Dual Infections with Pigmented and Albino Strains ofCryptococcus neoformansin Patients with or without Human Immunodeficiency Virus Infection in India

Abstract

Cryptococcus neoformans is an encapsulated yeast-like fungus of worldwide distribution. Melanin production is an important virulence factor of C. neoformans. We report the identification of distinct cryptococcal isolates with either pigmented or white colony phenotypes on L-dihydroxyphenylalanine agar plates in three patients who presented with meningitis to the All India Institute of Medical Sciences in India. Two of the patients were also infected with human immunodeficiency virus. Biochemical studies, India ink analysis, immunofluorescence with antibodies specific to capsular antigen, and serotyping confirmed that the melanotic and albino strains were C. neoformans serotypes A and D, respectively. Genotyping with M13 and [GACA]4 primers revealed that all the C. neoformans isolates were genetically different. The CNLAC1 gene associated with melanin production was identified in all the strains by PCR. Standard MIC testing revealed that the strains had similar susceptibilities to amphotericin B, but time-kill assays with the antifungal showed reduced susceptibility in melanin-producing strains. Infection studies with A/Jcr mice showed that the melanin-lacking yeast were less virulent than melanin-producing isolates. These findings indicate that these patients had dual infections with pigmented and albino strains of C. neoformans that were phenotypically and biologically different. Continued surveillance of primary isolates from patients with cryptococcosis by analyzing phenotypic differences and by molecular methods may reveal that mixed infections occur more commonly than is currently realized.

Evidence of a Role for Monocytes in Dissemination and Brain Invasion byCryptococcus neoformans

“…These experiments, together with other experiments involving various mutants or congenic strains (43,44), suggest that multiple strains can invade the brain concomitantly. These data support the idea that mixed infections can occur in humans (37) and that C. neoformans can use multiple means of crossing.…”

Differences in the cell wall architecture of melanin lacking and melanin producing Cryptococcus neoformans clinical isolates from India: an electron microscopic study

“…We previously showed that the melanin-lacking cells were able to produce laccase, although the enzyme contained a defective copper binding site that may have affected its ability to catalyze the formation of melanin. Additionally, we were previously unable to isolate insoluble black substances during melanin extraction of the albino isolates, (16). The lack of the melanin layer in the albino clinical isolates is consistent with the previous report of melanin lacking mutants of C. neoformans (15).…”

“…The deposition of melanin in the cell wall is associated with increased virulence and protection from antifungal compounds (16).…”

“…Colonies from primary isolates from 43 patients with cryptococcal meningitis were screened for melanin production on Caffeic acid ferric citrate agar medium (Himedia, India) at All India Institute of Medical Sciences (AIIMS) (16). In three patients melanized and white colonies were isolated.…”

“…However, the techniques have not been utilized to study the cell wall of melanin-lacking primary C. neoformans clinical isolates. We previously described three patients from each of whom distinct albino and melanotic isolates were identified in primary cultures of their CSF (16). The purpose of this study is to determine differences in the cell walls of these albino and pigmented C. neoformans isolates based on TEM analysis.…”