Serial dilutions of WHO, National Institute for Standards and Technology (NIST), and commercially produced (Exact/Bio-Rad) reference materials were also run by each assay as unknowns. The agreement of the clinical sample values was assessed as a group and in a pairwise manner. The commutability was estimated using both relativistic and quantitative means. The quantitative agreement across assays in the urine samples was within a single log10 unit across all assays, while the results from the plasma samples varied by 2 to 3 log10 IU/mL. The commutability showed a similar disparity between the matrices. Recalibration using international standards diminished the resulting discrepancies in some but not all cases. Differences in the sample matrix can affect the commutability and interassay agreement of quantitative BKPyV assays. Differences in commutability between matrices may largely be due to factors other than those such as amplicon size, previously described as important in the case of cytomegalovirus. Continued efforts to standardize viral load measurements must address multiple sources of variability and account for differences in assay systems, quantitative standards, and sample matrices.

Impact of Fragmentation on Commutability of Epstein-Barr Virus and Cytomegalovirus Quantitative Standards.

Despite the adaptation of international standards, quantitative viral load testing of transplant-associated viruses continues to be limited by interlaboratory disagreement. Studies have suggested that this disagreement and the poor commutability of standards may, in some cases, be linked to amplicon size and the fragmentation of circulating viral DNA. We evaluated target fragmentation as a cause of noncommutability and pretest fragmentation of quantitative standards as a potential means of increasing commutability and interassay agreement. Forty-two cytomegalovirus (CMV)-positive and 41 Epstein-Barr virus (EBV)-positive plasma samples, together with two different quantitative standards for each virus, were tested as unknowns using 10 different quantitative PCR assays at 5 different laboratories. Standards were tested both intact and after intentional fragmentation by ultrasonication. Quantitative agreement between methods was assessed, together with commutability, using multiple statistical approaches. Most assays yielded results within 0.5 log10 IU/ml of the mean for CMV, while for EBV a greater variability of up to 1.5 log10 IU/ml of the mean was shown. Commutability showed marked improvement following fragmentation of both CMV standards but not after fragmentation of the EBV standards. These findings confirm the impact of amplicon size and target fragmentation on commutability for CMV and suggest that for some (but not all) viruses, interlaboratory harmonization can be improved through the use of fragmented quantitative standards.

A Comprehensive Statistical Framework for Determination of Commutability, Accuracy, and Agreement in Clinical DNAemia Assays.

Despite advances in the standardization of quantitative DNAemia tests and efforts to better understand and characterize the performance of reference materials in different assays, it remains unclear how the commutability performance of reference materials is related to intra- and interassay agreement. Building upon previous work, we describe a comprehensive framework to determine the relationship of commutability with assay accuracy and agreement. The use of this framework is illustrated using previously generated data regarding the performance of four quantitative Epstein-Bar virus (EBV) PCR assays with the WHO and ABI standards as examples. The use of these statistical tools can link the performance characteristics of one or more assays with predetermined clinical decision limits and may help improve the development, validation, and clinical utility of such DNAemia tests.

Progress in Quantitative Viral Load Testing: Variability and Impact of the WHO Quantitative International Standards.

It has been hoped that the recent availability of WHO quantitative standards would improve interlaboratory agreement for viral load testing; however, insufficient data are available to evaluate whether this has been the case. Results from 554 laboratories participating in proficiency testing surveys for quantitative PCR assays of cytomegalovirus (CMV), Epstein-Barr virus (EBV), BK virus (BKV), adenovirus (ADV), and human herpesvirus 6 (HHV6) were evaluated to determine overall result variability and then were stratified by assay manufacturer. The impact of calibration to international units/ml (CMV and EBV) on variability was also determined. Viral loads showed a high degree of interlaboratory variability for all tested viruses, with interquartile ranges as high as 1.46 log10 copies/ml and the overall range for a given sample up to 5.66 log10 copies/ml. Some improvement in result variability was seen when international units were adopted. This was particularly the case for EBV viral load results. Variability in viral load results remains a challenge across all viruses tested here; introduction of international quantitative standards may help reduce variability and does so more or less markedly for certain viruses.

Quantitative Assessment of Commutability for Clinical Viral Load Testing Using a Digital PCR-Based Reference Standard.

Given recent advances in the development of quantitative standards, particularly WHO international standards, efforts to better understand the commutability of reference materials have been made. Existing approaches in evaluating commutability include prediction intervals and correspondence analysis; however, the results obtained from existing approaches may be ambiguous. We have developed a "deviation-from-ideal" (DFI) approach to evaluate commutability of standards and applied it to the assessment of Epstein-Bar virus (EBV) load testing in four quantitative PCR assays, treating digital PCR as a reference assay. We then discuss advantages and limitations of the DFI approach as well as experimental design to best evaluate the commutability of an assay in practice.

Factors contributing to variability of quantitative viral PCR results in proficiency testing samples: a multivariate analysis.

While viral load testing has gained widespread acceptance, a primary limitation remains the variability of results, particularly between different laboratories. While some work has demonstrated the importance of standardized quantitative control material in reducing this variability, little has been done to explore other important factors in the molecular amplification process. Results of 185 laboratories enrolled in the College of American Pathologists (CAP) 2009 viral load proficiency testing (PT) survey (VLS) were examined. This included 165 labs (89.2%) testing for cytomegalovirus (CMV), 99 (53.5%) for Epstein-Barr virus (EBV), and 64 (34.6%) for BK virus (BKV). At the time of PT, laboratories were asked a series of questions to characterize their testing methods. The responses to these questions were correlated to mean viral load (MVL) and result variability (RV). Contribution of individual factors to RV was estimated through analysis of variance (ANOVA) modeling and the use of backward selection of factors to fit those models. Selection of the quantitative calibrator, commercially prepared primers and probes, and amplification target gene were found most prominently associated with changes in MVL or RV for one or more of the viruses studied. Commercially prepared primers and probes and amplification target gene made the largest contribution to overall variability. Factors contributing to MVL and RV differed among viruses, as did relative contribution of each factor to overall variability. The marked variability seen in clinical quantitative viral load results is associated with multiple aspects of molecular testing design and performance. The reduction of such variability will require a multifaceted approach to improve the accuracy, reliability, and clinical utility of these important tests.

Interlaboratory comparison of epstein-barr virus viral load assays.

To assess interlaboratory variability in qualitative and quantitative Epstein-Barr virus (EBV) viral load (VL) testing, we distributed a panel of samples to 28 laboratories in the USA, Canada and Europe who performed testing using commercially available reagents (n = 12) or laboratory-developed assays (n = 18). The panel included two negatives, seven constructed samples using Namalwa and Molt-3 cell lines diluted in plasma (1.30-5.30 log(10) copies/mL) and three clinical plasma samples. Significant interlaboratory variation was observed for both actual (range 1.30-4.30 log(10) copies/mL) and self-reported (range, 1.70-3.30 log(10) copies/mL) lower limits of detection. The variation observed in reported results on individual samples ranged from 2.28 log(10) (minimum) to 4.14 log(10) (maximum). Variation was independent of dynamic range and use of commercial versus laboratory-developed assays. Overall, only 47.0% of all results fell within acceptable standards of variation: defined as the expected result +/- 0.50 log(10). Interlaboratory variability on replicate samples was significantly greater than intralaboratory variability (p < 0.0001). Kinetics of change in VL appears more relevant than absolute values and clinicians should understand the uncertainty associated with absolute VL values at their institutions. The creation of an international reference standard for EBV VL assay calibration would be an initial important step in quality improvement of this laboratory tool.

Human immunodeficiency virus in plasma and cervicovaginal secretions in Filipino women.

This study examined 30 HIV-infected women in Manila to assess the relationship between cervicovaginal and plasma HIV-1 viral load. An interview and gynaecologic examination was conducted and cervicovaginal lavage (CVL) and venous blood specimens were collected. HIV-1 RNA was detected in plasma samples of 24 patients (80%) and in CVL samples of 18 women (60%); 16 patients (53%) had detectable levels in both. CVL HIV-1 RNA was detectable in 75% of women (6/8) with plasma viral loads between 10,000 and 100,000 copies/mL and in 77% of women (10/13) with plasma viral loads higher than 100,000 copies/mL (P =0.0086). Among women with CD4 cell counts of less than 200, 200-500, and greater than 500/mm(3), CVL HIV-1 RNA was detected in 73%, 69%, and 17% of women, respectively (P =0.1428). HIV-1 RNA shedding in the genital tract was significantly associated with plasma viral load.

Cytomegalovirus (CMV) and human immunodeficiency virus (HIV) burden, CMV end-organ disease, and survival in subjects with advanced HIV infection (AIDS Clinical Trials Group Protocol 360).

We undertook a prospective study to analyze cytomegalovirus (CMV) end-organ disease (EOD) in subjects with advanced human immunodeficiency virus (HIV) infection. Of 403 individuals without prior CMV EOD who were followed up for a median of 151 weeks, 56 died and 21 developed CMV EOD. Twenty of the subjects with CMV EOD had CD4 cell counts of < or =50 cells/mm3 and HIV RNA level of >10,000 copies/mL of plasma at baseline; in these 20 subjects, an increase of CMV DNA level to greater than the quantification limits was associated with CMV EOD. A CD4 cell count of < or =100 cells/mm3 and an HIV RNA level of >10,000 copies/mL of plasma at baseline, a CMV DNA level of >200 copies/mL of blood during follow-up, or development of CMV EOD were all associated with decreased survival. HIV-infected subjects with CD4 cell counts of < or =50 cells/mm3 and HIV RNA levels of >10,000 copies/mL of plasma should have blood fractions screened for CMV DNA; if CMV DNA is detected, CMV prophylaxis might be considered.

Association between bacterial vaginosis and expression of human immunodeficiency virus type 1 RNA in the female genital tract.

We assessed the effect of lower genital tract infections on human immunodeficiency virus type 1 (HIV-1) RNA shedding in the female genital tract. Bacterial vaginosis was significantly associated with HIV-1 RNA expression in the female genital tract of HIV-infected women.

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.

Effect of highly active antiretroviral therapy on cervicovaginal HIV-1 RNA.

OBJECTIVES: To determine the frequency of cervicovaginal lavage and plasma HIV-1 RNA levels that are below detectable levels (< 400 copies/ml) among women on highly active antiretroviral therapy (HAART), non-HAART and on no therapy. To compare the effect of initiating HAART on the timing of HIV-1 RNA suppression in the blood plasma and genital tract among antiretroviral-naïve women. METHODS: Data were obtained from 205 HIV-infected women with paired plasma and cervicovaginal lavage viral load measurements. Seven antiretroviral-naïve women starting HAART had viral load measurements performed daily for one week, at 2 weeks and at 1 month after initiating therapy. Viral load quantification was carried out by nucleic acid sequence-based amplification assay. The lower limit of detection was 400 copies/ml. RESULTS: Plasma and cervicovaginal HIV-1 RNA was detectable in 71 and 26% of the women, respectively. Among women with plasma viral loads less than 400, 400-9999, and 10,000 copies/ml or over, genital tract HIV-1 RNA was detected in 3, 17 and 48%, respectively (P < 0.001). Fifty-one per cent of the women with CD4 cell counts of less than 200/mm3 had detectable cervicovaginal viral loads compared with 18% among women with CD4 cell counts of 200/mm3 or over (P < 0.001). Cervicovaginal HIV-1 RNA was less than 400 copies/ml in 85% of those on HAART, 69% of those on non-HAART and 69% of those on no therapy (P < 0.045). In seven antiretroviral-naïve women initiating HAART, cervicovaginal HIV-1 RNA decreased by 0.7-2.1 log10 within 1-14 days of starting therapy. CONCLUSION: The cervicovaginal HIV-1 RNA level was positively correlated with plasma HIV-1 RNA and negatively with the CD4 cell count. The use of HAART was significantly associated with below-detectable levels of HIV-1 RNA in both plasma and the genital tract. HIV-1 RNA suppression in the genital tract may occur rapidly after initiating therapy.

Bilateral acute retinal necrosis caused by cytomegalovirus in an immunocompromised patient.

PURPOSE: To report a case of bilateral acute retinal necrosis caused by cytomegalovirus. METHODS: A diagnostic vitrectomy was performed on a patient with non-Hodgkin lymphoma who presented with a bilateral, rapidly progressing necrotizing retinitis and uveitis. RESULTS: Immunohistochemical studies and polymerase chain reaction disclosed cytomegalovirus as the cause of retinitis. The patient was treated with intravitreal and intravenous ganciclovir. CONCLUSIONS: Although rare, cytomegalovirus may lead to an appearance identical to acute retinal necrosis and should be considered among the viral etiologies of this syndrome.

Vigorous HIV-1-specific CD4+ T cell responses associated with control of viremia.

Virus-specific CD4+ T helper lymphocytes are critical to the maintenance of effective immunity in a number of chronic viral infections, but are characteristically undetectable in chronic human immunodeficiency virus-type 1 (HIV-1) infection. In individuals who control viremia in the absence of antiviral therapy, polyclonal, persistent, and vigorous HIV-1-specific CD4+ T cell proliferative responses were present, resulting in the elaboration of interferon-gamma and antiviral beta chemokines. In persons with chronic infection, HIV-1-specific proliferative responses to p24 were inversely related to viral load. Strong HIV-1-specific proliferative responses were also detected following treatment of acutely infected persons with potent antiviral therapy. The HIV-1-specific helper cells are likely to be important in immunotherapeutic interventions and vaccine development.

Laboratory methods for quantitating HIV RNA.

AIDS: High plasma HIV RNA levels have been shown to be a strong predictor of rapid progression to AIDS after HIV seroconversion. Recently, three quantitation assays that are less expensive, easier to perform, and more reproducible than older methods were introduced: 1) Amplicor HIV Monitor Test (Roche Molecular Systems); 2) Nucleic Acid Sequence-Based Amplification (NASBA) (Organon Technika); and 3) Quantiplex HIV RNA assay, a branched DNA (bDNA) technology (Chiron Corporation). The Amplicor HIV Monitor Test requires 200 uL plasma samples for reverse transcription, polymerase chain reaction (PCR) and nonradioactive detection of amplified DNA. It has a three-log dynamic range with a lower limit of sensitivity of about 500 copies of HIV RNA/mL. The NASBA assay uses nonradioactive detection to measure RNA after nucleic acid isolation and amplification. NASBA is applicable to more body fluids and requires less plasma (100 uL) than the Amplicor test. Its clinical sensitivity is 400 molecules, and the assay is reproducible and interpretable over a four-log range. Quantiplex HIV RNA assay (bDNA) is a signal, rather than target, amplification method, allowing direct measurement of HIV RNA from 1 mL plasma specimens. Detection (via chemoluminescent substrate) requires hybridization of probes and branch DNA (bDNA) amplifier molecules to HIV RNA. The assay's dynamic range is currently 10,000 - 1,600,000 HIV RNA equivalents/mL, and it is about 20-fold less sensitive than the other two assays. Its detection ability is inversely related to the CD4 cell count.^ieng