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.

Phenotypic Switching of Cryptococcus neoformans and Cryptococcus gattii

“…First, relapse of cryptococcal meningitis results during persistent infection with a single infecting strain rather than re-infection with a new strain [24]. Second, serial Cryptococcus isolates from AIDS patients exhibit minor electrophoretic karyotype and they can also differ in growth rates, capsule size, or virulence in mice [23,25]. Additional changes that occur during chronic infection include stable alterations in cell membrane sterol composition and differences in the glucuronoxylomannan (GXM) structure of the capsule [26].…”

“…neoformans and var. grubii isolates were smooth in colony morphology whereas 100% of C. gattii were mucoid [25]. Switching in a smooth (SM) parent strain (RC2, serotype D) that switches to mucoid (MC) variants and reversibly in vitro and in vivo has been thoroughly investigated [30].…”

“…Several clinical case reports and epidemiologic studies have examined C. gattii strains from clinical isolates [31]. Clinical C. gattii strains were found to be less diverse in different studies [25,32] and often are typed as serotype B, MATalpha strains with a typical VGI molecular type [14]. C. gattii is predominantly clonal, and except for the newly emerged RG strain in Canada, the majority to C. gattii strains are not fertile and cannot generate haploid spores by monokaryotic fruiting [33].…”

Molecular diversity of serial Cryptococcus neoformans isolates from AIDS patients in the city of São Paulo, Brazil

“…1997, Calvo et al 2001, Horta et al 2002, Barreto de Oliveira et al 2004, Jain et al 2005, Litvintseva et al 2006, Almeida et al 2007). In general, molecular methods were employed to distinguish varieties and serotypes.…”

“…Some studies suggested that the majority of cases are due to persistence of the original infecting strain, while others suggested that the infections may result from acquisition of new strains. Mixed infections are also possible since environmental sites may contain more than one strain (Currie et al 1994, Barchiesi et al 1995, Haynes et al 1995, Chen et al 1996, Sullivan et al 1996, Sukroongreung et al 2001, Barreto de Oliveira et al 2004, Igreja et al 2004, Jain et al 2005. These controversial studies led us to suppose that all situations could occur in patients with different episodes.…”