Environmental distribution of Cryptococcus species and some other yeast-like fungi in India.

A study of environmental distribution revealed the occurrence of Cryptococcus neoformans and C. gattii in 9% and 3%, respectively, of 611 samples investigated. C. neoformans showed the highest isolation frequency from tree trunk hollows in Delhi (31%), whereas C. gattii occurred in 12% of the samples in Delhi and 5% in Rajasthan. In addition, Cryptococcus laurentii (=Papiliotrema laurentii), C. rajasthanensis (=Papiliotrema rajasthanensis), C. podzolicus (=Saitozyma podzolica) and C. flavescens (=Papiliotrema flavescens) occurred in 0.5% each. The recovery of C. flavescens and C. podzolicus was new findings for India. One more noteworthy finding was isolation of a new yeast, recently classified as Saitozyma cassiae sp. Novo. The previous strain of this yeast came from tree bark debris in South India. Our isolates came from decayed wood inside a trunk hollow of an Acacia tree in, Bharatpur Bird Sanctuary, Rajasthan. The isolations of novel strains of Cutaneotrichosporon moniliiforme from decayed wood of a Pinus tree was another significant finding. Phenotypically, they differed from T. moniliforme by being encapsulated cells, had melanin-like pigment production and were unable to assimilate d-manitol and d-melezitose. AFLP analysis showed a distinctive banding profile vis-a-vis the reference strains of T. moniliiforme and Cryptotrichosporon anacardii.

New clonal strain of Candida auris, Delhi, India.

A new clonal strain of Candida auris is an emerging etiologic agent of fungemia in Delhi, India. In 12 patients in 2 hospitals, it was resistant to fluconazole and genotypically distinct from isolates from South Korea and Japan, as revealed by M13 and amplified fragment length polymorphism typing.

Molecular characterization and in vitro antifungal susceptibility profile of Schizophyllum commune, an emerging basidiomycete in bronchopulmonary mycoses.

Schizophyllum commune (n = 30) showed lowest geometric mean MICs of isavuconazole (0.19 µg/ml), itraconazole (0.2 µg/ml), voriconazole (0.24 µg/ml), and amphotericin B (0.29 µg/ml) and high geometric mean MICs of fluconazole (19.39 µg/ml) and flucytosine (17.28 µg/ml). Five cases (of 8) of allergic bronchopulmonary mycosis that were treated with itraconazole had no recrudescence after 6 to 24 months of follow-up. One case each of invasive pulmonary mycosis and fungal ball were treated successfully with voriconazole and itraconazole.

Candida nivariensis as an etiologic agent of vulvovaginal candidiasis in a tertiary care hospital of New Delhi, India.

Candida nivariensis is a cryptic species, phenotypically indistinguishable from Candida glabrata and identified by molecular methods. Aside its isolation from broncho-alveolar lavage, we report for the first time the etiologic role of C. nivariensis in 4 patients with vulvovaginal candidiasis. Of 100 phenotypically identified C. glabrata isolates originating from vaginal swabs, 4 were identified as C. nivariensis by polymerase chain reaction and confirmed by sequencing. All of the C. nivariensis isolates exhibited white colonies on CHROMagar. Phylogenetic analysis revealed genotypic diversity in the C. nivariensis isolates originating from within or outside of India. Barring a solitary C. nivariensis isolate with MIC, 16 µg/mL of fluconazole, the rest were susceptible to voriconazole, itraconazole, posaconazole, isavuconazole, amphotericin B, and echinocandins. The patient with high fluconazole MIC did not respond to fluconazole therapy. It is suggested that the prevalence of this species is likely to be much higher than apparent from the sporadic published reports.

Blastomycosis in India: report of an imported case and current status.

We report a case of disseminated blastomycosis in a female resident of Delhi, who acquired the infection during travel to the USA, which was successfully treated with oral itraconazole. In addition, we present a critical literature review, indicating that blastomycosis is endemic in India but its areas of endemicity, prevalence, and the natural habitat of the etiologic agent, remain undetermined. The diagnosis of blastomycosis was made by examination of Gomori's methenamine silver stained sections of tissue obtained from a biopsy of a subcutaneous, abdominal nodular. These studies revealed thick-walled, broad-based budding yeast cells compatible with Blastomyces dermatitidis, and consistent with the isolation of the fungus in cultures inoculated with posterior auricular lymph node aspirate. Microscopically, the isolate had thin, septate hyphae and characteristic spherical to pyriform, smooth-walled microconidia. Its identity was confirmed by conversion to its typical yeast form on pea seed agar at 37°C and by DNA sequencing of ITS and BAD 1 promoter regions.

Isolation of multiple-triazole-resistant Aspergillus fumigatus strains carrying the TR/L98H mutations in the cyp51A gene in India.

OBJECTIVES: Azole resistance in Aspergillus fumigatus isolates impacts on the management of aspergillosis since azoles are primary agents used for prophylaxis and therapy. We report the emergence of resistance to triazoles in two A. fumigatus isolates from patients in Delhi, India. METHODS: One hundred and three A. fumigatus isolates, collected from 85 patients suspected of bronchopulmonary aspergillosis during 2005-10, were investigated for susceptibility to itraconazole, voriconazole, posaconazole and isavuconazole. We undertook a mixed-format real-time PCR assay for the detection of mutations leading to triazole resistance in A. fumigatus. The resistant isolates were compared with 25 Dutch TR/L98H-positive isolates by microsatellite analysis. RESULTS: Of the 103 A. fumigatus isolates tested, only 2 had high MIC values of itraconazole (>16 mg/L), voriconazole (2 mg/L), posaconazole (2 mg/L) and isavuconazole (8 mg/L). The resistant A. fumigatus isolates exhibited the TR/L98H genotype and showed identical patterns by microsatellite typing, but were different from 25 Dutch TR/L98H isolates. CONCLUSIONS: We report for the first time from India the occurrence of TR/L98H mutations in the cyp51A gene (responsible for reduced azole susceptibility) in two A. fumigatus isolates from patients with chronic respiratory disease who had not previously been exposed to azoles. The presence of TR/L98H is consistent with a route of resistance development through exposure to azole compounds in the environment. Given the emergence of azole resistance in environmental strains, continued surveillance of resistance in clinical A. fumigatus strains is desirable for successful therapy of aspergillosis.

Genetic differentiation, recombination and clonal expansion in environmental populations of Cryptococcus gattii in India.

Cryptococcus gattii is a ubiquitous eukaryotic pathogen capable of causing life-threatening infections in a wide variety of hosts, including both immunocompromised and immunocompetent humans. Since infections by C. gattii are predominantly obtained from environmental exposures, understanding environmental populations of this pathogen is critical, especially in countries like India with a large population and with environmental conditions conducive for the growth of C. gattii. In this study, we analysed 109 isolates of C. gattii obtained from hollows of nine tree species from eight geographic locations in India. Multilocus sequence typing was conducted for all isolates using nine gene fragments. All 109 isolates belonged to the VGI group and were mating type α. Population genetic analyses revealed limited evidence of recombination but unambiguous evidence for clonal reproduction and expansion. However, the observed clonal expansion has not obscured the significant genetic differentiation among populations from either different geographic areas or different host tree species. A positive correlation was observed between genetic distance and geographic distance. The results obtained here for environmental populations of C. gattii showed both similarities and differences with those of the closely related Cryptococcus neoformans var. grubii from similar locations and host tree species in India.

Bipolaris hawaiiensis as etiologic agent of allergic bronchopulmonary mycosis: first case in a paediatric patient.

Allergic bronchopulmonary mycosis (ABPM) is a worldwide hypersensitivity lung disease of multiple etiologies with Aspergillus fumigatus as the most common etiologic agent. We report the first instance of Bipolaris hawaiiensis causing ABPM in a paediatric patient. A six-year-old girl presented in June 2009 with productive cough, exertional dyspnoea, occasional wheezing, restricted air entry in left infra-scapular and infra-axillary areas, 7% eosinophils (absolute count 540/mm(3)) and total IgE 1051.3 IU/m in the sera. Bronchoscopy revealed narrowing of left main bronchus and mucoid impaction of the left lower lobe segmental bronchi. Cytological examination of BAL revealed few eosinophils, Charcot-Leyden crystals and mucus embedded hyphae. Examination of KOH wet mounts of repeated sputum and BAL specimens revealed septate, brownish hyphae and culture of the specimens resulted in the isolation of multiple colonies of a fungus later identified as B. hawaiiensis based on phenotypic characters and sequencing of internal transcribed spacer and D1/D2 regions of rDNA. In addition, (1-3)-ß-D-glucan was demonstrated in serum (316 pg/ml) by Fungitell kit, supportive of fungal infection/colonization. Histopathologic studies of a bronchial biopsy revealed necrotic debris, macrophage aggregates, lymphocytes, polymorphs and PAS positive hypae. The patient was administered oral itraconazole for 12 weeks, intravenous liposomal amphotericin B for one month, weekly bronchoscopic suctioning and voriconazole instillation, resulting in reduced mucopurulent secretions and considerable clinical improvement. A serum sample collected on 5 November demonstrated precipitins against antigens of the B. hawaiiensis isolate. In March 2010, intradermal skin testing revealed a strong, type I hypersensitivity (induration diam-12 mm) against B. hawaiiensis. The patient relapsed with wheezing and difficulty in respiration in April 2010. Considering the positive type I cutaneous hypersensitivity, the aforementioned laboratory and clinical observations, the patient was finally diagnosed as having ABPM and was successfully treated with oral prednisone. A high index of clinical suspicion with requisite investigations is crucial for early diagnosis and appropriate therapy of ABPM in order to prevent the late sequelae of irreversible broncho-pulmonary damage.

Application of hypertonic Sabouraud glucose agar for differentiation of Candida dubliniensis from Candida albicans.

An evaluation of hypertonic Sabouraud glucose agar (SGA) with 6.5% NaCl for phenotypic differentiation of Candida dubliniensis from Candida albicans is reported. Identity of the test fungi (C. albicans, 84; C. dubliniensis, 18) was based on their typical phenotypic characteristics and confirmed by a diagnostic polymerase chain reaction that targets the novel C. dubliniensis group I intron in the large ribosomal subunit. At 96 h of incubation at 28 °C, all of the 84 C. albicans isolates showed growth on hypertonic SGA contrary to the consistently negative results with the 20 C. dubliniensis isolates. In strong contrast, chlamydospore formation on Staib agar yielded 10 (11.9%) false-positive results and 74 (88%) of the test C. albicans isolates showed false-negative results at 45 °C. We conclude that hypertonic SGA with 6.5% NaCl can be recommended for wider application as a reliable and inexpensive medium for routine differentiation of C. dubliniensis from C. albicans.

Long-distance dispersal and recombination in environmental populations of Cryptococcus neoformans var. grubii from India.

The basidiomycete yeast Cryptococcus neoformans is a cause of significant morbidity and mortality in immunocompromised hosts throughout the world. The sporadic nature of the infection and the limited empirical evidence for direct human-to-human transmission have led to the belief that infections in humans are predominantly caused by the inhalation of basidiospores from environmental sources. Therefore, analysing the structure of environmental populations of C. neoformans can significantly increase our understanding of its ecology, evolution and epidemiology. Decaying wood is a rich source of organic and inorganic compounds and is known to be a suitable ecological niche for many micro-organisms, including C. neoformans. However, relatively little is known about the population structure of C. neoformans sampled from decaying wood. In this study, we analysed samples of C. neoformans var. grubii colonizing decaying wood in tree hollows of nine tree species in five geographical locations (Delhi, Bulandshahar, Hathras, Amritsar and Amrouli) in north-western India. Multilocus sequence typing was conducted using five gene fragments for each of 78 isolates. All isolates belonged to mating type alpha. Population-genetic analyses identified no evidence for significant differentiation among populations belonging to either different geographical areas or different host tree species. Interestingly, despite the lack of mating type a strains in our survey, we found unambiguous evidence for recombination in our population analyses. Our results are consistent with the hypothesis of long-distance dispersal and recombination in environmental populations of this species in India.

Antifungal susceptibility of Cryptococcus neoformans and Cryptococcus gattii isolates from decayed wood of trunk hollows of Ficus religiosa and Syzygium cumini trees in north-western India.

BACKGROUND: We present antifungal susceptibility data on environmental isolates of Cryptococcus neoformans (serotype A, n=117) and Cryptococcus gattii (serotype B, n=65) cultured from decayed wood of trunk hollows of Ficus religiosa and Syzygium cumini trees. METHODS: Susceptibilities to amphotericin B, fluconazole, ketoconazole, itraconazole and voriconazole were determined by using Etest. The MICs were read after 48 h as per the guidelines provided by the manufacturer. RESULTS: The MIC90s and susceptibility ranges for C. neoformans isolates were as follows: 0.094 (0.004-0.25) mg/L for amphotericin B, 4 (0.032-12) mg/L for fluconazole, 0.094 (0.004-0.75) mg/L for itraconazole, 0.064 (0.002-0.19) mg/L for ketoconazole, and 0.047 (0.006-0.125) mg/L for voriconazole, whereas for C. gattii isolates these were 0.125 (0.023-0.5) mg/L for amphotericin B, 8 (0.032-16) mg/L for fluconazole, 0.75 (0.006-2) mg/L for itraconazole, 0.125 (0.003-0.19) mg/L for ketoconazole, and 0.094 (0.004-0.125) mg/L for voriconazole. A comparison of the geometric means of MICs (mg/L) revealed that C. gattii was less susceptible than C. neoformans to amphotericin B (0.075 versus 0.051, P=0.0003), fluconazole (2.912 versus 2.316, P=0.003), itraconazole (0.198 versus 0.0344, P<0.0001), ketoconazole (0.072 versus 0.037, P<0.0001), and voriconazole (0.045 versus 0.023, P<0.0001). CONCLUSIONS: The antifungal susceptibility data obtained in this study indicate that the occurrence of primary resistance among environmental isolates of C. neoformans serotype A and C. gattii serotype B is rare, and serotype B isolates are less susceptible than serotype A isolates.