Aspergillus is a ubiquitous fungus causing a wide spectrum of infections and most threatening clinical manifestation being invasive aspergillosis is associated with high morbidity and mortality. Aspergillus species are easily misdiagnosed based on morphology making it difficult to ascertain the clinical and epidemiological peculiarities of the infections they cause. Also, these species may exhibit variable in vitro susceptibility to antifungal drugs; therefore, accurate identification of species is critical. The aim of the present study was to molecularly identify and to evaluate the MALDI-TOF spectral profile of cryptic Aspergillus species obtained from patients in a referral chest hospital, Delhi, India during 2011-2015.
The clinical specimens included bronchial aspirates, bronchoalveolar lavage, nasal washings, nasal polyps and cerebrospinal fluid from 5 hospitals in Delhi, India. Aspergillus species were preliminarily identified based on macro- and micro morphological characteristics on Czapek dox agar plates incubated at 28oC. Their identification were confirmed by sequencing β-tubulin and calmodulin genesand spectral profiles determined by MALDI-TOF MS. Antifungal susceptibility testing was performed using broth microdilution method (CLSI M38-A2).
Of 11,242 samples processed, 25.7% (n=2421) were positive for Aspergillus species, which included 46% (n=1113) A. flavus, 32.5% (n=786) A. fumigatus and 6.4% (n=155) A. terreus. The remaining 15% (n=367) were Aspergillus species. A selection of 79 Aspergillus spp. isolates were studied with molecular characterization, which included species, found to be atypical in terms of growth and sporulation. They were identified belonging to 24 species by β-tubulin and calmodulin sequencing. The species distribution included three isolates each of A. niveus, A. melleus, Eurotium amstelodami, A. sydowii followed by two each of A. fijiensis, A. tamarii, A. ochraceus, A. hortai, A. tritici, Neosartorya fischeri and A. aculeatus. Other species included A. chevalieri, A. clavatus, A. egyptiacus, A. unguis, A. wentii, Emericella corrugata, E. nidulans and its variety, E. dentata. Among the azoles, posaconazole (GM MIC, 0.06 μg/ml) showed excellent antifungal activity followed by itraconazole (GM MIC, 0.17 μg/ml), isavuconazole (GM MIC, 0.34 μg/ml) and voriconazole (GM MIC, 0.68 μg/ml). Among echinocandins micafungin and anidulafungin exhibited lower MICs than caspofungin against all species. Twelve (15%) isolates excluding A. terreus exhibited high MICs of amphotericin B (MIC ≥2μg/ml). Further 9 isolates (11%) had voriconazole MICs ≥2μg/ml. Also 4 and 3 isolates demonstrated isavuconazole and posaconazole MICs in the range of 4->8μg/ml, respectively. Of the 79 isolates, 62 were identified up to species level by MALDI with a score value of >2. The clinical profile of the majority of patients ranged from allergic, chronic and invasive aspergillosis including a solitary case of brain abscess. However, in 16% of cases Aspergillus species were isolated from respiratory specimens of pulmonary tuberculosis patients with damaged lungs.
The present report extends the spectrum of rare Aspergillus species involved in aspergillosis. Accurate molecular identification is taxonomically and clinically relevant as many of these species exhibit resistance to amphotericin B and voriconazole. Although Bruker MALDI-TOF database is limited to only 19 Aspergillus species, it correctly identified all the species present in the database.