Microbiological and Molecular Methods for Rapid Diagnosis in Systemic Mycoses
DOI:
https://doi.org/10.7546/CRABS.2024.11.10Keywords:
systemic fungal disease, Candida, Aspergillus, Cryptococcus, real-time PCRAbstract
The most frequently employed methodologies for molecular, antigenic, and antibody detection in body fluids (serum, plasma, bronchoalveolar lavage (BAL)) are enzyme-linked immunosorbent assay (ELISA-Platelia), indirect immunofluorescence (IIF), latex agglutination, and polymerase chain reaction (PCR, real-time PCR). Additionally, the examination of clinical materials from a target site may contribute to the identification of medically significant fungi.
The objective of this study was to present the principal laboratory diagnostic methods for the detection of the most prevalent causative agents of invasive fungal diseases, including Candidosis, Aspergillosis, and Cryptococcosis.
More than 200 clinical serum samples were examined serologically from 2018 to 2022 in the National Reference Laboratory “Mycoses” at the National Center of Infectious and Parasitic Diseases in Sofia. Additionally, 20 whole blood samples were analyzed by real-time PCR. Twenty-four clinical samples, including sputum and bronchoalveolar lavage (BAL), were examined culturally on Saburo agar. A total of 10 strains of Candida spp. and 9 strains of Aspergillus spp. were isolated. The antifungal sensitivity of the isolated strains was tested against the most commonly used antifungals, including fluconazole, itraconazole, voriconazole, nystatin, isavuconazole, caspofungin, anidulafungin, and posaconazole.
Three tests for Candida spp. and one for Cryptococcus spp. were positive by latex agglutination. By indirect immunofluorescence (IIF) in 56 examined sera, antibodies were found in seven samples for Candida spp. with a titer greater than 1:160, and 54 samples were positive for antibodies to Aspergillus spp. out of 182 examined serum samples.
A total of 25 serum samples were examined using the ELISA-Platelia test for the detection of Aspergillus antigen. Of these, five samples were found to be positive.
A further 20 samples were tested using real-time PCR to detect the presence of DNA from the most frequently isolated fungal strains from blood cultures. These included Candida albicans, Candida glabrata, Candida parapsilosis, Aspergillus fumigatus and Cryptococcus neoformans. Of the over 200 serum samples tested for the presence of fungi, only a small number yielded positive results. Similarly, testing 20 tubes of whole blood yielded negative results for the presence of fungi using real-time PCR.
The application of microbiological and molecular methods for the rapid diagnosis of systemic mycoses represents a practical approach for routine use in microbiological laboratories.
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