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.

Candida dubliniensis is a newly identified species of Candida that is phenotypically similar to but genetically distinct from C. albicans. This organism has been recovered with increasing frequency from the oral cavities of human immunodeficiency virus (HIV)-infected and AIDS patients and has been implicated as a causative agent of oral candidiasis and systemic disease. In the present study we characterized the molecular mechanisms of resistance to fluconazole (FLC) in C. dubliniensis clinical isolates from two different HIV-infected patients with oropharyngeal candidiasis. Isolates were identified to the species level by phenotypic and genotypic tests. DNA-typing techniques were used to assess strain identity. Antifungal susceptibility testing was performed by NCCLS techniques. Northern blotting analysis was used to monitor the expression of genes encoding lanosterol demethylase (ERG11) and efflux transporters (CDR and MDR1) in matched sets of C. dubliniensis-susceptible and -resistant isolates by using probes generated from their homologous C. albicans sequences. In addition, ERG11 genes were amplified by PCR, and their nucleotide sequences were determined in order to detect point mutations with a possible effect in the affinity for azoles. Decreasing susceptibilities to FLC were detected in C. dubliniensis isolates recovered from both patients during the course of treatment. FLC-resistant C. dubliniensis isolates from one patient demonstrated combined upregulation of the MDR1, CDR1, and ERG11 genes. Among the isolates from the second patient, all isolates showing decreased susceptibility to FLC demonstrated upregulation of MDR1, whereas the levels of mRNA for the ERG11 genes remained constant and the expression of CDR genes was negligible. Fourteen point mutations were found in the ERG11 genes of the isolates with decreased susceptibility to FLC. These data demonstrate that the development of azole resistance in C. dublinensis clinical isolates from HIV-infected patients treated with FLC is mediated by multiple molecular mechanisms of resistance, similar to the observations found in the case of C. albicans.

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Bordetella holmesii is a Gram-negative bacterium first identified in 1995. It can cause pertussis-like symptoms in humans. B. holmesii contains insertion sequences IS481 and IS1001, two frequently used targets in the PCR diagnosis of Bordetella pertussis and Bordetella parapertussis infections.

Cryptococcus neoformans comprises two varieties (neoformans and gattii) and four serotypes (A, B, C and D). Fertile isolates of both mating types have been identified in serotypes B, C and D; however, a fertile serotype A MATa strain has not been confirmed, although serotype A MATa strains will mate with serotype D MATa strains. Preliminary analysis of a recent Italian environmental isolate (IUM 96-2828) suggested that this strain was haploid, serotype A and MATa. In this study, IUM 96-2828 has been characterized in detail. A mating reaction between IUM 96-2828 and H99 (serotype A MATa) produced abundant spores with an equal distribution of MATa and MATa progeny, all of which were serotype A. Karyotypic analysis of F 1 spores revealed evidence of recombination, confirming that IUM 96-2828 was fertile. The MATa pheromone gene from IUM 96-2828 was sequenced and found to be most closely related to the serotype D MATa pheromone gene. Phylogenetic comparisons of other genes not linked to mating type also suggested IUM 96-2828 was most closely related to serotype A strains. Biochemical analysis showed that the carbon assimilation profiles of H99 and IUM 96-2828 were identical for 97 % (30/31) of the substrates while isozyme analysis showed 89 % (17/19) identity. Assays of major virulence factors found no difference between H99 and IUM 96-2828. Virulence studies using the mouse model demonstrated that IUM 96-2828 was virulent for mice, although it was less virulent than H99. These data strongly suggest that IUM 96-2828 is a true haploid serotype A MATa isolate that is fertile.