Development and validation of a multiplex real-time PCR assay for detection and quantification of Streptococcus pneumoniae in pediatric respiratory samples.

IMPORTANCE: Streptococcus pneumoniae (Spn) is the world's leading cause of lower respiratory tract infection morbidity and mortality in children. However, current clinical microbiological methods have disadvantages. Spn can be difficult to grow in laboratory conditions if a patient is pre-treated, and Spn antigen testing has unclear clinical utility in children. Syndromic panel testing is less cost-effective than targeted PCR if clinical suspicion is high for a single pathogen. Also, such testing entails a full, expensive validation for each panel target if used for multiple respiratory sources. Therefore, better diagnostic modalities are needed. Our study validates a multiplex PCR assay with three genomic targets for semi-quantitative and quantitative Spn molecular detection from lower respiratory sources for clinical testing and from upper respiratory sources for research investigation.

Qualitative detection of enterovirus D68 from PrimeStore® molecular transport medium: implications for home- and self-collection.

To ensure proper specimen handling for detecting pathogens, like Enterovirus D68 (EV-D68), from home- and self-collection, alternative techniques are needed to ensure safe transport and reliable testing. PrimeStore® Molecular Transport Medium (MTM) may be an option since it does not require cold storage and inactivates virus while preserving RNA for detection. The purpose of this validation study was to demonstrate the ability to detect EV-D68 via rRT-PCR in MTM. Using a quantified EV-D68 positive control standard, MTM limit of detection for EV-D68 RNA is 104 cp/mL and RNA remains stable up to 30 days unfrozen. Positive and negative residual respiratory specimens from the 2018 EV-D68 outbreak were used for clinical testing. There was an 80% positive and 100% negative agreement with samples in MTM compared to reference. This study demonstrates the feasibility of EV-D68 detection from respiratory specimens collected and stored in PrimeStore® MTM, with implications for home- and self-collection.

SARS-CoV-2 persistence in immunocompromised children.

OBJECTIVES: We evaluated the length of time immunocompromised children (ICC) remain positive for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), identified factors associated with viral persistence, and determined cycle threshold (CT ) values of children with viral persistence as a surrogate of viral load. METHODS: We conducted a retrospective cohort study of ICC at a pediatric hospital from March 2020 to March 2021. Immunocompromised status was defined as primary, secondary, or acquired due to medical comorbidities/immunosuppressive treatment. The primary outcome was time to first of two consecutive negative SARS-CoV-2 polymerase chain reaction (PCR) tests at least 24 hours apart. Testing of sequential clinical specimens from the same subject was conducted using the Centers for Disease Control (CDC) 2019-nCoV real-time reverse transcriptase (RT)-PCR Diagnostic Panel assay. Descriptive statistics, Kaplan-Meier curve median event times and log-rank tests were used to compare outcomes between groups. RESULTS: Ninety-one children met inclusion criteria. Median age was 15.5 years (interquartile range [IQR] 8-18), 64% were male, 58% were White, and 43% were Hispanic/Latinx. Most (67%) were tested in outpatient settings and 58% were asymptomatic. The median time to two negative tests was 42 days (IQR 25.0-55.0), with no differences in median time by illness presentation or level of immunosuppression. Seven children had more than one sample available for repeat testing, and five of seven (71%) children had initial CT values of <30 (moderate to high viral load); four children had CT values of <30, 3-4 weeks later, suggesting persistent moderate to high viral loads. CONCLUSIONS: Most ICC with SARS-CoV-2 infection had mild disease, with prolonged viral persistence >6 weeks and moderate to high viral load.