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Molecular epidemiology of clinical Cryptococcus neoformans strains from India.

ABSTRACT: Little is known about the molecular epidemiology of the human pathogenic fungus Cryptococcus neoformans in India, a country now in the midst of an epidemic of AIDS-related cryptococcosis. We studied 57 clinical isolates from several regions in India, of which 51 were C. neoformans var. grubii, 1 was C. neoformans var. neoformans, and 5 were C. neoformans var. gattii. This strain set included 18 additional sequential isolates from 14 patients. Strains were characterized phenotypically by measuring the polysaccharide capsule and by determining the MICs of standard antifungals. Molecular typing was performed by a PCR-based method using the minisatellite-specific core sequence (M13), by electrophoretic karyotyping, by restriction fragment length polymorphisms with the C. neoformans transposon 1 (TCN-1), and by URA5 DNA sequence analysis. Overall, Indian isolates were less heterogeneous than isolates from other regions and included a subset that clustered into one group based on URA5 DNA sequence analysis. In summary, our results demonstrate (i) differences in genetic diversity of C. neoformans isolates from India compared to isolates from other regions in the world; (ii) that DNA typing with the TCN-1 probe can adequately distinguish C. neoformans var. grubii strains; (iii) that TCN-1 sequences are absent in many C. neoformans var. gattii strains, supporting previous studies indicating that these strains have a limited geographical dispersal; and (iv) that human cryptococcal infection can be associated with microevolution of the infecting strain and by simultaneous coinfection with two distinct C. neoformans strains.

SUBMITTER: Jain N

PROVIDER: S-EPMC1287776 | biostudies-literature | 2005 Nov

REPOSITORIES: biostudies-literature

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Molecular epidemiology of clinical Cryptococcus neoformans strains from India.

Jain N N Wickes B L BL Keller S M SM Fu J J Casadevall A A Jain P P Ragan M A MA Banerjee U U Fries B C BC

Journal of clinical microbiology 20051101 11

Little is known about the molecular epidemiology of the human pathogenic fungus Cryptococcus neoformans in India, a country now in the midst of an epidemic of AIDS-related cryptococcosis. We studied 57 clinical isolates from several regions in India, of which 51 were C. neoformans var. grubii, 1 was C. neoformans var. neoformans, and 5 were C. neoformans var. gattii. This strain set included 18 additional sequential isolates from 14 patients. Strains were characterized phenotypically by measurin ...[more]

PMID: 16272511

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Project description:Abstract Poster session 2, September 22, 2022, 12:30 PM - 1:30 PM Objectives Cryptococcosis affects more than one million people per year worldwide. Despite the worldwide emergence of this ubiquitous infection, little is known about the global molecular epidemiology of this fungal pathogen. The ecological niche of the etiological agents of C. neoformans and C. gattii are not well established yet in various parts of India. Methodology The present study was performed on the C. neoformans isolated from the Eucalyptus terreticornis at Varanasi, India. A total of 265 samples including flowers, leaves, bark, and nearby soil of E. terreticornis. The fungal pathogens were identified by both the conventional and molecular methods. The isolates were also grown in Staib's and tobacco agar medium. In addition, all isolates were identified on the basis of different biochemical tests such as urease test and canavanine-glycine-bromothymol blue (CGB). Further, molecular characterization was carried out by using PCR and DNA sequence analyses. Results Of all the 265 samples, 11 were positive for C. neoformans. 8% (4/50) for E. tereticornis bark, 5% (3/60) for nearby soil, 6.66% (2/30) for debris, and 3.07% (2/65) for decayed wood from tree hollows. C. neoformans was not recovered from the leaves and flowers of E. tereticornis. The presence of brown colonies on Staib's and tobacco agar media confirmed that the isolated pathogen is Cryptococcus. Further the isolates of Cryptococcus were tested for urease production using Christensen urea citrate agar medium in which all isolates were found to hydrolyze urea rapidly and reddish pink color was obtained confirming it to be C. neoformans. All isolated yeast were negative for Canavanine glycinebromothymol blue (CGB) as they fail to produce a color change in the medium. Five strains were identified as C. neoformans var. grubii and one of the isolates was identified as C. neoformans var. neoformans. Molecular typing grouped the isolates into two major genotypes. Five were molecular type VNI (serotype A, var. grubii), one of the isolates was molecular type VNIV (serotype D, var. neoformans). Figure 3 shows the Gel electrophoresis image of ITS1- ITS4 amplicons. The nucleotide sequences were submitted in NCBI GenBank with accession numbers MZ824412, MZ848145, MZ823611, MZ882370, MZ882372, and MZ817986. Conclusion In spite of a major Eucalyptus growing region of India, there is no report of C. neoformans/C. gattii-Eucalyptus association from Eastern Uttar Pradesh. Therefore, the current study would certainly be helpful in the establishment of molecular epidemiology of Cryptococcus in this area.

Project description:BackgroundCryptoccocal meningitis continues to present high incidence among AIDS patients. The treatment of choice is the synergistic combination of flucytosine (5-FC) with amphotericin B deoxycholate (AmBd) or its lipid formulations. However, 5-FC is unavailable in many countries and AmB demands hospitalization. The combination of AmB with the fungistatic fluconazole (FLC) or the use of high FLC daily doses alone became the choice. Nonetheless, sterilization of cerebrospinal fluid is delayed with FLC monotherapy, mainly with high fungal burden. These findings suggest the search for new antifungal compounds, such as liriodenine.MethodsLiriodenine antifungal activity was evaluated by three procedures: determining the minimum inhibitory concentration (MIC) on 30 strains of the Cryptococcus neoformans (C. neoformans) complex and 30 of the Cryptococcus gattii (C. gattii) complex, using EUCAST methodology and amphotericin B deoxycholate as control; performing the time-kill methodology in two strains of the C. neoformans complex and one of the C. gattii complex; and injury to cryptococcal cells, evaluated by transmission electron microscopy (TEM). Liriodenine absorption and safety at 0.75 and 1.50 mg.kg-1 doses were evaluated in BALB/c mice.ResultsLiriodenine MICs ranged from 3.9 to 62.5 μg.mL-1 for both species complexes, with no differences between them. Time-kill methodology confirmed its concentration-dependent fungicidal effect, killing all the strains below the limit of detection (33 CFU.mL-1) at the highest liriodenine concentration (32-fold MIC), with predominant activity during the first 48 hours. Liriodenine induced severe Cryptococcus alterations - cytoplasm with intense rarefaction and/or degradation, injury of organelles, and presence of vacuoles. Liriodenine was better absorbed at lower doses, with no histopathological alterations on the digestive tract.ConclusionThe fungicidal activity confirmed by time-kill methodology, the intense Cryptococcus injury observed by TEM, the absorption after gavage administration, and the safety at the tested doses indicate that the liriodenine molecule is a promising drug lead for development of anticryptococcal agents.

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Molecular epidemiology of clinical Cryptococcus neoformans strains from India.

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Molecular epidemiology of clinical Cryptococcus neoformans strains from India.

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