Project description:Limited understanding of the immunopathogenesis of human herpesvirus 6B (HHV-6B) has prevented its acceptance as a pulmonary pathogen after hematopoietic cell transplantation (HCT). We conducted a prospective multicenter study of patients undergoing bronchoalveolar lavage (BAL) for pneumonia after allogeneic HCT. We tested blood and BAL fluid (BALF) for HHV-6B DNA and mRNA transcripts associated with lytic infection and performed RNA-seq on paired blood. Among 116 participants, HHV-6B DNA was detected in 37% of BALs, 49% of which had HHV-6B mRNA detection. We established an HHV-6B DNA threshold (≥2.3 log10copies/ml in BALF) that was highly predictive of HHV-6B mRNA detection and increased risk for death from respiratory failure (adjusted HR, 2.35; 95% CI, 1.08-5.11). Participants with HHV-6B DNA in BALF exhibited distinct host gene expression signatures, notable for enriched interferon signaling pathways in participants clinically diagnosed with idiopathic pneumonia. These data implicate HHV-6B as a pulmonary pathogen after allogeneic HCT.
Project description:Diagnostic primer extension assay to serotype Streptococcus pneumoniae. Assay validation. Background: Monitoring of Streptococcus pneumoniae serotype epidemiology is essential since serotype replacement is a concern when introducing new polysaccharide-conjugate vaccines. To simplify S. pneumoniae serotyping, a novel PCR-based automated microarray assay was developed to assist in the tracking of the serotypes. Results: Autolysin (lytA), pneumolysin (ply) and eight genes located in the capsular operon (cps) were amplified using multiplex PCR. This step was followed by a tagged fluorescent primer extension step targeting serotype-specific polymorphisms. The tagged primers were then hybridized to a microarray. Results were exported to an expert system that transforms genetic typing data into capsular serotype identification. The assay was validated on 166 cultured S. pneumoniae samples from 63 different serotypes as determined by the Quellung method. In addition, the assay was tested on clinical specimens including 43 cerebrospinal fluid samples from patients with meningitidis and 59 nasopharyngeal aspirates from bacterial pneumonia patients. The assay presented with no cross-reactivity for 24 relevant bacterial species found in these types of samples. The limit of detection for serotyping and S. pneumoniae detection was 100 genome equivalent per reaction. Conclusion: This automated assay is amenable to clinical testing and does not require any culturing of the samples. The assay will be useful for the evaluation of serotype prevalence changes after new conjugate vaccines introduction.
