Diversity among clinical isolates of Histoplasma capsulatum detected by polymerase chain reaction with arbitrary primers

Abstract

Clinical isolates of the fhngal respiratory and systemic pathogen Histoplasma capsum have been placed in several different classes by using genomic restriction fragent length polymorphisms (RFLPs), but in general have not been distinguished further. We report here that a polymerase chain reaction (PCR)-based DNA fingerprinting method that has been termed arbitrary primer or random amplified polymorphic DNA (RAPD) PCR can distinguish among isolates in a single RF1LP class. In this method, arbitrarily chosen oligonucleotides are used to prime DNA synthesis from genomic sites that they fortuitously match, or almost match, to generate strain-specific arrays of DNA frgments. Each of 29 isolates of RFLP class 2, the group endemic in the American Midwest, was distinguished by using just three arbitrary primers. In contrast, laboratory-derived S and E colony morphology variants of two strains were not disfinguished from their R parents by using 18 such primers. Thus, the clinical isolates of H. capsua are quite diverse, but their genomes remain stable during laboratory culture. These outcomes suggest new possibilities for epidemiological analysis and studies of fungal populations in infected hosts.The fungal respiratory and systemic pathogen Histoplasma capsulatum grows in rich soils and rotting organic matter in moist temperate and tropical regions around the world and infects humans and animals when they inhale its conidia or hyphal fragments (for reviews, see references 4, 6, 16, and 24). Once inhaled, the fungus changes from a free-living mycelium to a host-adapted yeast form, is ingested by pulmonary macrophages, proliferates within them, and spreads to other tissues as the macrophages migrate. A disseminated, often life-threatening form of disease occurs in immunodeficient persons, including those with AIDS and various cancers, very young children, and elderly people, and in those with pulmonary lesions such as are caused by heavy smoking. Infections of immunocompetent people may cause mild influenza-like symptoms or be clinically inapparent, but they can also result in long-term carriage of the organism and a potential for reactivation years after the initial exposure. In these infections, activated macrophages ingest the fungal cells and suppress their growth (unlike nonactivated macrophages) but do not eradicate them completely; calcified lesions also form in the lungs and other tissues, probably because of localized fungal growth and then restriction by host defenses. Survival of fungi in calcifications and in macrophages and other cell types would contribute to long-term latency (4).An ability to distinguish individual strains of H. capsulatum with sensitivity and ease would contribute to epidemiological studies and analyses of infection mechanisms. in mitochondrial DNA and in several nuclear genes allowed H. capsulatum isolates to be divided into six "classes" that seem to reflect geographical distribution or virulence levels (7,(19)(20)(21). In particular, most isolates from immunocompetent adults in the Am...

Genetic Diversity of Histoplasma capsulatum Strains Isolated from Soil, Animals, and Clinical Specimens in Rio de Janeiro State, Brazil, by a PCR-Based Random Amplified Polymorphic DNA Assay

“…Analysis of our findings showed that the H. capsulatum isolates included in this study had 80 to 100% similarity and that despite this genetic similarity, they could be separated into four to six different genotypes. The primers used for RAPD analysis were chosen based on a previous study (8). Although the choice of other primers with higher discriminatory power could be applied to this study, many arbitrary oligonucleotides can serve as informative primers.…”

“…Broad groupings can be defined using hybridization with the yps3 probe (7), with additional discrimination provided by applying a mitochondrial DNA probe to the same Southern blots (15). Random amplification of polymorphic DNA (RAPD) is capable of revealing individual strain subtypes (8). More recently, another typing method has been described that is based on nucleotide sequence variation in the internal transcribed spacer regions of the rRNA gene, where Indianapolis isolates were classified into four types (4).…”

Genotyping Techniques for Determining the Diversity of Microorganisms

“…It has been successfully applied in the genetic differentiation of Salmonella (Tikoo, 2001), E. coli (Renqua-Mangia et al, 2004), A. baumannii (Reboli et al, 1994), S. marcescens (Debast et al, 1995), Proteus mirabilis (Bingen et al, 1993), Enterobacter cloacae (Hou et al, 1997), Haemophilus somnus (Myers et al, 1993), Leptospira species (Ralph et al, 1993), L. pneumophilia (Tram et al, 1990, Van Belkum et al, 1993, B. pseudomallei (Haase et al, 1995, Leelayuwet et al, 2000, Aeromonas salmoniciola (Miyata et al, 1995), Aeromonas hydrophila (Miyata et al, 1995), Vibrio cholerae (Leal et al, 2004), S. aureus (Saulnier et al, 1993), Lactobacillus plantarum (Elegado et al, 2004;Lawrence et al, 1993), Bacillus cereus (Svensson et al, 2004), Listeria monocytogenes (Hiroshi et al, 2007), Candida albicans (Lehmann et al, 1992, Robert et al, 1995, Histoplasma capsulatum (Kersulyte et al, 1992) and Cryptococcus neoformans (Yamamoto et al, 1995). RAPD is highly discriminatory and therefore especially useful in the investigation of short term or local outbreaks of disease.…”

Phylogenetic relationships of Pleurotus species based on RAPD analysis

“…(Berg et al, 1994;Kim et al, 1995). RAPD markers distinguished between the cultivated mushroom (Khush et al, 1992) and between clinical the fungal pathogens (Kersulyte et al, 1992). RAPD markers identified different populations and species of arbiscularmycohorizal fungi.…”