Multicenter evaluation of BioCode GPP for syndromic molecular detection of gastrointestinal pathogens from stool specimens.

Acute gastroenteritis (AGE) is a leading cause of morbidity and mortality worldwide across all age groups that disproportionally affects young children in low- and middle-income countries and immunocompromised patients in high-income countries. Regional outbreaks of AGE are typically detected by traditional microbiological detection methods that target limited organisms and are associated with low sensitivity and lengthy time-to-results. Combined, these may result in repeat testing, imprecise or delayed treatment, and delayed recognition of outbreaks. We conducted a multi-site prospective study comparing the BioCode Gastrointestinal Pathogen Panel (BioCode GPP) for the detection of 17 common bacterial, viral, and protozoan causes of gastroenteritis with reference methods, including stool culture, enzyme immunoassays, pathogen-specific PCR assays, and sequencing. One thousand five hundred fifty-eight residual, de-identified stool samples (unpreserved stool and stool in Cary-Blair transport medium) were enrolled and tested for 11 bacterial, 3 viral, and 3 protozoan pathogens. BioCode GPP and reference methods were positive for 392 (25.2%) and 283 (18.2%) samples, respectively (P < 0.0001). In this study, the BioCode GPP and reference methods detected 69 and 65 specimens positive for Clostridioides difficile, 51 and 48 for enteroaggregative Escherichia coli, 33 and 27 for enterotoxigenic E. coli, 50 and 47 for norovirus GI/GII, and 30 and 22 for rotavirus A, respectively. The BioCode GPP showed good positive and negative agreements for each pathogen ranging from 89.5% to 100%, with overall sensitivity and specificity of 96.1% and 99.7%, post adjudication. The BioCode GPP detected >1 pathogens in 49 samples, representing 12.5% of the total 392 positive specimens. IMPORTANCE: This study highlights performance of a novel technology for timely and accurate detection and differentiation of 17 common bacterial, viral, and protozoan causes of gastroenteritis. Utilizing molecular tests such as the BioCode Gastrointestinal Pathogen Panel may improve the detection of gastrointestinal pathogens and provide actionable results, particularly for patient populations at most risk.

Accelerated evolution of SARS-CoV-2 in free-ranging white-tailed deer.

The zoonotic origin of the COVID-19 pandemic virus highlights the need to fill the vast gaps in our knowledge of SARS-CoV-2 ecology and evolution in non-human hosts. Here, we detected that SARS-CoV-2 was introduced from humans into white-tailed deer more than 30 times in Ohio, USA during November 2021-March 2022. Subsequently, deer-to-deer transmission persisted for 2-8 months, disseminating across hundreds of kilometers. Newly developed Bayesian phylogenetic methods quantified how SARS-CoV-2 evolution is not only three-times faster in white-tailed deer compared to the rate observed in humans but also driven by different mutational biases and selection pressures. The long-term effect of this accelerated evolutionary rate remains to be seen as no critical phenotypic changes were observed in our animal models using white-tailed deer origin viruses. Still, SARS-CoV-2 has transmitted in white-tailed deer populations for a relatively short duration, and the risk of future changes may have serious consequences for humans and livestock.

The Clinical Performance of the BioCode Respiratory Pathogen Panel for the Detection of Viruses and Bacteria from Nasopharyngeal Swabs.

Early detection of microbial pathogens causing respiratory tract infection plays a crucial role in clinical management. The BioCode Respiratory Pathogen Panel (BioCode RPP) utilizes reverse transcriptase PCR (RT-PCR) in combination with barcoded magnetic beads to amplify, detect, and identify respiratory pathogens. This panel qualitatively detects and identifies 14 viruses, including influenza virus A with H1 pdm09, H1, and H3 subtyping; influenza B; respiratory syncytial virus (RSV); human metapneumovirus; parainfluenza virus 1; parainfluenza virus 2; parainfluenza virus 3; parainfluenza virus 4; coronavirus (229E, NL63, OC43, and HKU1); adenovirus; and human rhinovirus/enterovirus, and 3 bacteria, including Chlamydia pneumoniae, Mycoplasma pneumoniae, and Bordetella pertussis. Reproducibility, which was assessed with contrived specimens containing 12 targets at 3 clinical sites, with 2 operators at each site for 5 days, was 99.4% for Flu A H3 and Flu B, 98.9% for RSV, and 100% for the remaining 9 targets assayed. A multicenter clinical trial evaluated the performance of the BioCode RPP with 2,647 nasopharyngeal swab specimens from 5 geographically distinct sites and revealed comparable performance between the BioCode RPP and FilmArray Respiratory Panel (FA-RP). Specifically, the positive percent agreements (PPAs) for various pathogens ranged between 80.8% and 100% compared with the FA-RP (1.7 and 2.0). Negative percent agreement ranged from 98.4% to 100% for BioCode RPP. The BioCode RPP also offers scalable automated testing capability of up to 96 specimens in a single run with total sample-to-result time under 5 h. The invalid rate of the BioCode RPP on initial testing was 1.0% (26/2,649). IMPORTANCE Early detection of microbial pathogens causing respiratory tract infection plays a crucial role in clinical management. The BioCode Respiratory Pathogen Panel (BioCode RPP) is a high-throughput test that utilizes RT-PCR in combination with barcoded magnetic beads to amplify, detect, and identify 17 respiratory pathogens, including 14 viruses and 3 bacteria. This study summarizes data generated from a multicenter clinical trial evaluating the performance of the BioCode RPP on 2,647 nasopharyngeal swab specimens from five geographically distinct sites.

Accelerated evolution of SARS-CoV-2 in free-ranging white-tailed deer.

While SARS-CoV-2 has sporadically infected a wide range of animal species worldwide1, the virus has been repeatedly and frequently detected in white-tailed deer in North America2â€"7. The zoonotic origins of this pandemic virus highlight the need to fill the vast gaps in our knowledge of SARS-CoV-2 ecology and evolution in non-human hosts. Here, we detected SARS-CoV-2 was introduced from humans into white-tailed deer more than 30 times in Ohio, USA during November 2021-March 2022. Subsequently, deer-to-deer transmission persisted for 2-8 months, which disseminated across hundreds of kilometers. We discovered that alpha and delta variants evolved in white-tailed deer at three-times the rate observed in humans. Newly developed Bayesian phylogenetic methods quantified how SARS-CoV-2 evolution is not only faster in white-tailed deer but driven by different mutational biases and selection pressures. White-tailed deer are not just short-term recipients of human viral diversity but serve as reservoirs for alpha and other variants to evolve in new directions after going extinct in humans. The long-term effect of this accelerated evolutionary rate remains to be seen as no critical phenotypic changes were observed in our animal model experiments using viruses isolated from white-tailed deer. Still, SARS-CoV-2 viruses have transmitted in white-tailed deer populations for a relatively short duration, and the risk of future changes may have serious consequences for humans and livestock.

Comparing molecular quantification of herpes simplex virus (HSV) in cerebrospinal fluid (CSF) with quantitative structural and functional disease severity in patients with HSV encephalitis (HSVE): Implications for improved therapeutic approaches.

BACKGROUND: Herpes Simplex Virus encephalitis (HSVE) is a devastating disease of all ages. Rigorous studies correlating viral load with neuroradiological and clinical severity have not been performed, particularly in neonates. Understanding these relationships may improve therapies. OBJECTIVES: To correlate molecularly quantified HSV in cerebrospinal fluid (CSF) and disease severity. STUDY DESIGN: HSV loads (VL) were evaluated by real-time PCR from the CSF of 33 patients (20 neonates, 5 children, 8 adults) with HSVE. We studied relationships between CSF VL and structural and volumetric brain abnormalities (MRI); hospital morbidity; and discharge and long-term (>3 month) clinical outcomes. RESULTS: Initial CSF VL did not differ in neonates vs non-neonates (median 4.6 vs 5.1 log10 copies/mL, p = 0.75). Initial CSF VL was higher in neonates with HSV-2 vs HSV-1 (median 4.8 vs 3.2 log10 copies/mL, respectively, p = 0.02). Persistently detectable DNA in CSF despite acyclovir trended towards higher odds of unfavorable outcome at discharge for neonates [0.87 (CI 0.75-1), p = 0.07]. Initial VL correlated with higher CSF protein concentrations for the cohort and for neonates (p = 0.03 and 0.01, respectively), but not with lesion volume or subarachnoid exposure of involved brain (p all >0.05), hospital morbidity (p all >0.05), nor with higher odds of unfavorable discharge or long-term outcomes for the cohort [OR = 0.9(CI 0.5-1.6), p = 0.72; OR = 1.0(CI 0.5-1.8), p = 0.9] or for neonates [OR = 1.3(CI 0.5-3.3), p = 0.57; OR = 2.3(CI 0.7-8), p = 0.2]. CONCLUSIONS: Initial HSV VL did not predict neuroradiological or clinical outcomes in patients with HSVE, suggesting host inflammatory factors contribute to disease in treated patients with good viral clearance.

Use of real-time semiquantitative PCR data in management of a neonatal intensive care unit adenovirus outbreak.

OBJECTIVE: To describe an adenovirus outbreak in a neonatal intensive care unit (NICU), including the use of qualitative and semiquantitative real-time polymerase chain reaction (qPCR) data to inform the outbreak response. DESIGN: Mixed prospective and retrospective observational study. SETTING: A level IV NICU in the southeastern United States.PatientsTwo adenovirus cases were identified in a NICU. Screening of all inpatients with qPCR on nasopharyngeal specimens revealed 11 additional cases.InterventionsOutbreak response procedures, including enhanced infection control policies, were instituted. Serial qPCR studies were used to screen for new infections among exposed infants and to monitor viral clearance among cases. Changes to retinopathy of prematurity (ROP) exam procedures were made after an association was noted in those patients. At the end of the outbreak, a retrospective review allowed for comparison of clinical factors between the infected and uninfected groups. RESULTS: There were no new cases among patients after outbreak identification. One adenovirus-infected patient died; the others recovered their clinical baselines. The ROP exams were associated with an increased risk of infection (odds ratio [OR], 84.6; 95% confidence interval [CI], 4.5-1,601). The duration of the outbreak response was 33 days, and the previously described second wave of cases after the end of the outbreak did not occur. Revisions to infection control policies remained in effect following the outbreak. CONCLUSIONS: Retinopathy of prematurity exams are potential mechanisms of adenovirus transmission, and autoclaved or single-use instruments should be used to minimize this risk. Real-time molecular diagnostic and quantification data guided outbreak response procedures, which rapidly contained and fully terminated a NICU adenovirus outbreak.

Rhinovirus Viremia in Patients Hospitalized With Community-Acquired Pneumonia.

Background: Rhinoviruses (RVs) are ubiquitous respiratory pathogens that often cause mild or subclinical infections. Molecular detection of RVs from the upper respiratory tract can be prolonged, complicating etiologic association in persons with severe lower respiratory tract infections. Little is known about RV viremia and its value as a diagnostic indicator in persons hospitalized with community-acquired pneumonia (CAP). Methods: Sera from RV-positive children and adults hospitalized with CAP were tested for RV by real-time reverse-transcription polymerase chain reaction. Rhinovirus species and type were determined by partial genome sequencing. Results: Overall, 57 of 570 (10%) RV-positive patients were viremic, and all were children aged <10 years (n = 57/375; 15.2%). Although RV-A was the most common RV species detected from respiratory specimens (48.8%), almost all viremias were RV-C (98.2%). Viremic patients had fewer codetected pathogens and were more likely to have chest retractions, wheezing, and a history of underlying asthma/reactive airway disease than patients without viremia. Conclusions: More than 1 out of 7 RV-infected children aged <10 years hospitalized with CAP were viremic. In contrast with other RV species, RV-C infections were highly associated with viremia and were usually the only respiratory pathogen identified, suggesting that RV-C viremia may be an important diagnostic indicator in pediatric pneumonia.

Serology Enhances Molecular Diagnosis of Respiratory Virus Infections Other than Influenza in Children and Adults Hospitalized with Community-Acquired Pneumonia.

Both molecular and serological assays have been used previously to determine the etiology of community-acquired pneumonia (CAP). However, the extent to which these methods are correlated and the added diagnostic value of serology for respiratory viruses other than influenza virus have not been fully evaluated. Using data from patients enrolled in the Centers for Disease Control and Prevention (CDC) Etiology of Pneumonia in the Community (EPIC) study, we compared real-time reverse transcription-PCR (RT-PCR) and serology for the diagnosis of respiratory syncytial virus (RSV), human metapneumovirus (HMPV), parainfluenza virus 1 to 3 (PIV1, PIV2, and PIV3), and adenovirus (AdV) infections. Of 5,126 patients enrolled, RT-PCR and serology test results were available for 2,023, including 1,087 children below the age of 18 years and 936 adults. For RSV, 287 (14.2%) patients were positive by RT-PCR and 234 (11.6%) were positive by serology; for HMPV, 172 (8.5%) tested positive by RT-PCR and 147 (7.3%) by serology; for the PIVs, 94 (4.6%) tested positive by RT-PCR and 92 (4.6%) by serology; and for AdV, 111 (5.5%) tested positive by RT-PCR and 62 (3.1%) by serology. RT-PCR provided the highest number of positive detections overall, but serology increased diagnostic yield for RSV (by 11.8%), HMPV (by 25.0%), AdV (by 32.4%), and PIV (by 48.9%). The method concordance estimated by Cohen's kappa coefficient (κ) ranged from good (for RSV; κ = 0.73) to fair (for AdV; κ = 0.27). Heterotypic seroresponses observed between PIVs and persistent low-level AdV shedding may account for the higher method discordance observed with each of these viruses. Serology can be a helpful adjunct to RT-PCR for research-based assessment of the etiologic contribution of respiratory viruses other than influenza virus to CAP.

Identification of Bacterial and Viral Codetections With Mycoplasma pneumoniae Using the TaqMan Array Card in Patients Hospitalized With Community-Acquired Pneumonia.

Mycoplasma pneumoniae was detected in a number of patients with community-acquired pneumonia in a recent prospective study. To assess whether other pathogens were also detected in these patients, TaqMan Array Cards were used to test 216 M pneumoniae-positive respiratory specimens for 25 additional viral and bacterial respiratory pathogens. It is interesting to note that 1 or more codetections, predominantly bacterial, were identified in approximately 60% of specimens, with codetections being more common in children.

Multicenter Evaluation of Meridian Bioscience HSV 1&2 Molecular Assay for Detection of Herpes Simplex Virus 1 and 2 from Clinical Cutaneous and Mucocutaneous Specimens.

Herpes simplex virus (HSV) causes acute and relapsing symptoms characterized by ulcerative lesions. Laboratory diagnosis of HSV in cutaneous or mucocutaneous lesions has historically been performed with the use of viral cell culture systems; however, these tests are laborious and suffer decreased sensitivity for advanced-stage lesions. The recent availability of FDA-cleared moderately complex assays has resulted in the increased use of molecular diagnostics for the routine detection of HSV in superficial swab specimens. We performed a clinical evaluation of the recently FDA-cleared illumigene HSV 1&2 loop-mediated isothermal amplification (LAMP) assay (Meridian Bioscience, Cincinnati OH) for the detection and differentiation of HSV-1 and HSV-2 in cutaneous and mucocutaneous swab specimens. A total of 1,153 clinical swab specimens were collected and tested at 7 different clinical centers. Each specimen was tested for the presence of HSV-1 and HSV-2 using the illumigene assay, and results were compared to those of the enzyme-linked virus-inducible system (ELVIS) as the reference method. Overall, the illumigene assay demonstrated a sensitivity and specificity of 94.8% and 95.5%, respectively, for the detection of HSV-1. Detection of HSV-2 was similar, with a sensitivity of 98.9% and a specificity of 95.5%. Discrepant analysis was performed using an alternative molecular test (AmpliVue HSV1+2 assay; Quidel Molecular, San Diego, CA) on 91/99 specimens that were recorded as false positive (FP) or false negative (FN) compared to the reference method. In total, 57/78 (73%) FP and 9/13 (69%) FN illumigene results were supported by the AmpliVue result. The illumigene HSV 1&2 assay demonstrated high sensitivity and specificity to detect and differentiate HSV in clinical specimens and identified 57 additional specimens that were positive for HSV compared to culture. The use of LAMP eliminates the need for the cycling of temperatures and provides results in less than 60 min, with approximately 2 min of hands-on time per specimen.

Molecular Detection and Characterization of Mycoplasma pneumoniae Among Patients Hospitalized With Community-Acquired Pneumonia in the United States.

Background. Mycoplasma pneumoniae is a common cause of community-acquired pneumonia (CAP). The molecular characteristics of M pneumoniae detected in patients hospitalized with CAP in the United States are poorly described. Methods. We performed molecular characterization of M pneumoniae in nasopharyngeal/oropharyngeal swabs from children and adults hospitalized with CAP in the Centers for Disease Control and Prevention Etiology of Pneumonia in the Community (EPIC) study, including P1 typing, multilocus variable-number tandem-repeat analysis (MLVA), and macrolide susceptibility genotyping. Results. Of 216 M pneumoniae polymerase chain reaction-positive specimens, 40 (18.5%) were obtained from adults and 176 (81.5%) from children. P1 type distribution differed between adults (64% type 1 and 36% type 2) and children (84% type 1, 13% type 2, and 3% variant) (P < .05) and among sites (P < .01). Significant differences in the proportions of MLVA types 4/5/7/2 and 3/5/6/2 were also observed by age group (P < .01) and site (P < .01). A macrolide-resistant genotype was identified in 7 (3.5%) specimens, 5 of which were from patients who had recently received macrolide therapy. No significant differences in clinical characteristics were identified among patients with various strain types or between macrolide-resistant and -sensitive M pneumoniae infections. Conclusions. The P1 type 1 genotype and MLVA type 4/5/7/2 predominated, but there were differences between children and adults and among sites. Macrolide resistance was rare. Differences in strain types did not appear to be associated with differences in clinical outcomes. Whole genome sequencing of M pneumoniae may help identify better ways to characterize strains.

Community-acquired pneumonia requiring hospitalization among U.S. children.

BACKGROUND: Incidence estimates of hospitalizations for community-acquired pneumonia among children in the United States that are based on prospective data collection are limited. Updated estimates of pneumonia that has been confirmed radiographically and with the use of current laboratory diagnostic tests are needed. METHODS: We conducted active population-based surveillance for community-acquired pneumonia requiring hospitalization among children younger than 18 years of age in three hospitals in Memphis, Nashville, and Salt Lake City. We excluded children with recent hospitalization or severe immunosuppression. Blood and respiratory specimens were systematically collected for pathogen detection with the use of multiple methods. Chest radiographs were reviewed independently by study radiologists. RESULTS: From January 2010 through June 2012, we enrolled 2638 of 3803 eligible children (69%), 2358 of whom (89%) had radiographic evidence of pneumonia. The median age of the children was 2 years (interquartile range, 1 to 6); 497 of 2358 children (21%) required intensive care, and 3 (<1%) died. Among 2222 children with radiographic evidence of pneumonia and with specimens available for bacterial and viral testing, a viral or bacterial pathogen was detected in 1802 (81%), one or more viruses in 1472 (66%), bacteria in 175 (8%), and both bacterial and viral pathogens in 155 (7%). The annual incidence of pneumonia was 15.7 cases per 10,000 children (95% confidence interval [CI], 14.9 to 16.5), with the highest rate among children younger than 2 years of age (62.2 cases per 10,000 children; 95% CI, 57.6 to 67.1). Respiratory syncytial virus was more common among children younger than 5 years of age than among older children (37% vs. 8%), as were adenovirus (15% vs. 3%) and human metapneumovirus (15% vs. 8%). Mycoplasma pneumoniae was more common among children 5 years of age or older than among younger children (19% vs. 3%). CONCLUSIONS: The burden of hospitalization for children with community-acquired pneumonia was highest among the very young, with respiratory viruses the most commonly detected causes of pneumonia. (Funded by the Influenza Division of the National Center for Immunization and Respiratory Diseases.).

Comparison of the Simplexa™ Flu A/B & RSV kit (nucleic acid extraction-dependent assay) and the Prodessa ProFlu+™ assay for detecting influenza and respiratory syncytial viruses.

The relative performance of 2 widely used reverse transcription polymerase chain reaction (RT-PCR) assays, the Focus diagnostics Simplexa™ Flu A/B & RSV kit (nucleic acid extraction-dependent assay) and the Prodessa Proflu+™ assay, was evaluated using 735 prospectively and retrospectively collected nasopharyngeal swab specimens. Overall, the assays showed positive and negative agreements of 100% and 99.7% for influenza A, 98.1% and 99.9% for influenza B, and 99.3% and 99.5% for respiratory syncytial virus. The relative analytical sensitivity of the 2 assays was also similar.

Site-specific clinical evaluation of the Luminex xTAG gastrointestinal pathogen panel for detection of infectious gastroenteritis in fecal specimens.

We evaluate the clinical performance of the Luminex xTAG gastrointestinal (GI) pathogen in vitro diagnostic (IVD) assay in a comparison between clinical and public health laboratories. The site reproducibility study showed 98.7% sensitivity with high positive and negative agreement values (96.2% and 99.8%, respectively), while assay performance against confirmatory methods resulted in 96.4% sensitivity with similar positive and negative agreement values (90.1% and 99.5%, respectively). High-throughput detection of multiple GI pathogens improved turnaround time, consolidated laboratory workflow, and simplified stool culture practices, thus reducing the overall cost and number of specimens processed.

Multi-center evaluation of the adenovirus R-gene US assay for the detection of adenovirus in respiratory samples.

BACKGROUND: Adenoviruses (AdV) cause a variety of upper and lower respiratory tract infections, with the potential for severe outcomes, especially in persons with immune suppression or other underlying diseases. The ADENOVIRUS US R-gene (AdV R-gene, Argene/bioMérieux) is a FDA cleared real-time PCR assay that utilizes primers and fluorescent probes that target a conserved region of the hexon gene and an internal control DNA. OBJECTIVES: This prospective multi-center study evaluated the clinical performance of AdV R-gene for AdV detection in respiratory specimens from symptomatic patients of all ages. STUDY DESIGN: Nucleic acids from nasopharyngeal washes/aspirates (NPW/A; n=393) and NP flocked swabs (NPS, Copan) (n=1183) were extracted using NucliSENS easyMAG (bioMérieux) and AdV R-gene PCR was performed using the SmartCycler (Cepheid). AdV R-gene results were compared to R-Mix culture (Quidel/Diagnostic Hybrids). For a subset of samples (n=946) AdV R-gene and R-Mix results were also compared to A549 cell culture. RESULTS: In first intention analysis for NPS the AdV R-gene positive percent agreement (PPA), and negative percent agreement (NPA) were 91.7% and 96.2%, respectively, and for NPW/A were 100% and 94.4%, respectively, compared to R-Mix culture. In second intention analysis, discordant samples only were tested with an AdV real-time PCR assay (Viracor-IBT Labs) and amplicon sequencing. For NPS, the sensitivity, specificity, PPV and NPV for AdV R-gene were 98.9%, 100%, 100%, and 99.9%, respectively and for R-Mix culture were 51.7%, 99.7%, 93.8%, and 96.3%, respectively. For NPW/A, the sensitivity, specificity, PPV and NPV for AdV R-gene were 100%, 99.7%, 97.6%, and 100%, respectively, and for R-Mix culture were 52.5%, 100%, 100%, and 94.9%, respectively. Overall, AdV was detected by AdV R-gene and R-Mix in 7.4% and 4.1% NPS, respectively, and in 10.7% and 5.3% NPW/A, respectively. Children 5yr and younger had the highest rates of AdV infections. In a subset of specimens (n=946) the sensitivity of AdV R-gene, R-Mix, and A549 cell culture were 95.0%, 55.4% and 66.3%. CONCLUSIONS: AdV R-gene is sensitive and specific for the detection of AdV in NPW/A and NPS samples. AdV R-gene is simple to use and provides a rapid time to results (within 2.5-3h).

C20209T prothrombin gene mutation associated deep venous thrombosis in a hemodialysis patient.

Venous thromboembolism (VTE) represents the formation of a blood clot in one of the deep veins of human body. The significant morbidity and mortality rates associated with VTE have spurred increasing investigations seeking to identify causative factors for this complex condition. While the most frequent causes of an inherited hypercoagulable state are the Factor V Leiden mutation and the prothrombin gene mutation, polymerase chain reaction (PCR) analysis has helped to identify other rare causes of inherited VTE. We report a case of a recurrent deep venous thrombosis in an end-stage renal disease patient. All laboratory tests for hypercoagulable states were normal. However, PCR analysis detected a rare polymorphism of prothrombin gene mutation at position C20209T, instead of G20210A. The patient was treated successfully with a high dose of warfarin to maintain adequate anti-coagulation during the 2-year follow-up.

Influenza and respiratory syncytial virus detection in clinical specimens without nucleic acid extraction using FOCUS direct disc assay is substantially equivalent to the traditional methods and the FOCUS nucleic acid extraction-dependent RT-PCR assay.

In this study, we evaluated FOCUS diagnostic's Flu A/B & RSV direct kit (Direct Disc assay), designed to detect influenza (FLU) and respiratory syncytial viruses (RSV) directly in clinical specimens without nucleic acid extraction. This novel 'sample-to-answer', nucleic acid extraction-independent assay uses a unique disc to process, amplify, and detect viral targets in up to 8 specimens at a time. The performance of this assay for detecting FLU and RSV viruses was compared to the traditional methods (culture and/or direct florescent antibody testing) using 945 nasopharyngeal swab specimens. In addition, a total of 150 consecutive clinical specimens positive for FLU (FLU A=50, FLU B=50) or RSV (n=50) were tested in parallel using the novel Direct Disc assay and FOCUS diagnostic's nucleic acid extraction-dependent assay to assess their relative performance. Compared to the traditional methods, the overall (prospective+retrospective) positive/negative percent agreement was determined to be 96.6%/98.1% for FLU A, 98.4%/99.9% for FLU B, and 99.3%/98.8% for RSV. Compared to the nucleic acid extraction-dependent assay, the positive percent agreement was 90% (n=45/50) for FLU A, 92% (n=46/50) for FLU B, and 98% (n=49/50) for RSV. Overall, the Direct Disc assay showed good agreement with both traditional methods and nucleic acid extraction-dependent assay. Although we encountered some failures compared to the nucleic acid extraction-dependent assay, these limitations must be balanced against the substantial advantages of the extraction-free nature of this assay and rapid turnaround time.

Comparison of a real-time reverse transcriptase PCR assay and a culture technique for quantitative assessment of viral load in children naturally infected with respiratory syncytial virus.

Respiratory syncytial virus (RSV) is the most common cause of lower respiratory infection of children. Understanding RSV pathogenesis and evaluating interventions requires quantitative RSV testing. Previous studies have used the plaque assay technique. Real-time reverse transcriptase PCR (RTrtPCR) offers possible greater sensitivity, stability after freeze/thaw, and lower cost, thus facilitating multicenter studies. We developed RTrtPCR assays based upon the RSV N and F genes. The N-gene assay detected greater RSV quantity and was further evaluated. Standard curves utilized both extractions from RSV culture supernatants of known quantity and cloned purified copies of the target DNA. In vitro, the ratio of RSV subgroup A (RSV-A) genome copies to PFU was 153:1. A total of 462 samples collected quantitatively from 259 children were analyzed in duplicate by RTrtPCR. Results were compared with those of RSV plaque assays performed on fresh aliquots from the same children. Duplicate RTrtPCR results were highly correlated (r2 = 0.9964). The mean viral load from nasal washes obtained on the first study day was 5.75 +/- standard error of the mean 0.09 log PFU equivalents (PFUe)/ml. Viral load by RTrtPCR correlated with plaque assay results (r2 = 0.158; P < 0.0001). Within individuals, upper and lower respiratory tract secretions contained similar viral concentrations. RSV-A-infected children had 1.17 log PFUe higher viral loads than did those with RSV-B (P < 0.0001). RSV quantification by RTrtPCR of the N gene is precise and has significant, though limited, correlation with quantitative culture. The utility of the RTrtPCR quantification technique for clinical studies would be solidified after its correlation with RSV disease severity is established.

Classification of pediatric acute lymphoblastic leukemia by gene expression profiling.

Contemporary treatment of pediatric acute lymphoblastic leukemia (ALL) requires the assignment of patients to specific risk groups. We have recently demonstrated that expression profiling of leukemic blasts can accurately identify the known prognostic subtypes of ALL, including T-cell lineage ALL (T-ALL), E2A-PBX1, TEL-AML1, MLL rearrangements, BCR-ABL, and hyperdiploid karyotypes with more than 50 chromosomes. As the next step toward developing this methodology into a frontline diagnostic tool, we have now analyzed leukemic blasts from 132 diagnostic samples using higher density oligonucleotide arrays that allow the interrogation of most of the identified genes in the human genome. Nearly 60% of the newly identified subtype discriminating genes are novel markers not identified in our previous study, and thus should provide new insights into the altered biology underlying these leukemias. Moreover, a proportion of the newly selected genes are highly ranked as class discriminators, and when incorporated into class-predicting algorithms resulted in an overall diagnostic accuracy of 97%. The performance of an array containing the identified discriminating genes should now be assessed in frontline clinical trials in order to determine the accuracy, practicality, and cost effectiveness of this methodology in the clinical setting.

Real-time polymerase chain reaction as an aid for the detection of SYT-SSX1 and SYT-SSX2 transcripts in fresh and archival pediatric synovial sarcoma specimens: report of 25 cases from St. Jude Children's Research Hospital.

Synovial sarcoma is the most common nonrhabdomyosarcomatous soft tissue sarcoma in children and adolescents and is characterized by a reciprocal t(X;18)(p11;q11) which results in the fusion of the SYT gene on chromosome 18q11 to either of two closely related genes, SSX1 (Xp11.23) or SSX2 (Xp11.21). Detection of this translocation or its resultant gene fusion by molecular methods is helpful in the pathologic diagnosis of synovial sarcoma, especially in poorly differentiated tumors. This study was designed to evaluate the utility of a real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay to detect and distinguish SYT-SSX1 and SYT-SSX2 fusions in fresh and archival specimens of synovial sarcoma in pediatric patients seen at St. Jude Children's Research Hospital. In addition, the clinicopathologic features of the tumors with SYT-SSX1 vs. SYT-SSX2 fusions were compared. The 25 patients studied had a median age of 13 years 9 months (range 5 to 19 years). Estimates of survival and event-free survival at 5 years were 78.7 +/- 10.5% and 56.2 +/- 13.2%, respectively. Seventeen (68%) tumors were monophasic, eight (32%) were biphasic. Seven tumors contained poorly differentiated areas. Positive results for either SYT-SSX1 or SYT-SSX2 were obtained in 21/25 (84%) cases. Three cases did not have a detectable gene fusion and one had no amplifiable RNA. SYT-SSX1 transcripts were found in 18/24 (75%) of the tumors while SYT-SSX2 transcripts were identified in 3/24 (12.5%). All of the poorly differentiated tumors and seven out of eight tumors from patients who developed lung metastases had an SYT-SSX1 fusion transcript. Real-time PCR is useful in detecting and distinguishing SYT-SSX1 from SYT-SSX2 gene fusions in synovial sarcoma. Valuable aspects of this methodology are the applicability to both frozen and formalin-fixed samples, decreased labor costs, and the rapidity of results. In addition, distinguishing SYT-SSX1 from SYT-SSX2 fusions with these methods allow for prospective collection of information that may clarify issues of prognostic relevance.