Subject(s)
COVID-19 , Middle East Respiratory Syndrome Coronavirus , Adolescent , Humans , Reinfection , SARS-CoV-2/geneticsABSTRACT
CONTEXT: The incidence of infections due to diverse Candida species is increasing, with correspondingly different antifungal susceptibility patterns. Routine yeast identification methods cause significant delays in appropriate patient management. OBJECTIVE: A DNA pyrosequencing strategy was evaluated for identification of pathogenic Candida species associated with human infections. DESIGN: Clinical (n = 51) and commercial (n = 9) Candida isolates were identified in a blinded, parallel study consisting of routine fungal cultures and biochemical analyses in comparison with DNA pyrosequencing. RESULTS: DNA pyrosequencing yielded species-level identification of all 60 Candida isolates, and sequencing interpretations agreed in all cases with results of biochemical and morphologic testing. Different Candida species were identified, such as C. albicans, C. dubliniensis, C. glabrata, C. guilliermondii, C. krusei, C. lusitaniae, C. parapsilosis, and C. tropicalis. Automated and manual approaches to DNA sequence interpretation, each coupled with the Identifire identification software, demonstrated 100% agreement with respect to Candida species identification. Twenty-one isolates yielded intraspecies DNA sequence differences (90%-98% nucleic acid sequence identity) by automated interpretation. Sequence differences resulted from single-nucleotide polymorphisms or single-base additions/deletions, in addition to interpretative challenges in homopolymeric tracts. CONCLUSION: DNA pyrosequencing coupled with automated DNA sequence alignment provides a practical approach for accurate and timely identification of Candida pathogens. Relatively rapid and facile genotypic studies by DNA pyrosequencing matched the effectiveness of extensive biochemical/morphologic studies for yeast identification.
Subject(s)
Candida/genetics , Candida/pathogenicity , Candidiasis/diagnosis , DNA, Fungal/genetics , Sequence Analysis, DNA/methods , Adolescent , Adult , Base Sequence , Candida/classification , Candidiasis/pathology , Child , Child, Preschool , Genotype , Humans , Infant , Infant, Newborn , Molecular Sequence Data , Mycological Typing Techniques , Phenotype , Polymorphism, Single Nucleotide/geneticsABSTRACT
Sole reliance on biochemical methods can limit the clinical microbiology laboratory's ability to identify bacterial pathogens. This study describes the incorporation of DNA pyrosequencing-based identification for routine pathogen identification of atypical clinical isolates in a large children's hospital. The assay capitalized on the highly conserved nature of 16S rRNA genes by positioning amplification and sequencing primers in conserved target sequences flanking the variable V1 and V3 regions. A total of 414 isolates of 312 pediatric patients were tested by DNA pyrosequencing during the time period from December 2003 to July 2006. Seventy-eight different genera were specified by DNA pyrosequencing, and isolates were derived from diverse specimen types. By integrating DNA sequencing of bacterial pathogens with conventional microbiologic methods, isolates that lacked a definitive identification by biochemical testing yielded genus- or species-level identifications in approximately 90% of cases by pyrosequencing. Improvements incorporated into the assay process during the period of clinical testing included software enhancements, improvements in sequencing reagents, and refinements in database search strategies. Coupled with isolation by bacteriologic culture and biochemical testing, DNA pyrosequencing-based bacterial identification was a valuable tool that markedly improved bacterial pathogen identification in a pediatric hospital setting.
