RESUMEN
Candida inconspicua and Candida (Pichia) norvegensis are two emerging pathogenic species that exhibit reduced susceptibility to azole derivatives. Conventional (biochemical) approaches do not readily differentiate between the two species. The first aim of this work was to analyze the performance of biochemical, proteomic (matrix-assisted laser desorption ionization-time of flight [MALDI-TOF]), and molecular approaches in the precise identification of these species. These results then led us to sequence 3 genomic loci, i.e., the internal transcribed spacer (ITS) region of the ribosomal DNA (rDNA), the D1/D2 domain of the 28S rDNA, and the elongation factor 1α (EF-1α) gene, either directly or following cloning, of 13 clinical isolates and 9 reference strains belonging to the 5 species included in the Pichia cactophila clade, namely, Pichia cactophila, Pichia insulana, C. inconspicua, C. norvegensis, and P. pseudocactophila. Finally, isolates of C. inconspicua were challenged for sexual reproduction on the appropriate medium. Our results show that EF-1α sequencing and proteic profiling by MALDI-TOF are the two most efficient approaches to distinguish between C. norvegensis and C. inconspicua. As a characteristic of the P. cactophila clade, we found multiple alleles of the rDNA regions in certain strains belonging to the tested species, making ITS or D1/D2 sequencing not appropriate for identification. Whatever the method of identification, including MALDI-TOF and EF-1α sequencing, none could differentiate C. inconspicua from P. cactophila. The results of phylogenetic analysis and the generation of asci from pure cultures of all C. inconspicua strains both support the identification of P. cactophila as the teleomorph of C. inconspicua.
Asunto(s)
Candida/clasificación , Cruzamientos Genéticos , Orden Génico , Candida/química , Candida/genética , Candida/metabolismo , Candidiasis/microbiología , Análisis por Conglomerados , ADN de Hongos/química , ADN de Hongos/genética , ADN Ribosómico/química , ADN Ribosómico/genética , ADN Espaciador Ribosómico/química , ADN Espaciador Ribosómico/genética , Humanos , Datos de Secuencia Molecular , Técnicas de Tipificación Micológica , Factor 1 de Elongación Peptídica/genética , Filogenia , Proteoma/análisis , ARN Ribosómico 28S/genética , Análisis de Secuencia de ADN , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Antibody detection is a key diagnostic tool for noninvasive aspergillosis (NIA) such as allergic bronchopulmonary aspergillosis and chronic pulmonary aspergillosis. Specific immunoprecipitin detection (IPD) is considered as the reference but lacks standardization and is time-consuming. To evaluate the performance of a new anti-Aspergillus fumigatus IgG enzyme immunoassay (EIA) kit using a recombinant A. fumigatus antigen (Bio-Rad), a retrospective study was performed on 551 sera collected from patients with a definite diagnosis of NIA (group 1; n = 64), bronchial Aspergillus colonization (group 2; n = 26), and probable aerial Aspergillus contamination (group 3; n = 44); from patients suspected of NIA with negative serological and mycological investigations (group 4; n = 49); and from a group of 222 patients not suspected of NIA (group 5). The EIA exhibited excellent reproducibility with coefficients of variation below 10%. Agreement with IPD was calculated between 62.5 and 84.4% according to the group of patients with Cohen's kappa coefficient at 0.6196 ± 0.077. Taking as reference a composite status including clinical, radiological, mycological, and serological data, sensitivity (group 1) and specificity (other groups) were calculated between 90.2 and 93.8% and 54.3 and 100%, respectively. Lower specificity was observed for patients with Aspergillus colonization. However, Yule Q coefficients estimating the correlation between EIA result and the definite diagnosis of NIA were calculated between 0.97 and 0.98. The method is a highly useful screening tool for the diagnosis of NIA, reducing the need for confirmatory IPD tests.