Randall T. Hayden

Comparison of Droplet Digital PCR to Real-Time PCR for Quantitative Detection of Cytomegalovirus

ABSTRACT Quantitative real-time PCR (QRT-PCR) has been widely implemented for clinical viral load testing, but a lack of standardization and relatively poor precision have hindered its usefulness. Digital PCR offers highly precise, direct quantification without requiring a calibration curve. Performance characteristics of real-time PCR were compared to those of droplet digital PCR (ddPCR) for cytomegalovirus (CMV) load testing. Tenfold serial dilutions of the World Health Organization (WHO) and the National Institute of Standards and Technology (NIST) CMV quantitative standards were tested, together with the AcroMetrix CMV tc panel (Life Technologies, Carlsbad, CA) and 50 human plasma specimens. Each method was evaluated using all three standards for quantitative linearity, lower limit of detection (LOD), and accuracy. Quantitative correlation, mean viral load, and variability were compared. Real-time PCR showed somewhat higher sensitivity than ddPCR (LODs, 3 log10 versus 4 log10 copies/ml and IU/ml for NIST and WHO standards, respectively). Both methods showed a high degree of linearity and quantitative correlation for standards (R 2 ≥ 0.98 in each of 6 regression models) and clinical samples (R 2 = 0.93) across their detectable ranges. For higher concentrations, ddPCR showed less variability than QRT-PCR for the WHO standards and AcroMetrix standards (P < 0.05). QRT-PCR showed less variability and greater sensitivity than did ddPCR in clinical samples. Both digital and real-time PCR provide accurate CMV load data over a wide linear dynamic range. Digital PCR may provide an opportunity to reduce the quantitative variability currently seen using real-time PCR, but methods need to be further optimized to match the sensitivity of real-time PCR.

Multiplexed, Real-Time PCR for Quantitative Detection of Human Adenovirus

ABSTRACT Adenovirus infection is becoming increasingly recognized as a cause of morbidity and mortality in the immunosuppressed patient population. While early detection and quantitation of adenovirus in peripheral blood has been suggested as a means of directing and monitoring antiviral therapy in these patients, few methods have been published, particularly with respect to viral quantitation. A multiplexed real-time PCR assay was developed that can quantitatively detect a wide range of known serotypes of human adenovirus, including all of subgroups A to C. This assay was compared to a qualitative, Southern blot-based PCR assay by using 45 peripheral blood specimens from 16 patients. There was 100% concordance between the two tests in terms of qualitative results. The real-time assay detected adenovirus in patient samples at levels from <200 to 266,681 copies/ml of blood. By using control viral samples, sensitivity was demonstrated to less than 10 copies of viral genome per reaction and quantitative linearity was demonstrated from 10 to 106 copies of input viral DNA. Equivalent sensitivity and linearity were demonstrated for 15 different reference serotypes of adenovirus. Eleven other viral serotypes have complete target region sequence homology to one or more of the strains tested. No cross-reactivity was noted with other commonly isolated viral species. Sequence analysis showed no significant homology with any other human pathogens (bacterial or viral). This assay allows rapid, sensitive, and specific quantitation of adenovirus and may have a significant impact on the care of immunocompromised patients at risk for disseminated viral infection.

Rapid immune reconstitution after a reduced‐intensity conditioning regimen and a CD3‐depleted haploidentical stem cell graft for paediatric refractory haematological malignancies

The main obstacles to successful haploidentical haematopoietic stem cell transplantation from a mismatched family member donor are delayed immune reconstitution, vulnerability to infections and severe graft‐versus‐host disease (GvHD). We designed a reduced‐intensity conditioning regimen that excluded total body irradiation and anti‐thymocyte globulin in order to expedite immune reconstitution after a CD3‐depleted haploidentical stem cell transplant. This protocol was used to treat 22 paediatric patients with refractory haematological malignancies. After transplantation, 91% of the patients achieved full donor chimaerism. They also showed rapid recovery of CD3+ T‐cells, T‐cell receptor (TCR) excision circle counts, TCRβ repertoire diversity and natural killer (NK)‐cells during the first 4 months post‐transplantation, compared with those results from a group of patients treated with a myeloablative conditioning regimen. The incidence and extent of viremia were limited and no lethal infection was seen. Only 9% of patients had grade 3 acute GvHD, while 27% patients had grade 1 and another 27% had grade 2 acute GvHD. This well‐tolerated regimen appears to accelerate immune recovery and shorten the duration of early post‐transplant immunodeficiency, thereby reducing susceptibility to viral infections. Rapid T‐cell reconstitution, retention of NK‐cells in the graft and induction of low grade GvHD may also enhance the potential anti‐cancer immune effect.

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

ABSTRACT 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.

Clinical Utility of Viral Load in Management of Cytomegalovirus Infection after Solid Organ Transplantation

SUMMARY The negative impact of cytomegalovirus (CMV) infection on transplant outcomes warrants efforts toward improving its prevention, diagnosis, and treatment. During the last 2 decades, significant breakthroughs in diagnostic virology have facilitated remarkable improvements in CMV disease management. During this period, CMV nucleic acid amplification testing (NAT) evolved to become one of the most commonly performed tests in clinical virology laboratories. NAT provides a means for rapid and sensitive diagnosis of CMV infection in transplant recipients. Viral quantification also introduced several principles of CMV disease management. Specifically, viral load has been utilized (i) for prognostication of CMV disease, (ii) to guide preemptive therapy, (iii) to assess the efficacy of antiviral treatment, (iv) to guide the duration of treatment, and (v) to indicate the risk of clinical relapse or antiviral drug resistance. However, there remain important limitations that require further optimization, including the interassay variability in viral load reporting, which has limited the generation of standardized viral load thresholds for various clinical indications. The recent introduction of an international reference standard should advance the major goal of uniform viral load reporting and interpretation. However, it has also become apparent that other aspects of NAT should be standardized, including sample selection, nucleic acid extraction, amplification, detection, and calibration, among others. This review article synthesizes the vast amount of information on CMV NAT and provides a timely review of the clinical utility of viral load testing in the management of CMV in solid organ transplant recipients. Current limitations are highlighted, and avenues for further research are suggested to optimize the clinical application of NAT in the management of CMV after transplantation.

Galactomannan antigenemia in pediatric oncology patients with invasive aspergillosis.

Background: Diagnosing invasive aspergillosis is difficult but might be improved by detection of circulating galactomannan. Although galactomannan antigenemia has been well studied in the detection of invasive aspergillosis in adult patients, little is known about the expression of circulating galactomannan in immunocompromised children with invasive aspergillosis. Methods: We studied the expression of galactomannan antigen by enzyme immunoassay (EIA) in 990 serum samples from 56 pediatric oncology patients (ages 3 months to 18 years) of whom 17 had proven or probable invasive aspergillosis defined by the European Organization for Research and Treatment of Cancer-Mycoses Study Group criteria. Any sample with a galactomannan EIA Galactomannan index value of ≥0.5 was considered positive. Results: At least 1 serum sample was positive for 11 of 17 pediatric oncology patients (65.7% sensitivity, 95% confidence interval: 38.3–85.7) with invasive aspergillosis. Galactomannan EIA was positive in 99 of 304 samples from patients with proven or probable invasive aspergillosis, and 7 of 686 (1.0%) samples from 39 control subjects resulted in a positive galactomannan EIA result. At least 1 sample tested positive in 5 of the 39 controls (12.8%, 95% confidence interval: 4.3–27.4). No significant association between accuracy and patient age was observed. Among the 7 evaluable galactomannan-positive patients with IA, the galactomannan EIA produced a positive result before clinical or radiographic evidence of infection in 6 cases, with a lead-time to diagnosis ranging from 1 day to 34 days (median: 10 days). In the remaining case, a positive galactomannan was observed on the same day as diagnosis by non-EIA methods. Conclusions: The presence of circulating galactomannan is predictive of invasive aspergillosis in most pediatric oncology patients. Galactomannan antigenemia may precede clinical, microbiologic, or radiographic evidence of invasive aspergillosis.

Comparison of Various Blood Compartments and Reporting Units for the Detection and Quantification of Epstein-Barr Virus in Peripheral Blood

ABSTRACT Epstein-Barr virus (EBV) infection is associated with a broad spectrum of disease. While quantification of EBV nucleic acid in the peripheral blood has been demonstrated to be useful for diagnosis and patient care, the optimal sample type and reporting format for such testing remain uncertain. Using quantitative real-time PCR (QRT-PCR), we evaluated EBV in whole blood (WB), peripheral blood mononuclear cells (PBMC), and plasma in 249 samples from 122 patients. In WB and PBMC, results were reported both in viral copies/ml and in copies/μg of total DNA. Trendings of quantitative values over time among the different sample types were compared. The sensitivities of QRT-PCR using WB and that using PBMC did not differ significantly (P = 0.33), and both were more sensitive than plasma alone (P < 0.0001). EBV viral load results from WB and PBMC paired sample types also showed a significant correlation (P < 0.05), as did results reported in copies/ml and copies/μg DNA for both WB and PBMC (R2 > 0.93). EBV viral loads detected using WB and PBMC trended very closely for the few patients who had multiple positive samples available for analysis. WB and PBMC show comparable sensitivities and a close quantitative correlation when assayed for EBV by QRT-PCR. The close correlation between copies/ml and copies/μg DNA also suggests that normalization to cell number or genomic DNA in cellular specimens may not be necessary.

Timeline, epidemiology, and risk factors for bacterial, fungal, and viral infections in children and adolescents after allogeneic hematopoietic stem cell transplantation.

Advances made in the field of hematopoietic stem cell transplantations (HSCT) over the past 20 years may have had an impact on the distribution of posttransplantation infections. We sought to retrospectively analyze the epidemiology and risk factors for bacterial, fungal, and viral infections in children after allogeneic HSCT in a cohort of 759 children who underwent allogeneic HSCT in a single institution between 1990 and 2009. The association between infections and risk factors of interest at 0 to 30 days, 31 to 100 days, and 101 days to 2 years posttransplantation was evaluated using logistic regression. Difference among the subtypes within each category was studied. There were 243 matched-related donors, 239 matched-unrelated donors (MUDs), and 176 haploidentical donor transplantations. Era of transplantation (0-30 days), peripheral blood stem cell product, acute graft-versus-host disease (aGVHD; 31-100 days), and chronic GVHD (cGVHD; 101-730 days) were associated with higher risk for bacterial infections at the respective time periods. Patients with aGVHD (31-100 days), cGVHD, and older age (101-730 days) were at higher risk for fungal infections. Cytomegalovirus (CMV) donor/recipient (D/R) serostatus (0-100 days), era of transplantation, MUD HSCT (31-100 days), and cGVHD (101-730 days), influenced viral infections. Gram-positive outnumbered gram-negative bacterial infections; aspergillosis and candidemia were equally prevalent in all time periods. Haploidentical donor HSCT was not associated with an increased risk of infections. There seems to be a continuum in the timeline of infections posttransplantation, with bacterial, fungal, and viral infections prevalent in all time periods, particularly late after the transplantation, the risk affected by GVHD, CMV, D/R status, product type, older age, and use of unrelated donors.

Can Multidrug-Resistant Candida auris Be Reliably Identified in Clinical Microbiology Laboratories?

Candida auris, an emerging multidrug-resistant yeast associated with a high mortality rate, has been increasingly reported outside the United States to cause outbreaks in hospital settings ([1][1]). Although this organism is rare in the United States, its prevalence may be underestimated because of

Diagnosis of catheter-related bloodstream infections among pediatric oncology patients lacking a peripheral culture, using differential time to detection.

Background: Current methods for in situ diagnosis of catheter-related bloodstream infections require concurrent collection of central venous catheter (CVC) and peripheral vein (PV) blood cultures. Both the pain and inconvenience of PV cultures are undesirable. Methods: A prospective study was conducted (August 2002 to March 2004) to assess the accuracy of diagnosing catheter-related bloodstream infections based on the difference in time to detection of blood cultures drawn concurrently from 2 lumens of a multilumen CVC. This difference in time to detection between 2 lumens was compared with results of the standard criterion with paired CVC and PV blood cultures. Results: Twenty-one infectious episodes were categorized as catheter-related bloodstream infections and 38 as non-catheter-related bloodstream infections. With a cutoff in difference in time to detection between 2 lumens of ≥180 minutes, the sensitivity of this test to diagnose a catheter-related bloodstream infection was 61% (95% confidence interval, 39–80%) and the specificity was 94% (95% confidence interval, 82–99%). In 4 of 7 episodes with false-negative results, the colony counts in cultures from both lumens were >400 colony-forming units/mL (maximal value reported), indicating the limitation of this method when both lumens of the catheter are colonized. With the pretest probability of catheter-related bloodstream infections ranging from 28% to 54%, the positive predictive value of a difference in time to detection between 2 lumens of ≥180 minutes for diagnosis of catheter-related bloodstream infections ranged from 81% to 93% and the negative predictive value ranged from 67% to 86%. Conclusion: Within the context of its limitations, this novel method provides an alternative for diagnosing catheter-related bloodstream infections among patients with a CVC, without PV cultures.