Digital PCR to Measure SARS-CoV-2 RNA, Variants, and Outcomes in Youth.

BACKGROUND: The role of SARS-CoV-2 viral load in predicting contagiousness, disease severity, transmissibility, and clinical decision-making continues to be an area of great interest. However, most studies have been in adults and have evaluated SARS-CoV-2 loads using cycle thresholds (Ct) values, which are not standardized preventing consistent interpretation critical to understanding clinical impact and utility. Here, a quantitative SARS-CoV-2 reverse-transcription digital PCR (RT-dPCR) assay normalized to WHO International Units was applied to children at risk of severe disease diagnosed with COVID-19 at St. Jude Children's Research Hospital between March 28, 2020, and January 31, 2022. METHODS: Demographic and clinical information from children, adolescents, and young adults treated at St. Jude Children's Research Hospital were abstracted from medical records. Respiratory samples underwent SARS-CoV-2 RNA quantitation by RT-dPCR targeting N1 and N2 genes, with sequencing to determine the genetic lineage of infecting virus. RESULTS: Four hundred and sixty-two patients aged 0-24 years (median 11 years old) were included during the study period. Most patients were infected by the omicron variant (43.72%), followed by ancestral strain (22.29%), delta (13.20%), and alpha (2.16%). Viral load at presentation ranged from 2.49 to 9.14 log10 IU/mL, and higher viral RNA loads were associated with symptoms (OR 1.32; CI 95% 1.16-1.49) and respiratory disease (OR 1.23; CI 95% 1.07-1.41). Viral load did not differ by SARS-CoV-2 variant, vaccination status, age, or baseline diagnosis. CONCLUSIONS: SARS-CoV-2 RNA loads predict the presence of symptomatic and respiratory diseases. The use of standardized, quantitative methods is feasible, allows for replication, and comparisons across institutions, and has the potential to facilitate consensus quantitative thresholds for risk stratification and treatment.

Clinical Correlation of Adenoviral Load in the Respiratory Tract Measured by Digital PCR in Immunocompromised Children.

Immunocompromised patients can have life-threatening adenoviral infection. Viral load in blood and stool is commonly used to guide antiviral therapy. We developed and evaluated a digital polymerase chain reaction assay to quantify human adenovirus in the respiratory tract and showed that higher peak load correlates with disseminated infection, mechanical ventilation, and death.

Prediction of Symptomatic Severe Acute Respiratory Syndrome Coronavirus 2 Infection by Quantitative Digital Polymerase Chain Reaction Normalized to International Units.

Although numerous studies have evaluated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection using cycle threshold (Ct) values as a surrogate of viral ribonucleic acid (RNA) load, few studies have used standardized, quantitative methods. We validated a quantitative SARS-CoV-2 digital polymerase chain reaction assay normalized to World Health Organization International Units and correlated viral RNA load with symptoms and disease severity.

A research framework of land use transition in Suzhou City coupled with land use structure and landscape multifunctionality.

Under the premise of facing land-use sustainable development goals, clarifying the process and trend of regional land-use transition (LUT) is of considerable significance to the direction of national land-use optimization in the future. Suzhou City is not only an economically developed area in China but also a leading area of economic transformation and development, which embodies the changing process of regional development path since China's reform and opening up. This paper constructed an integrated research framework of micro-individual land use structure and macro-mixed landscape multifunctionality. It used spatial analysis technology to deeply analyze the LUT process of Suzhou, and quantified change characteristics of land use structure and function in Suzhou from 2000 to 2015. For structure, Suzhou has undergone a large-scale transition during the study period, mainly from farmland to construction land, in which transition speed and degree are at a high level until the trend slows down after 2010. For function, the number of high values of landscape multifunctionality gradually increases. Still, the scope of high-value areas progressively reduces by urban expansion constraints; the multifunctionality around urban expansion area gradually weakens. Besides, forest land, grassland, and other ecological land have the most significant number of land use functions. The comprehensive transition of land use structure and function can give a summary as a circle-layer dynamic change process of urban development. Transition hotspots can be divided into five specific regions of land management and finally realize comprehensive development zoning of urban and rural areas at the township level. LUT research framework based on structure-function coupling will provide ideas for land management mode transformation and contribute to sustainable land spatial planning strategy formulation.

A framework for assessing carbon effect of land consolidation with life cycle assessment: A case study in China.

Ecological transformation is an inevitable trend for the development of land consolidation (LC) worldwide, and the research on carbon effect of LC is an important theoretical basis for promoting the construction of Eco-LC. However, there is currently a lack of analysis of the carbon effect based on the whole process of LC, ignoring the stage elements and temporal factors. This study applied Life Cycle Assessment (LCA) method to construct a research framework and accounting system for carbon footprint assessment of LC, and explored the carbon effect in a typical land consolidation project area (LCPA) of China. Results showed that: (a) The carbon effect of the project area was characterized as carbon emission during the whole life cycle of LC. Carbon footprint before and after LC was 3.251 tCE·ha-1·a-1 and 2.401 tCE·ha-1·a-1 respectively. The carbon storage reduced and the carbon footprint is declined by 0.850 tCE·ha-1·a-1. (b) Carbon effect varied among different stages of LC. The Benefit Period (BP) was the only stage that was manifested as carbon absorption (-14.65%), while all the other stages were manifested as carbon emission. Among them, as to the carbon emission, the Construction Period (CP) played a decisive role with the most proportion (102.74%), followed by DP and RP, and the carbon effect of PP was negligible. (c) The dominant factors of carbon effect at different stages were also different. During CP, cement contributed the most to the carbon emission in this case. During RP, carbon sequestration effect of cropland proved to be the most significant. During RP, the carbon sequestration effect of cultivated land and the carbon emission effect of unused land were the most prominent. During BP, the carbon sequestration capacity of farmland ecosystems proved to be greater than the carbon emissions from agricultural activities. This study contributes to providing certain theoretical guidance and method reference for the realization of Low-Carbon LC project planning, with this comprehensive and reliable method.

A new framework of land use efficiency for the coordination among food, economy and ecology in regional development.

Great challenges regarding land use conflicts in rapid urbanization call for deeper research on land use efficiency (LUE) from the perspective of sustainable land use for the coordination among food security, economic development, and ecological protection. This study firstly develops a new framework of LUE based upon the expectations in land use and the coordination among three sub-categories in food production, economic development, and ecological protection, then, uses the coupling coordination degree model to quantify the spatial differentiation characteristics and coupling coordination relationships among three sub-categories, and finally uses the multivariable linear regression and geographical detectors to analyze the impact factors of sub-category efficiency. The framework is applied to Jiangsu Province in eastern China by using ten indicators (i.e., cultivated land quality, grain output, multiple cropping index, average GDP per km2, population density, proportion of industry and service industry, vegetation cover index, water conservation index, soil retention index, and carbon sequestration index) in terms of food production, economy, and ecology analysis at the county level. Compared with expectations, the LUE of Jiangsu in food production, economic development, and ecological protection is 54.15%, 85.56%, and 54.95%, respectively, indicating that Jiangsu has great potential for sustainable land use. The coupling coordination degree in land use generally synchronizes with the coupling degree, accounting for 65.34% of the province's area, of which 75.00% are in lower-coupling & lower-coordination, medium-coupling & medium-coordination. Among all the factors, proportion of industry and service industry, population density, multiple cropping index, average GDP per km2, and water conservation index have the most important roles in the coordinated development of land use sub-systems. Therefore, we suggest land use/urban management need to implement more integrated planning and differentiated strategies to stimulate land use potential and maintain efficient and sustainable land use.

Clinical correlation of influenza and respiratory syncytial virus load measured by digital PCR.

Acute respiratory tract infections are a major cause of respiratory morbidity and mortality in pediatric patients worldwide. However, accurate viral and immunologic markers to predict clinical outcomes of this patient population are still lacking. Droplet digital PCR assays for influenza and respiratory syncytial virus (RSV) were designed and performed in 64 respiratory samples from 23 patients with influenza virus infection and 73 samples from 19 patients with RSV infection. Samples of patients with hematologic malignancies, solid tumors, or sickle cell disease were included. Clinical information from institutional medical records was reviewed to assess disease severity. Samples from patients with fever or respiratory symptoms had a significantly higher viral loads than those from asymptomatic patients. Samples from patients with influenza virus and RSV infection collected at presentation had significantly higher viral loads than those collected from patients after completing a course of oseltamivir or ribavirin, respectively. RSV loads correlated positively with clinical symptoms in patients ≤5 years of age, whereas influenza viral loads were associated with clinical symptoms, irrespective of age. Patients receiving antivirals for influenza and RSV had a significant reduction in viral loads after completing therapy. Digital PCR offers an effective method to monitor the efficacy of antiviral treatment for respiratory tract infections in immunocompromised hosts.

Satellite data indicates multidimensional variation of agricultural production in land consolidation area.

Land consolidation (LC) is an innovative way to improve agricultural production. Spatiotemporal pattern of agricultural production in land consolidation area (LCA) is difficult to quantify with limited field observations and survey data. Satellite data has advantages on recording vegetation status changes frequently, which is very supportive of estimating variation of agricultural production. In this paper, we used Net Primary Productivity (NPP), Normal Difference Vegetation Index (NDVI), and Multiple Band Drought Index (MBDI) from satellite data, to examine five attributes (irrigation capacity, multiple cropping index, crop phenology, farmland productivity, and production stability) of agricultural production after land consolidation (LC) at a site in China. Results show that there were no significant spatial differences in irrigation capacity for farmland in few years after LC due to consistent climatic conditions and uniform irrigation and drainage system. Multiple cropping index shows a pattern of "first reducing, then growing, last reducing", which may result from the disturbed "water-soil" environment and weak farmers' intention. Interannual variation of spatial distribution of phenology for the second-season crop is larger than that for the first-season crop since LC implementation adjusts short-term land use and management. With the improvement of production conditions and balanced distribution of production elements, farmland productivity has been improved and its differences among various farmland patches imply a reducing trend. Production in LCA is slightly less stable than that in the control area (TCA) where LC is not carried out because of limited and short-term effect from LC. We concluded that satellite data presents variation of agricultural production in LCA from different dimensions of time, space and attributes. Multidimensional variation of agricultural production is decided by several factors, including climate condition, LC activity, and farmers' intention.

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).

Epidemiology of Diarrheal Illness in Pediatric Oncology Patients.

BACKGROUND: Diarrhea is common in children with cancer, but this has not been systematically studied to date. METHODS: Remnant stool samples collected between January 2010 and June 2011 from pediatric oncology patients with diarrhea were tested for bacterial, viral, and parasitic enteropathogens using a combination of standard-of-care (SOC) diagnostic tests, including broad-range, real-time polymerase chain reaction (PCR) assays for adenoviruses, astroviruses, and sapoviruses and 2 commercially available multiplexed PCR assays. Corresponding demographic and clinical data were abstracted from patients' medical records. RESULTS: One hundred fourteen episodes of diarrhea in 93 patients (median age, 3.7 years; range, 0.2-18.8) were included in the study. No patients died, but morbidity was significant. A total of 158 potential pathogens were detected in 114 diarrhea episodes, with >1 organism in one third of these; the most common were Clostridium difficile, noroviruses, adenoviruses, and astroviruses. Clostridium difficile, in combination with norovirus or adenovirus, was most common when >1 pathogen was detected. When both studies were obtained, SOC and broadly multiplexed PCR tests were concordant in 64 episodes (56%). Forty-five pathogens (28%) were identified retrospectively by broadly multiplexed PCR assays only. A total of 19 (13%) were detected by SOC real-time PCR assays but not by either commercially available multiplexed PCR assay. CONCLUSIONS: Most pediatric oncology patients in this study had 1 or more potential infectious causes for their diarrhea. Additional studies are warranted to understand the natural history of gastroenteritis in this patient population. Although broadly multiplexed PCR assays offer some advantages over conventional testing, there may be disadvantages to their use for the diagnosis of infectious gastroenteritis that are unique to pediatric oncology patients.

Comparative Evaluation of Four Real-Time PCR Methods for the Quantitative Detection of Epstein-Barr Virus from Whole Blood Specimens.

Monitoring of Epstein-Barr virus (EBV) load in immunocompromised patients has become integral to their care. An increasing number of reagents are available for quantitative detection of EBV; however, there are little published comparative data. Four real-time PCR systems (one using laboratory-developed reagents and three using analyte-specific reagents) were compared with one another for detection of EBV from whole blood. Whole blood specimens seeded with EBV were used to determine quantitative linearity, analytical measurement range, lower limit of detection, and CV for each assay. Retrospective testing of 198 clinical samples was performed in parallel with all methods; results were compared to determine relative quantitative and qualitative performance. All assays showed similar performance. No significant difference was found in limit of detection (3.12-3.49 log10 copies/mL; P = 0.37). A strong qualitative correlation was seen with all assays that used clinical samples (positive detection rates of 89.5%-95.8%). Quantitative correlation of clinical samples across assays was also seen in pairwise regression analysis, with R(2) ranging from 0.83 to 0.95. Normalizing clinical sample results to IU/mL did not alter the quantitative correlation between assays. Quantitative EBV detection by real-time PCR can be performed over a wide linear dynamic range, using three different commercially available reagents and laboratory-developed methods. EBV was detected with comparable sensitivity and quantitative correlation for all assays.

Comparative Evaluation of Broad-Panel PCR Assays for the Detection of Gastrointestinal Pathogens in Pediatric Oncology Patients.

Broadly multiplexed molecular amplification assays offer an unprecedented ability to diagnose gastrointestinal infection in immunocompromised patients. However, little data are available to compare the performance of such systems in this population. A total of 436 stool samples were collected from 199 predominantly immunocompromised pediatric oncology patients. Remnant samples were tested in parallel with the use of the premarket (investigational use only) versions of two broadly multiplexed PCR assays (BioFire and Luminex), and the results of samples corresponding to the first episode per patient were compared with those from laboratory-developed molecular assays, culture, and antigen detection. Overall performance of the multiplexed systems was comparable, with BioFire and Luminex detecting 94 and 99 positives (P = 0.34), respectively. Stratifying by analyte, BioFire assay detected 51 samples positive for Clostridium difficile, whereas Luminex assay detected 60 (P = 0.01). Biofire and Luminex detected 28 and 38 norovirus-positive samples (P = 0.002), respectively. Astrovirus- and adenovirus-positive samples were detected in higher numbers by in-house PCR than by BioFire; the same was observed for adenovirus with Luminex. Differences observed with other analytes were minimal, did not reach statistical significance, or lacked the numbers needed to detect a difference between systems. Broadly multiplexed PCR offers an effective means of detecting a variety of gastrointestinal pathogens in pediatric oncology patients, with assay performance comparable among the tests examined.

Impact of adenoviral stool load on adenoviremia in pediatric hematopoietic stem cell transplant recipients.

BACKGROUND: Adenoviremia adversely affects prognosis in the post-hematopoietic stem cell transplant setting. METHODS: We sought to determine retrospectively the cutoff load of adenovirus in the stool as a predictor of adenoviremia, in children who underwent an allogeneic hematopoietic stem cell transplant. The prevalence of sapovirus, norovirus and astrovirus in the stool was also studied. RESULTS: The study cohort consisted of 117 patients, of which 71 (60%) had diarrhea. Adenovirus was detected in the stool in 39 of 71 (55%) patients. Age ≤10 years (P = 0.05; odds ratio: 2.57; 95% confidence interval: 0.98-6.75) and male sex (P = 0.04; odds ratio: 2.67; 95% confidence interval: 1.02-6.99) increased risk for detection of adenovirus in stool on univariate analysis. Coinfections with enteric pathogens were infrequent. Viral load >10 copies/g stool predicted adenoviremia with a sensitivity and specificity of 82%. Sapovirus, norovirus and astrovirus were detected in 3, 4 and 1 patient, respectively. CONCLUSIONS: Quantitative detection of adenovirus in stool may have implications for preemptive therapy. Testing for other enteric viruses may have implications for infection control.

Quantitative multiplexed detection of common pulmonary fungal pathogens by labeled primer polymerase chain reaction.

CONTEXT: Invasive fungal infections are an important cause of morbidity and mortality among immunocompromised patients. OBJECTIVE: To design and evaluate a multiplexed assay aimed at quantitative detection and differentiation of the 5 molds that are most commonly responsible for pulmonary infections. DESIGN: Using labeled primer polymerase chain reaction chemistry, an assay was designed to target the 5.8S and 28S ribosomal RNA genes of Aspergillus spp, Fusarium spp, Scedosporium spp, and members of the order Mucorales ( Rhizopus oryzae , Rhizopus microsporus, Cunninghamella bertholletiae, Mucor circinelloides, Lichtheimia corymbifera, and Rhizomucor pusillus). This assay was split into 2 multiplexed reactions and was evaluated using both samples seeded with purified nucleic acid from 42 well-characterized clinical fungal isolates and 105 archived samples (47 blood [45%], 42 bronchoalveolar lavage fluid [40%], and 16 tissue [15%]) collected from rabbit models of invasive pulmonary fungal infections. RESULTS: Assay detection sensitivity was less than 25 copies of the target sequence per reaction for Aspergillus spp, 5 copies for Fusarium spp and Scedosporium spp, and 10 copies for the Mucorales. The assay showed quantitative linearity from 5 × 10(1) to 5 × 10(5) copies of target sequence per reaction. Sensitivities and specificities for bronchoalveolar lavage fluid, tissue, and blood samples were 0.86 and 0.99, 0.60 and 1.00, and 0.46 and 1.00, respectively. CONCLUSIONS: Labeled primer polymerase chain reaction permits rapid, quantitative detection and differentiation of common agents of invasive fungal infection. The assay described herein shows promise for clinical implementation that may have a significant effect on the rapid diagnosis and treatment of patients' severe infections caused by these pulmonary fungal pathogens.

Prospective evaluation for respiratory pathogens in children with sickle cell disease and acute respiratory illness.

BACKGROUND: Human rhinovirus (HRV), human coronavirus (hCoV), human bocavirus (hBoV), and human metapneumovirus (hMPV) infections in children with sickle cell disease have not been well studied. PROCEDURE: Nasopharyngeal wash specimens were prospectively collected from 60 children with sickle cell disease and acute respiratory illness, over a 1-year period. Samples were tested with multiplexed-PCR, using an automated system for nine respiratory viruses, Chlamydophila pneumoniae, Mycoplasma pneumoniae, and Bordetella pertussis. Clinical characteristics and distribution of respiratory viruses in patients with and without acute chest syndrome (ACS) were evaluated. RESULTS: A respiratory virus was detected in 47 (78%) patients. Nine (15%) patients had ACS; a respiratory virus was detected in all of them. The demographic characteristics of patients with and without ACS were similar. HRV was the most common virus, detected in 29 of 47 (62%) patients. Logistic regression showed no association between ACS and detection of HRV, hCoV, hBoV, hMPV, and other respiratory pathogens. Co-infection with at least one additional respiratory virus was seen in 14 (30%) infected patients, and was not significantly higher in patients with ACS (P = 0.10). Co-infections with more than two respiratory viruses were seen in seven patients, all in patients without ACS. Bacterial pathogens were not detected. CONCLUSION: HRV was the most common virus detected in children with sickle cell disease and acute respiratory illness, and was not associated with increased morbidity. Larger prospective studies with asymptomatic controls are needed to study the association of these emerging respiratory viruses with ACS in children with sickle cell disease.

Comparison of two multiplexed PCR assays for the detection of HSV-1, HSV-2, and VZV with extracted and unextracted cutaneous and mucosal specimens.

BACKGROUND: Several analyte specific reagents (ASRs) are available for the detection and differentiation of HSV-1, HSV-2, and VZV in clinical specimens. However, there is limited data on the test performance of these reagents used in multiplexed PCR assays. OBJECTIVE: This study compared the performance of two multiplexed ASR sets for detection of HSV-1, HSV-2, and VZV in dermal specimens. STUDY DESIGN: Two commercially available ASRs were combined to produce multiplexed PCR assays for simultaneous detection of HSV-1, HSV-2, and VZV. Seeded samples were used to determine the limit of detection (LOD) for each assay. Patient samples (n=156) were tested in duplicate and results for each method compared to the reference standard of culture. Both extracted and unextracted specimens were used in the study. RESULTS: Both multiplexed PCR assays showed similar test performance, with minimal LOD differences observed. The LOD was 10(3) copies/mL for HSV-1 and HSV-2 using the Focus assay compared to 5×10(3) copies/mL and 2×10(4) copies/mL, respectively for the EraGen assay. Both assays showed equal performance for VZV with a LOD of 5×10(3) copies/mL. Analytical specificity testing showed no cross reactivity with other selected DNA viruses. Both assays showed similar performance when clinical samples were tested using both extracted and unextracted specimens. CONCLUSION: Commercially available ASRs can be successfully multiplexed for the PCR detection of HSV-1, HSV-2, and VZV using dermal specimens. Either direct testing or nucleic acid extracted specimens can be used with similar performance in these assays.

PCR followed by electrospray ionization mass spectrometry for broad-range identification of fungal pathogens.

Invasive fungal infections are a significant cause of morbidity and mortality among immunocompromised patients. Early and accurate identification of these pathogens is central to direct therapy and to improve overall outcome. PCR coupled with electrospray ionization mass spectrometry (PCR/ESI-MS) was evaluated as a novel means for identification of fungal pathogens. Using a database grounded by 60 ATCC reference strains, a total of 394 clinical fungal isolates (264 molds and 130 yeasts) were analyzed by PCR/ESI-MS; results were compared to phenotypic identification, and discrepant results were sequence confirmed. PCR/ESI-MS identified 81.4% of molds to either the genus or species level, with concordance rates of 89.7% and 87.4%, respectively, to phenotypic identification. Likewise, PCR/ESI-MS was able to identify 98.4% of yeasts to either the genus or species level, agreeing with 100% of phenotypic results at both the genus and species level. PCR/ESI-MS performed best with Aspergillus and Candida isolates, generating species-level identification in 94.4% and 99.2% of isolates, respectively. PCR/ESI-MS is a promising new technology for broad-range detection and identification of medically important fungal pathogens that cause invasive mycoses.

Detection of respiratory viruses in asymptomatic children undergoing allogeneic hematopoietic cell transplantation.

Detection of respiratory viruses by molecular methods, in children without respiratory symptoms undergoing hematopoietic cell transplantation (HCT), has not been well described. A prospective study of 33 asymptomatic children detected respiratory viruses in 8 of 33 (24%) patients before HCT. Human rhinovirus (HRV) was detected in five patients, and human adenovirus (hADV) in three patients. Two additional patients shed HRV, and one shed human coronavirus (hCoV), post-HCT. Two patients had co-infections. Of the 11 asymptomatic patients where respiratory virus was detected, 3 (27%) later developed an upper respiratory tract infection, from the same virus.

Prospective detection of respiratory pathogens in symptomatic children with cancer.

BACKGROUND: : The data on human rhinovirus, coronavirus, bocavirus, metapneumovirus, Chlamydophila pneumoniae, Mycoplasma pneumoniae and Bordetella pertussis infections in children with cancer is limited. METHODS: : We sought to determine prospectively the prevalence of respiratory pathogens in these children, using multiplexed-polymerase chain reaction. RESULTS: : We enrolled 253 children with upper or lower respiratory tract infection (LRTI) during a 1-year period. A respiratory virus was detected in 193 (76%) patients; 156 (81%) patients had upper respiratory tract infection. Human rhinovirus was the most common virus detected in 97 (62%) and 24 (65%) patients with upper respiratory tract infection and LRTI, respectively. Leukemia or lymphoma was the most common underlying diagnosis in 95 (49%) patients followed by solid tumor 47 (24%), posthematopoietic stem cell transplant 28 (15%) and brain tumor in 23 (12%) patients. By multiple logistic regression analysis, human bocavirus was the most commonly detected respiratory virus in patients with LRTI (P = 0.008; odds ratio, 4.52; 95% confidence interval: 1.48-13.79). Coinfection with >1 virus was present in 47 (24%) patients, and did not increase the risk for LRTI. Two (0.7%) patients succumbed to LRTI from parainfluenza virus-3 and respiratory syncytial virus/human rhinovirus infection, respectively. C. pneumoniae and M. pneumoniae were detected in 4 and 3 patients, respectively. CONCLUSIONS: : Human rhinovirus was the most common virus detected in children with cancer and posthematopoietic stem cell transplant hospitalized with an acute respiratory illness, and was not associated with increased morbidity. Prospective studies with viral load determination and asymptomatic controls are needed to study the association of these emerging respiratory viruses with LRTI in children with cancer and posthematopoietic stem cell transplant.

Evaluation of multiplexed PCR and liquid-phase array for identification of respiratory fungal pathogens.

Invasive fungal infections are the cause of serious morbidity and high mortality in immunocompromised patients. Early laboratory diagnostic options remain limited; however, rapid detection and accurate identification may improve outcome. Herein, multiplexed PCR followed by liquid-phase array was evaluated for detection and identification of common respiratory fungal pathogens, including Aspergillus fumigatus, Rhizopus microsporus, Scedosporium apiospermum and Fusarium solani. The limit of detection ranged 0.1-1 ng of DNA, depending on the fungus being tested. Primer cross-reactivity was seen for some fungi: Aspergillus flavus primers detected Aspergillus oryzae; Scedosporium apiospermum primers detected Paecilomyces lilacinus, and Aspergillus terreus primers detected S. apiospermum. PCR followed by liquid-phase array is potentially useful for the identification of clinically relevant fungal pathogens.