PCR-RFLP detection and species identification of fungal pathogens in patients with febrile neutropenia
Language English Country England, Great Britain Media print
Document type Journal Article, Research Support, Non-U.S. Gov't
PubMed
14686984
DOI
10.1111/j.1469-0691.2003.00719.x
PII: S1198-743X(14)63226-2
Knihovny.cz E-resources
- MeSH
- Child MeSH
- DNA, Fungal chemistry genetics MeSH
- Fever microbiology MeSH
- Immunocompromised Host MeSH
- Fungi genetics isolation & purification MeSH
- Infant MeSH
- Humans MeSH
- DNA, Ribosomal Spacer chemistry genetics MeSH
- Adolescent MeSH
- Mycoses microbiology MeSH
- Neutropenia microbiology MeSH
- Polymerase Chain Reaction methods MeSH
- Polymorphism, Restriction Fragment Length MeSH
- Child, Preschool MeSH
- RNA, Ribosomal, 5.8S chemistry genetics MeSH
- RNA, Ribosomal chemistry genetics MeSH
- Sensitivity and Specificity MeSH
- Check Tag
- Child MeSH
- Infant MeSH
- Humans MeSH
- Adolescent MeSH
- Male MeSH
- Child, Preschool MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- DNA, Fungal MeSH
- DNA, Ribosomal Spacer MeSH
- RNA, Ribosomal, 5.8S MeSH
- RNA, Ribosomal MeSH
- RNA, ribosomal, 25S MeSH Browser
OBJECTIVE: To assess the usefulness of polymerase chain reaction (PCR) assays in the diagnosis of fungal infections in immunocompromised patients. METHODS: A rapid and sensitive PCR-based assay for the detection and identification of fungal pathogens was designed and applicability of this method was investigated in a group of children with cancer and febrile neutropenia (FN). RESULTS: The ITS2 sequences and adjacent regions of 40 fungal pathogens were analyzed and primers for detection of all analyzed fungal species were designed. Amplification product length polymorphism (APLP) and restriction fragment length polymorphism (RFLP) generated genus- or species-specific patterns. The sensitivity of the method was approximately three cells of Candida albicans per 1 mL of blood. The results were available within 8 h after sample collection. The method was tested on 53 blood samples and one lung biopsy sample from 24 children with cancer and febrile neutropenia (FN). The PCR assay detected fungal DNA in 25 clinical samples from ten patients. Blood cultures were positive in only five samples, while another two blood-culture negative patients had positive cultures from throat swabs. The remaining 14 patients were both culture- and PCR-negative. Culture-isolated strains matched completely those obtained by PCR-APLP-RFLP identification. The identity of fungal species was confirmed by direct sequencing of amplified products. CONCLUSION: Our results suggest that PCR-APLP-RFLP assays can be useful in the diagnosis of fungal infections in immunocompromised patients.
References provided by Crossref.org
Rapid identification of medically important Candida isolates using high resolution melting analysis
