Molecular characterization of Candida spp . isolates from patients with bloodstream infections

Introduction: The aim of this study was to conduct an epidemiological study comparing the genetic similarity of yeasts isolated from blood cultures. Methods: Random amplifi cation of polymorphic DNA (RAPD) techniques were used for the Candida samples obtained from patients at the Hospital Universitário da Universidade Federal do Mato Grosso do Sul (HU/UFMS) in Campo Grande, State of Mato Grosso do Sul, Brazil, from 1998-2000. Results: The most frequently isolated species was Candida albicans (45.8%). DNA amplifi cation from genomic yeast isolates indicated a genetic similarity of over 90%. Conclusions: The RAPD profi les obtained were able to differentiate between the isolated Candida species, thereby suggesting that the method might be useful in epidemiological studies.

Invasive candidiasis is the fourth leading cause of bloodstream infection in cases of nosocomial infection by fungi.Candida albicans is the most often isolated species, accounting for approximately 50% of cases 1 .Molecular techniques have been performed to aid epidemiological studies 2,3 .
This retrospective study was conducted using clinical samples obtained from the mycology collection of the laboratory of the Hospital Universitário da Universidade Federal do Mato Grosso do Sul (HU/UFMS) in Campo Grande, State of Mato Grosso do Sul, Brazil.The yeasts were previously detected in blood cultures using an automated system, BACTTEC TM FX (BD, New Jersey,USA) as a routine hospital practice, and the three most frequently isolated species were selected for genotypic analysis.
The study was approved by the University Federal do Mato Grosso do Sul Ethics Committee.The molecular tests were conducted in the Laboratory of Animal Health at the Brazilian Enterprise for Agricultural Research (EMBRAPA) according to the protocol described by Valério et al. 3 .The dendrogram was created using GelCompar II (Applied Maths, Kortrijk, Belgium) in the Enterobacteria Laboratory of Fundação Oswaldo Cruz, RJ.Similarity was evaluated using Pearson's coeffi cient.For purposes of epidemiological analysis, the strains that showed > 90% similarity or had a dendrogram showing a single insertion or inclusion were considered similar or belonging to the same clone 4 .
The electrophoretic profi les of the Candida albicans and Candida parapsilosis complex strains demonstrated a predominance of profi le A (Figures 1 and 2).
The frequency of systemic infections caused by Candida has increased considerably, particularly in patients hospitalized in critical areas because of the use of immunosuppressive agents, invasive medical procedures, and underlying diseases that may contribute to yeast proliferation 2,5 .Among infecting yeasts, C. albicans is the most common in cases of hospital-acquired infections in several countries 1 .
The present results demonstrate that the method was limited to detecting intra-specifi c polymorphisms within clones of the same species using only one primer pair 2,3 .The profi les obtained using random amplifi cation of polymorphic DNA (RAPD) were able to differentiate between the Candida species, but few differences between strains of the same species were observed.Nevertheless, the dendrograms showed a genetic similarity > 90%, suggesting that the method might be useful for epidemiological studies and detection during hospital outbreaks.

A B A B
The authors thank Fundect-MS, Embrapa Beef Cattle, and the UFMS Dean for Research for their fi nancial support.

FIGURE 1
FIGURE 2 -(A) Agarose gel electrophoresis (1.8%) following random amplifi cation of polymorphic DNA-polymerase chain reaction (RAPD-PCR) using the primer 5'd [ACGACCGACA] -3 'indicated different genetic patterns for the Candida parapsilosis isolates that had caused bloodstream infections.Line Y: 100-pb ladder (Invitrogen); Line 1: ATCC 22019; Line 2-21: the samples isolated from infections (B) Dendrograms showing the genotypic profi le of Candida parapsilosis isolates.Clinical samples were obtained from patients at the University Hospital in Campo Grande, State of Mato Grosso do Sul, Brazil.