T. Kowshik

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.

Decayed wood of Syzygium cumini and Ficus religiosa living trees in Delhi/New Delhi metropolitan area as natural habitat of Cryptococcus neoformans.

The isolation is reported of Cryptococcus neoformans var. gattii and C. n. var. neoformans from decayed wood inside trunk hollows of Syzygium cumini and of C. n. var. neoformans from Ficus religiosa trees in the Delhi/New Delhi metropolitan area. Fourteen of sixty-six (21%) S. cumini trees investigated proved to be positive, seven for each variety. The two varieties never co-occurred in the same hollow. C. n. var. neoformans was also isolated from three of seventeen Ficus religiosa-trees. Two of these isolates originated from decayed wood and one from bark. The C. n. var. gattii and C. n. var. neoformans isolates belonged to serotype B and serotype A, respectively. The data strongly supported colonization of S. cumini by both varieties and of F. religiosa trees by C. n. var. neoformans. Evidence of this was found by repeated isolations. For example, in 36/44 (82%) samples for C. n. var. gattii and 22/27 (81%) samples for C. n. var. neoformans, and by a high population density in the tested wood debris (maximally 6 x 10(5) colony-forming units per gram [c.f.u./g] for C. n. var. gattii and 8 x 10(4) c.f.u./g for C. n. var. neoformans). No eucalypt trees were seen near the positive S. cumini and F. religiosa trees. The densities of C. neoformans in these trees exceeded those found previously in Eucalyptus camaldulensis and in other tree species more rarely reported to be sources of C. neoformans in India. S. cumini and F. religiosa appear not to have been reported to date as sources for either C. n. var. gattii or C n. var. neoformans. Our results add to the recently emerging evidence that the natural habitat of C. n. var. gattii and C. n. var. neoformans is not specific to woody or other debris of particular tree species, but instead is more generalized.

The expanding host tree species spectrum of Cryptococcus gattii and Cryptococcus neoformans and their isolations from surrounding soil in India.

This study reports the widespread prevalence of Cryptococcus neoformans and Cryptococcus gattii in decayed wood inside trunk hollows of 14 species representing 12 families of trees and from soil near the base of various host trees from Delhi and several places in the Indian states of Uttar Pradesh, Haryana, Tamil Nadu and Chandigarh Union Territory. Of the 311 trees from which samples were obtained, 64 (20.5%) were found to contain strains of the C. neoformans species complex. The number of trees positive for C. neoformans var grubii (serotypeA) was 51 (16.3%), for C. gattii (serotype B) 24 (7.7%) and for both C. neoformans and C. gattii 11 (3.5%). The overall prevalence of C. neoformans species complex in decayed wood samples was 19.9% (111/556). There was no obvious correlation between the prevalence of these two yeast species and the species of host trees. The data on prevalence of C. gattii (24%) and C. neoformans (26%) in soil around the base of some host trees indicated that soil is another important ecologic niche for these two Cryptococcus species in India. Among our sampled tree species, eight and six were recorded for the first time as hosts for C. neoformans var grubii and C. gattii, respectively. A longitudinal surveillance of 8 host tree species over 0.7 to 2.5 years indicated long term colonization of Polyalthia longifolia, Mimusops elengi and Manilkara hexandra trees by C. gattii and/or C. neoformans. The mating type was determined for 153 of the isolates, including 98 strains of serotype A and 55 of serotype B and all proved to be mating type alpha (MAT alpha). Our observations document the rapidly expanding spectrum of host tree species for C. gattii and C. neoformans and indicate that decayed woods of many tree species are potentially suitable ecological niches for both pathogens.

Distribution of Cryptococcus gattii and Cryptococcus neoformans in decayed trunk wood of Syzygium cumini trees in north-western India

The aim of this study is to report the regional distribution of Cryptococcus. gattii and Cryptococcus. neoformans in decayed wood inside trunk hollows of Syzygium cumini trees (Java plum, Indian black berry) investigated in Amritsar (Panjab), Meerut Cantt. and Bulandshahr (Uttar Pradesh) and Delhi, in north-western India. Two hundred and seventeen wood samples collected from 74 S. cumini trees were investigated. This includes 7 known positive S. cumini trees in Delhi subjected to a mycological surveillance for perennial colonization by C. gattii and C. neoformans. Cryptococcus gattii showed the highest prevalence (89%) in S. cumini trees in Delhi, followed by 27%, 12.5% and 9% prevalence in Bulandshahr, Amritsar City and Meerut Cantt., respectively. In contrast, C. neoformans had the highest prevalence (54%) in Amritsar, followed by 44% in Delhi, 9% in Bulandshahr and 0% in Meerut Cantt. Furthermore, 44% of the S. cumini trees in Delhi, 9% in Bulandshahr and 8% in Amritsar were concomitantly colonized by both C. gattii and C. neoformans. A mycological surveillance over 4.8-5.2 years of 7 selected S. cumini trees in Delhi revealed perennial colonization by both the Cryptococcus species. In addition, air samples taken close to the decayed trunk hollows of 4 of the perennially colonized S. cumini trees contained strains of the C. neoformans species complex. Of a random sample of 48 isolates serotyped, 26 (54%) were C. neoformans, serotype A, and 22 (46%) C. gattii, serotype B. Determination of mating type alleles was done in 44 of the isolates, comprising 31 of C. neoformans, serotype A and 13 of C.gattii, serotype B. All of them proved to be mating type alpha (MATalpha). The data on high prevalence, fungal population density, perennial colonization and aerial isolations indicate that decayed wood in trunk hollows of S. cumini trees is to-date the main well documented primary environmental niche of C. gattii and C. neoformans in north-western India. Attention is drawn to the likely health hazard posed by the environmental reservoirs of C. gattii and C. neoformans occurring in tree trunk hollows in proximity to human and animal habitations.

Efficacy of swabbing versus a conventional technique for isolation of Cryptococcus neoformans from decayed wood in tree trunk hollows

The efficacy of swabbing versus a conventional sedimentation technique was evaluated for sampling of decayed wood in tree trunk hollows for isolation of Cryptococcus neoformans. Of 52 samples of decayed wood, bark or other plant debris originating from 35 living trees, 42 wood samples yielded C. neoformans. The positive samples included 40 collected from 31 Syzygium cumini trees growing along roadsides in Old Delhi, whereas the remaining two were from inside tree trunk fissures of Ficus religiosa in a New Delhi locality. The number of wood samples found positive by swabbing was 40 (95%) as opposed to 32 (76%) by the conventional technique, and this difference was statistically significant (P < 0.01). Also, the conventional technique showed 24% false-negative results, which was in striking contrast to only 5% by swabbing. Furthermore, swabbing yielded a significantly higher C. neoformans mean colony count than did the conventional technique (P < 0.005), thus highlighting greater efficacy of the former technique. The overall prevalence of C. neoformans in the S. cumini trees investigated was 84% (26/31 trees) which is the highest as yet reported from any tree species in India. Varietal identification and serotyping was done with 33 of the C. neoformans isolates, 31 of which came from 23 tree trunk hollows of S. cumini and two from the tree trunk fissures of F. religiosa. Among the S. cumini isolates, 26 were identified as C. neoformans var. gattii (all serotype B except two untypeable ones) and five as C. neoformans var. neoformans, serotype A (= C. neoformans var. grubii). Both of the F. religiosa isolates belonged to C. n. var. neoformans, serotype A. Being a more efficacious, simple, less time-consuming and less hazardous technique, swabbing is recommended for wider use in order to further elucidate the ecology of C. neoformans.

Cryptococcus randhawai sp. nov., a novel anamorphic basidiomycetous yeast isolated from tree trunk hollow of Ficus religiosa (peepal tree) from New Delhi, India.

A novel anamorphic Cryptococcus species is described, which was isolated in New Delhi (India) from decaying wood of a tree trunk hollow of Ficus religiosa. On the basis of sequence analysis of the D1/D2 domains of the 26S rRNA gene and the internally transcribed spacer (ITS)-1 and ITS-2 region sequences, the isolate belonged to the Cryptococcus albidus cluster (Filobasidiales, Tremellomycetes) and was closely related to Cryptococcus saitoi, Cryptococcus cerealis and Cryptococcus friedmannii with 98% sequence identity. Phenotypically, the species differed from C. saitoi with respect to growth temperature (up to 37oC), presence of a thin capsule, ability to grow in the absence of vitamins, and inability to assimilate citrate and ethylamine. With respect to C. friedmannii, it differed in growth temperature, ability to assimilate lactose, raffinose, l-rhamnose, myo-inositol, and inability to utilize citrate. Furthermore, our isolate also differed from C. cerealis in growth temperature, presence of capsule and inability to assimilate l-sorbose. In view of the above phenotypic differences and unique rDNA sequences, we consider that our isolate represents a new species of Cryptococcus, and therefore, a new species, Cryptococcus randhawai is proposed for this taxon. The type strain J11/2002 has been deposited in the culture collection of the Centraalbureau voor Schimmelcultures (CBS10160) and CABI Biosciences (IMI 393306).

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.

Seasonal variations in the prevalence of Cryptococcus neoformans var. grubii and Cryptococcus gattii in decayed wood inside trunk hollows of diverse tree species in north-western India: a retrospective study

This study presents a 7-year retrospective analysis of seasonal variations in the prevalence of Cryptococcus neoformans var. grubii and Cryptococcus gattii in decayed wood inside trunk hollows of 518 trees belonging to 20 species in north-western India during 2000-2007. Of the 1,439 wood samples investigated, 406 (28.2%) were found to be positive for the Cryptococcus neoformans species complex which included 247 samples from which C. neoformans var. grubii was recovered and 171 which yielded C. gattii. While both of the pathogens were isolated through all the seasons, the overall prevalence of C. neoformans var. grubii was significantly higher (17.2%) than that of C. gattii serotype B (11.9%, P < 0.0001), indicating that decayed wood was as good, if not better, a natural habitat of C. neoformans var. grubii as that of C. gattii. The highest recovery of both yeasts was in the autumn, followed by that in the summer. For C. gattii, the lowest prevalence occurred during the winter and for C. neoformans var. grubii during the rainy season. The low prevalence of C. gattii during winter is similar to that reported from Bogota, Colombia, where C. gattii had a low population density in bark samples but it was not found in decayed wood of trunk hollows investigated during the period of January and February. The prevalence of C. neoformans var. grubii was significantly lower in the rainy season than in the other portions of the year. This finding is similar to the reported low isolation frequency (4%) of C. neoformans var. grubii from chicken feces in the rainy season in northern Thailand. Further investigations are warranted to determine the clinical significance of seasonal variations in the prevalence of C. neoformans var. grubii and C. gattii in decayed trunk wood of various trees in climatically divergent regions of India.

Cryptococcus neoformans serotype A and Cryptococcus gattii serotype B isolates differ in their susceptibilities to fluconazole and voriconazole

This study presents antifungal susceptibility data for environmental isolates of Cryptococcus neoformans serotype A (n=32) and Cryptococcus gattii serotype B (n=18) to fluconazole and voriconazole employing disc diffusion and Etest methods. The disc diffusion test was performed on Mueller-Hinton agar as recommended by the Clinical and Laboratory Standards Institute (CLSI). For comparison, the disc diffusion test and Etest were also performed on RPMI-1640 agar supplemented with 2% glucose. The plates were incubated at 35 degrees C and read after 48h. Comparison of geometric mean inhibition zone diameters revealed that C. gattii isolates were significantly less susceptible than C. neoformans isolates to fluconazole (P=0.001) and voriconazole (P<0.0001). Similar results were obtained on RPMI agar by disc diffusion test and Etest, showing significantly reduced susceptibility for C. gattii isolates. Notwithstanding differences in the susceptibilities of the two species to fluconazole and voriconazole, they appeared susceptible according to the CLSI breakpoints recommended for some Candida spp. To what extent these differences in the susceptibilities of C. neoformans and C. gattii impact on the therapeutic management of cryptococcosis is unclear, although some studies have reported less favourable response in cases caused by the latter species.

Sporotrichosis in India: first authentic case report from the north-western region and a critical literature review

The first case of sporotrichosis from north-western region is reported along with a critical review of the work done on this disease in India. The patient, a 55-year-old housewife with multiple lymphocutaneous lesions on her left arm, had never travelled to north-eastern India where sporotrichosis occurs endemically. She was a resident of Ranikhet, District Nainital, Uttar Pradesh (U.P.), situated about 235 km north-east of Delhi. The diagnosis was established by demonstration of Sporothrix schenckii through direct microscopy and culture of pus collected from multiple suppurative lesions, verification of the dimorphic character of the fungus in vitro and its pathogenicity to white mice. She was treated successfully with oral administration of potassium iodide for 6 weeks.