Choanephora infundibulifera Rhinosinusitis in Man with Acute Lymphoblastic Leukemia, Tennessee, USA.

Choanephora infundibulifera is a member of the Mucorales order of fungi. The species is associated with plants as a saprophyte or parasite and may be responsible for spoilage or disease but is an uncommon cause of human infection. We describe C. infundibulifera rhinosinusitis in a young man with leukemia in Tennessee, USA.

Microbiota Predict Infections and Acute Graft-Versus-Host Disease After Pediatric Allogeneic Hematopoietic Stem Cell Transplantation.

BACKGROUND: Despite preventive measures, infections continue to pose significant risks to pediatric allogeneic hematopoietic cell transplantation (allo-HCT) recipients. The gut microbiota has been linked to clinical outcomes following adult allo-HCT. This study evaluated whether similar disruptions or differing microbiota patterns were associated with infection risk in pediatric allo-HCT. METHODS: In a prospective observational study, fecal samples were obtained from 74 children before conditioning and upon neutrophil recovery. Microbiome signatures identified through sequencing were examined for their associations with infections or acute graft-versus-host disease (aGVHD) in the first-year post-HCT using Cox proportional hazards analysis. RESULTS: Microbiome disruption in adults, did not predict infection risk in pediatric allo-HCT. Unique microbiota signatures were associated with different infections or aGVHD. A ratio of strict and facultative anaerobes (eg, Lachnoclostridium, Parabacteroides) prior to conditioning predicted bacteremia risk (Cox hazard ratio [HR], 3.89). A distinct ratio of oral (eg, Rothia, Veillonella) to intestinal anaerobes (eg, Anaerobutyricum, Romboutsia) at neutrophil recovery predicted likelihood of bacterial infections (Cox HR, 1.81) and viral enterocolitis (Cox HR, 1.96). CONCLUSIONS: Interactions between medical interventions, pediatric hosts, and microbial communities contribute to microbiota signatures that predict infections. Further multicenter study is necessary to validate the generalizability of these ratios as biomarkers.

Molecular Epidemiology and Genetic Relatedness of Clostridioides difficile Isolates in Pediatric Oncology and Transplant Patients Using Whole Genome Sequencing.

BACKGROUND: The incidence of Clostridioides difficile infection (CDI) has been rising among hospitalized children, with poor understanding of genomic variability of C. difficile isolates in this population. METHODS: This was a retrospective cohort study of CDI in inpatient and outpatient pediatric oncology and cell transplant patients (POTPs) in 2016 and 2017. CDI cases were identified by positive C. difficile toxin polymerase chain reaction tests. Retrieved residual stool specimens were cultured anaerobically and toxin-producing C. difficile isolates underwent whole genome sequencing (WGS) followed by core genome multilocus sequence typing. Plausible time and location epidemiologic links among the closely related strains were evaluated to identify potential transmission events. RESULTS: Among 226 CDI episodes in 157 patients, 202 stool samples were cultured and had positive cytotoxicity tests. Sequencing identified 33 different strain types in 162 (80%) isolates. Thirty-nine (28%) patients had multiple episodes of CDI, and 31 clusters of related isolates were identified, 15 (47%) of which involved exclusively multiple specimens from the same patient. For the 16 clusters involving multiple patients, epidemiologic investigation revealed only 2 (12.5%) clusters with potential transmission events. CONCLUSIONS: WGS identified a highly diverse group of C. difficile isolates among POTPs with CDI. Although WGS identified clusters of closely related isolates in multiple patients, epidemiologic investigation of shared inpatient exposures identified potential transmission in only 2 clusters. Clostridioides difficile transmission was uncommon in this population. More than 70% of new CDI reinfections in POTPs are actually recurrences caused by a previous CDI strain.

A conceptual framework for nomenclatural stability and validity of medically important fungi: a proposed global consensus guideline for fungal name changes supported by ABP, ASM, CLSI, ECMM, ESCMID-EFISG, EUCAST-AFST, FDLC, IDSA, ISHAM, MMSA, and MSGERC.

The rapid pace of name changes of medically important fungi is creating challenges for clinical laboratories and clinicians involved in patient care. We describe two sources of name change which have different drivers, at the species versus the genus level. Some suggestions are made here to reduce the number of name changes. We urge taxonomists to provide diagnostic markers of taxonomic novelties. Given the instability of phylogenetic trees due to variable taxon sampling, we advocate to maintain genera at the largest possible size. Reporting of identified species in complexes or series should where possible comprise both the name of the overarching species and that of the molecular sibling, often cryptic species. Because the use of different names for the same species will be unavoidable for many years to come, an open access online database of the names of all medically important fungi, with proper nomenclatural designation and synonymy, is essential. We further recommend that while taxonomic discovery continues, the adaptation of new name changes by clinical laboratories and clinicians be reviewed routinely by a standing committee for validation and stability over time, with reference to an open access database, wherein reasons for changes are listed in a transparent way.

Evolution of vancomycin-resistant Enterococcus faecium during colonization and infection in immunocompromised pediatric patients.

Patients with hematological malignancies or undergoing hematopoietic stem cell transplantation are vulnerable to colonization and infection with multidrug-resistant organisms, including vancomycin-resistant Enterococcus faecium (VREfm). Over a 10-y period, we collected and sequenced the genomes of 110 VREfm isolates from gastrointestinal and blood cultures of 24 pediatric patients undergoing chemotherapy or hematopoietic stem cell transplantation for hematological malignancy at St. Jude Children's Research Hospital. We used patient-specific reference genomes to identify variants that arose over time in subsequent gastrointestinal and blood isolates from each patient and analyzed these variants for insight into how VREfm adapted during colonization and bloodstream infection within each patient. Variants were enriched in genes involved in carbohydrate metabolism, and phenotypic analysis identified associated differences in carbohydrate utilization among isolates. In particular, a Y585C mutation in the sorbitol operon transcriptional regulator gutR was associated with increased bacterial growth in the presence of sorbitol. We also found differences in biofilm-formation capability between isolates and observed that increased biofilm formation correlated with mutations in the putative E. faecium capsular polysaccharide (cps) biosynthetic locus, with different mutations arising independently in distinct genetic backgrounds. Isolates with cps mutations showed improved survival following exposure to lysozyme, suggesting a possible reason for the selection of capsule-lacking bacteria. Finally, we observed mutations conferring increased tolerance of linezolid and daptomycin in patients who were treated with these antibiotics. Overall, this study documents known and previously undescribed ways that VREfm evolve during intestinal colonization and subsequent bloodstream infection in immunocompromised pediatric patients.

Multicenter Prospective Study of Biomarkers for Diagnosis of Invasive Candidiasis in Children and Adolescents.

BACKGROUND: Diagnosis of invasive candidiasis (IC) relies on insensitive cultures; the relative utility of fungal biomarkers in children is unclear. METHODS: This multinational observational cohort study enrolled patients aged >120 days and <18 years with concern for IC from 1 January 2015 to 26 September 2019 at 25 centers. Blood collected at onset of symptoms was tested using T2Candida, Fungitell (1→3)-ß-D-glucan, Platelia Candida Antigen (Ag) Plus, and Platelia Candida Antibody (Ab) Plus assays. Operating characteristics were determined for each biomarker, and assays meeting a defined threshold considered in combination. Sterile site cultures were the reference standard. RESULTS: Five hundred participants were enrolled at 22 centers in 3 countries, and IC was diagnosed in 13 (2.6%). Thirteen additional blood specimens were collected and successfully spiked with Candida species, to achieve a 5.0% event rate. Valid T2Candida, Fungitell, Platelia Candida Ag Plus, and Platelia Candida Ab Plus assay results were available for 438, 467, 473, and 473 specimens, respectively. Operating characteristics for T2Candida were most optimal for detecting IC due to any Candida species, with results as follows: sensitivity, 80.0% (95% confidence interval, 59.3%-93.2%), specificity 97.1% (95.0%-98.5%), positive predictive value, 62.5% (43.7%-78.9%), and negative predictive value, 98.8% (97.2%-99.6%). Only T2Candida and Platelia Candida Ag Plus assays met the threshold for combination testing. Positive result for either yielded the following results: sensitivity, 86.4% (95% confidence interval, 65.1%- 97.1%); specificity, 94.7% (92.0%-96.7%); positive predictive value, 47.5% (31.5%-63.9%); and negative predictive value, 99.2% (97.7%-99.8%). CONCLUSIONS: T2Candida alone or in combination with Platelia Candida Ag Plus may be beneficial for rapid detection of Candida species in children with concern for IC. CLINICAL TRIALS REGISTRATION: NCT02220790.

Incorporating G6PD genotyping to identify patients with G6PD deficiency.

Glucose-6-phosphate-dehydrogenase (G6PD) deficiency is a common X-linked enzyme disorder associated with hemolytic anemia after exposure to fava beans or certain medications. Activity testing is the gold standard for detecting G6PD deficiency; however, this test is affected by various hematologic parameters. Clinical G6PD genotyping is now included in pharmacogenetic arrays and clinical sequencing efforts and may be reconciled with activity results. Patients (n = 1391) enrolled on an institutional pharmacogenetic testing protocol underwent clinical G6PD genotyping for 164 G6PD variants. An algorithm accounting for known interferences with the activity assay is proposed. We developed clinical decision support alerts to inform prescribers when high-risk medications were prescribed, warning of gene-drug interactions and recommending therapy alteration. Of 1391 patients with genotype results, 1334 (95.9%) patients were predicted to have normal G6PD activity, 30 (2.1%) were predicted to have variable G6PD activity and 27 (2%) were predicted to have deficient G6PD activity. Of the 417 patients with a normal genotype and an activity result, 415 (99.5%) had a concordant normal G6PD phenotype. Of the 21 patients with a deficient genotype and an activity result, 18 (85.7%) had a concordant deficient activity result. Genotyping reassigned phenotype in five patients with discordant genotype and activity results: three switched from normal to deficient, and two switched from deficient to normal. G6PD activity and genotyping are two independent testing methods that can be used in conjunction to assign a more informed G6PD phenotype than either method alone.

Clinical Metagenomic Sequencing for Diagnosis of Meningitis and Encephalitis.

BACKGROUND: Metagenomic next-generation sequencing (NGS) of cerebrospinal fluid (CSF) has the potential to identify a broad range of pathogens in a single test. METHODS: In a 1-year, multicenter, prospective study, we investigated the usefulness of metagenomic NGS of CSF for the diagnosis of infectious meningitis and encephalitis in hospitalized patients. All positive tests for pathogens on metagenomic NGS were confirmed by orthogonal laboratory testing. Physician feedback was elicited by teleconferences with a clinical microbial sequencing board and by surveys. Clinical effect was evaluated by retrospective chart review. RESULTS: We enrolled 204 pediatric and adult patients at eight hospitals. Patients were severely ill: 48.5% had been admitted to the intensive care unit, and the 30-day mortality among all study patients was 11.3%. A total of 58 infections of the nervous system were diagnosed in 57 patients (27.9%). Among these 58 infections, metagenomic NGS identified 13 (22%) that were not identified by clinical testing at the source hospital. Among the remaining 45 infections (78%), metagenomic NGS made concurrent diagnoses in 19. Of the 26 infections not identified by metagenomic NGS, 11 were diagnosed by serologic testing only, 7 were diagnosed from tissue samples other than CSF, and 8 were negative on metagenomic NGS owing to low titers of pathogens in CSF. A total of 8 of 13 diagnoses made solely by metagenomic NGS had a likely clinical effect, with 7 of 13 guiding treatment. CONCLUSIONS: Routine microbiologic testing is often insufficient to detect all neuroinvasive pathogens. In this study, metagenomic NGS of CSF obtained from patients with meningitis or encephalitis improved diagnosis of neurologic infections and provided actionable information in some cases. (Funded by the National Institutes of Health and others; PDAID ClinicalTrials.gov number, NCT02910037.).

covidscreen: a web app and R Package for assessing asymptomatic COVID-19 testing strategies.

BACKGROUND: COVID-19 has caused over 305 million infections and nearly 5.5 million deaths globally. With complete eradication unlikely, organizations will need to evaluate their risk and the benefits of mitigation strategies, including the effects of regular asymptomatic testing. We developed a web application and R package that provides estimates and visualizations to aid the assessment of organizational infection risk and testing benefits to facilitate decision-making, which combines internal and community information with malleable assumptions. RESULTS: Our web application, covidscreen, presents estimated values of risk metrics in an intuitive graphical format. It shows the current expected number of active, primarily community-acquired infections among employees in an organization. It calculates and explains the absolute and relative risk reduction of an intervention, relative to the baseline scenario, and shows the value of testing vaccinated and unvaccinated employees. In addition, the web interface allows users to profile risk over a chosen range of input values. The performance and output are illustrated using simulations and a real-world example from the employee testing program of a pediatric oncology specialty hospital. CONCLUSIONS: As the COVID-19 pandemic continues to evolve, covidscreen can assist organizations in making informed decisions about whether to incorporate covid test based screening as part of their on-campus risk-mitigation strategy. The web application, R package, and source code are freely available online (see "Availability of data and materials").

Vancomycin Heteroresistance and Clinical Outcomes in Bloodstream Infections Caused by Coagulase-Negative Staphylococci.

Coagulase-negative staphylococci (CoNS) are a common etiology of serious and recurrent infections in immunocompromised patients. Although most isolates appear susceptible to vancomycin, a single strain might have a subpopulation of resistant bacteria. This phenomenon is termed heteroresistance and may adversely affect the response to treatment. A retrospective cohort study was performed of pediatric patients with leukemia treated at St. Jude Children's Research Hospital who developed CoNS central line-associated bloodstream infection (CLABSI). Available isolates were sequenced and tested for vancomycin heteroresistance by population analysis profiling. Risk factors for heteroresistance and the association of heteroresistance with treatment failure (death or relapse of infection) or poor clinical response to vancomycin therapy (treatment failure or persistent bacteremia after vancomycin initiation) were evaluated. For 65 participants with CoNS CLABSI, 62 initial isolates were evaluable, of which 24 (39%) were vancomycin heteroresistant. All heteroresistant isolates were of Staphylococcus epidermidis and comprised multiple sequence types. Participants with heteroresistant bacteria had more exposure to vancomycin prophylaxis (P = 0.026) during the 60 days prior to infection. Of the 40 participants evaluable for clinical outcomes, heteroresistance increased the risk of treatment failure (P = 0.012) and poor clinical response (P = 0.001). This effect persisted after controlling for identified confounders. These data indicate that vancomycin heteroresistance is common in CoNS isolates from CLABSIs in pediatric patients with leukemia and is associated with poor clinical outcomes. Validation of these findings in an independent cohort and evaluation of alternative antibiotic therapy in patients with heteroresistant infections have the potential to improve care for serious CoNS infections.

Gut Microbiome Composition Predicts Infection Risk During Chemotherapy in Children With Acute Lymphoblastic Leukemia.

Background: Myelosuppression-related infections remain important causes of morbidity and mortality in children with acute lymphoblastic leukemia (ALL). Methods: By analyzing fecal samples collected at diagnosis and after each of the initial 3 phases of chemotherapy, we evaluated the role of gut microbiota in predicting infections in 199 children with newly diagnosed ALL. The bacterial 16S rRNA gene was analyzed by high-depth sequencing to determine the diversity and composition of the microbiome. Results: After the induction and reinduction I phases of chemotherapy, microbial diversity decreased significantly relative to the prechemotherapy value. After chemotherapy, the relative abundance of certain bacterial taxa (eg, Bacteroidetes) decreased significantly, whereas that of other taxa (eg, Clostridiaceae and Streptococcaceae) increased. A baseline gut microbiome characterized by Proteobacteria predicted febrile neutropenia. Adjusting for the chemotherapy phase and ALL risk level, Enterococcaceae dominance (relative abundance ≥30%) predicted significantly greater risk of subsequent febrile neutropenia and diarrheal illness, whereas Streptococcaceae dominance predicted significantly greater risk of subsequent diarrheal illness. Conclusions: In children undergoing therapy for newly diagnosed ALL, the relative abundance of Proteobacteria before chemotherapy initiation predicts development of febrile neutropenia, and domination of the gut microbiota by Enterococcaceae or Streptococcaceae at any time during chemotherapy predicts infection in subsequent phases of chemotherapy. Clinical Trial Registration: NCT00549848.

Does Size Matter? Comparison of Extraction Yields for Different-Sized DNA Fragments by Seven Different Routine and Four New Circulating Cell-Free Extraction Methods.

An element essential for PCR detection of microbial agents in many sample types is the extraction step, designed to purify nucleic acids. Despite the importance of this step, yields have not been extensively compared across methods to determine whether the method used contributes to quantitative differences and the lack of commutability seen with existing clinical methods. This may in part explain why plasma and blood viral load assays have proven difficult to standardize. Also, studies have identified small DNA fragments of <200 bp in plasma (cell-free DNA [cfDNA]), which may include significant quantities of viral DNA. Our study evaluated extraction yields for 11 commercially available extraction methods, including 4 new methods designed to isolate cfDNA. Solutions of DNA fragments with sizes ranging from 50 to 1,500 bp were extracted, and then the eluates were tested by droplet digital PCR to determine the DNA fragment yield for each method. The results demonstrated a wide range of extraction yields across the variety of methods/instruments used, with the 50- and 100-bp fragment sizes showing especially inconsistent quantitative results and poor yields of less than 20%. Slightly higher, more consistent yields were seen with 2 of the 4 circulating cell-free extraction kits. These results demonstrate a significant need for further evaluation of nucleic acid yields across the variety of extraction platforms and highlight the poor extraction yields of small DNA fragments by existing methods. Further work is necessary to determine the impact of this inconsistency across instruments and the relevance of the low yields for smaller DNA fragments in clinical virology testing.

Selective T-cell depletion targeting CD45RA reduces viremia and enhances early T-cell recovery compared with CD3-targeted T-cell depletion.

BACKGROUND: T-cell depletion (TCD) effectively reduces severe graft-versus-host disease in recipients of HLA-mismatched allografts. However, TCD is associated with delayed immune recovery and increased infections. We hypothesized that specific depletion of CD45RA+ naive T cells, rather than broad depletion of CD3+ T cells, can preserve memory-immunity in the allografts and confer protection against important viral infections in the early post-transplant period. METHODS: Sixty-seven patients who received TCD haploidentical donor transplantation for hematologic malignancy on 3 consecutive trials were analyzed. RESULTS: Patients receiving CD45RA-depleted donor grafts had 2000-fold more donor T cells infused, significantly higher T-cell counts at Day +30 post transplant (550/µL vs 10/µL; P < .001), and higher T-cell diversity by Vbeta spectratyping at Day +100 (P < .001). Importantly, these recipients experienced a significant reduction in both the incidence (P = .002) and duration (P = .02) of any viremia (cytomegalovirus, Epstein-Barr virus, or adenovirus) in the first 6 months post transplant. Specifically, recipients of CD3-depleted grafts were more likely to experience adenovirus viremia (27% vs 4%, P = .02). CONCLUSION: CD45RA-depletion provided a large number of donor memory T cells to the recipients and was associated with enhanced early T-cell recovery and protection against viremia.

Levofloxacin Prophylaxis During Induction Therapy for Pediatric Acute Lymphoblastic Leukemia.

BACKGROUND: Infection is the most important cause of treatment-related morbidity and mortality in pediatric patients treated for acute lymphoblastic leukemia (ALL). Although routine in adults with leukemia, antibacterial prophylaxis is controversial in pediatrics because of insufficient evidence for its efficacy or antibiotic choice and concerns about promoting antibiotic resistance and Clostridium difficile infection. METHODS: This was a single-center, observational cohort study of patients with newly diagnosed ALL, comparing prospectively collected infection-related outcomes in patients who received no prophylaxis, levofloxacin prophylaxis, or other prophylaxis during induction therapy on the total XVI study. A propensity score-weighted logistic regression model was used to adjust for confounders. RESULTS: Of 344 included patients, 173 received no prophylaxis, 69 received levofloxacin prophylaxis, and 102 received other prophylaxis regimens. Patients receiving prophylaxis had longer duration of neutropenia. Prophylaxis reduced the odds of febrile neutropenia, likely bacterial infection, and bloodstream infection by ≥70%. Levofloxacin prophylaxis alone reduced these infections, but it also reduced cephalosporin, aminoglycoside, and vancomycin exposure and reduced the odds of C. difficile infection by >95%. No increase in breakthrough infections with antibiotic-resistant organisms was seen, but this cannot be excluded. CONCLUSIONS: This is the largest study to date of antibacterial prophylaxis during induction therapy for pediatric ALL and the first to include a broad-spectrum fluoroquinolone. Prophylaxis prevented febrile neutropenia and systemic infection. Levofloxacin prophylaxis also minimized the use of treatment antibiotics and drastically reduced C. difficile infection. Although long-term antibiotic-resistance monitoring is needed, these data support using targeted prophylaxis with levofloxacin in children undergoing induction chemotherapy for ALL. CLINICAL TRIALS REGISTRATION: NCT00549848.

Persistent Infections with Diverse Co-Circulating Astroviruses in Pediatric Oncology Patients, Memphis, Tennessee, USA.

Human astroviruses are a major cause of pediatric gastroenteritis, especially in immunocompromised children. We conducted a retrospective study to demonstrate that diverse astrovirus genotypes can co-circulate in pediatric oncology patients. A subset of cases is associated with long-term virus shedding (range 17-183 days).

Are We There Yet? Impact of the First International Standard for Cytomegalovirus DNA on the Harmonization of Results Reported on Plasma Samples.

BACKGROUND: Interassay harmonization of cytomegalovirus (CMV) DNA measurement is important for infection management. Uncertainty exists regarding the result harmonization achievable in patient plasma samples using quantitative polymerase chain reaction (qPCR) assays with calibrators now traceable to the First World Health Organization International Standard (IS) for CMV DNA. METHOD: Serial dilutions of the IS and a blinded panel of 40 genotyped CMV DNA-positive pooled plasma samples and 10 negative plasma samples were tested by 6 laboratories using 10 qPCR assays calibrated to the IS. Each clinical sample was constructed using plasma from a single unique transplant recipient. RESULTS: The variance for individual CMV DNA-positive samples was greater for clinical samples (median, 1.50 [range, 1.22-2.82] log10 IU/mL) than for IS dilutions (median, 0.94 [range, 0.69-1.35] log10 IU/mL) (P < .001); 58.9% of all clinical sample results and 93.6% of IS dilution results fell within ±0.5 log10 IU/mL of the mean viral load of each sample. Result variability was not impacted by either genotype or quantitative levels of CMV DNA. Testing procedure differences can significantly influence results, even when analyte-specific reagents are identical. For clinical samples, all assays demonstrated result bias (P < .008). Assays with amplicon sizes ≤86 bp had significantly higher results compared to assays with larger amplicon sizes (≥105 bp) (P < .001). CONCLUSIONS: The variability in CMV DNA results reported on individual samples has been reduced by the IS, but ongoing clinically relevant variability persists, preventing meaningful interassay result comparison.

Rapid Antimicrobial Susceptibility Testing Using Forward Laser Light Scatter Technology.

The delayed reporting of antimicrobial susceptibility testing remains a limiting factor in clinical decision-making in the treatment of bacterial infection. This study evaluates the use of forward laser light scatter (FLLS) to measure bacterial growth for the early determination of antimicrobial susceptibility. Three isolates each (two clinical isolates and one reference strain) of Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa were tested in triplicate using two commercial antimicrobial testing systems, the Vitek2 and the MicroScan MIC panel, to challenge the BacterioScan FLLS. The BacterioScan FLLS showed a high degree of categorical concordance with the commercial methods. Pairwise comparison with each commercial system serving as a reference standard showed 88.9% agreement with MicroScan (two minor errors) and 72.2% agreement with Vitek (five minor errors). FLLS using the BacterioScan system shows promise as a novel method for the rapid and accurate determination of antimicrobial susceptibility.

Clinical utility of viral load in management of cytomegalovirus infection after solid organ transplantation.

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.