Rev. Soc. Bras. Med. Trop. vol.46 número6

786

Address to: Dra _{Paula Cristina Niz Xavier. Rua Itacaja 06/Casa 49, Res. Guarani,}

79092-400 Campo Grande, MS, Brasil. Phone: 55 67 9283-6620.

e-mail: paulaxavier80@yahoo.com.br Received 16 October 2010

Accepted 25 July 2012

Revista da Sociedade Brasileira de Medicina Tropical 46(6):786-787, Nov-Dec, 2013

http://dx.doi.org/10.1590/0037-8682-1718-2013

Short Communication

Molecular characterization of

Candida

spp. isolates from

patients with bloodstream infections

Paula Cristhina Niz Xavier

_{, Marilene Rodrigues Chang}

_,

Claudete Rodrigues Paula

_{, Flávia Emi Matsumoto}

_{, Marise Dutra Asensi}

_,

Maria de Fátima Cepa Matos

_{and Renato Andreotti}

[1]. Programa de Pós-Graduação, Universidade Federal do Mato Grosso do Sul, Campo Grande, MS. [2]. Departamento de Farmácia e Bioquímica, Universidade Federal de Mato Grosso do Sul, Campo Grande, MS. [3]. Departamento de Microbiologia, Instituto de Ciências Biológicas, Universidade de São Paulo, São Paulo, SP. [4]. Departamento de Enterobactérias, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, RJ. [5]. Empresa Brasileira de Pesquisa Agropecuária, Gado de Corte, Campo Grande, MS.

ABSTRACT

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.

Keywords: Candida. Polymerase chain reaction. Random amplifi cation of polymorphic DNA.

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 cases1_{. Molecular techniques have} been performed to aid epidemiological studies2,3_.

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, BACTTECTM _{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 clone4_.

The species implicated in the infectious episodes were

Candida albicans, 44 (45.8%), Candida parapsilosis complex 33, (34.4%), Candida tropicalis, 14 (14.6%) and Candida glabrata,

5 (5.2%).

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 proliferation2,5_{. Among infecting yeasts,}

C. albicans is the most common in cases of hospital-acquired infections in several countries1_.

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 pair2,3_{. The} profi les obtained using random amplifi cation of polymorphic DNA

Xavier PCN et al - Molecular characterization of Candida spp. isolates

REFERENCES

The authors declare that there is no confl ict of interest.

CONFLICT OF INTEREST

Pearson correlation [0.0% - 100.0%]_{R A P D 3}

80 90 100

1000bp 1000bp

500bp 500bp

100bp 100bp

Primer 5'd[ACGACCGACA]-3

194H2/04 - L12 667H/04 - L14 154H1/99 - L19 863H4/05 - L15 66H8/99 - L3 ATCC 22019 - L1 614H11/05 - L13 25H3/00 - L18 530H11/01 - L20 988H10/00 - L21 262H12/00 - L4 887H9/06 - L7 60H1/04 - L9 790H6/04 - L11 126H10/01 - L8 539H8/00 - L5 62H10/06 - L6 989H5/05 - L16 910H2/06 - L17 1000H1/04 - L10 698H3/99 - L2 Y 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 171819 20 21 Y

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 bloodstream 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.

1. Maluche ME, Santos JI. Candida sp. e infecções hospitalares: aspectos epidemiológicos e laboratoriais. RBAC 2008; 40:65-67.

Pearson correlation [0.0% - 100.0%] R A P D 6 80 90 100

1000bp 1000bp

500bp 500bp

100bp 100bp

Primer 5'd[ACGACCGACA]-3

Y _{1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 171819 20 21 22 23 24 25 26 27} Y 714H12/05 – L12_{788H4/05 – L13}

817H6/04 – L11 852H2/02 – L8 735H10/03 – L9 85H8/04 – L10 716H4/01 – L7 422H8/05 – L5 591H11/01 – L6 113H3/98 – L2 10H10/00 – L3 ATCC 10231 – L1 78H7/01 – L4 314H5/06 – L22 814H1/06 – L23 992H5/03 – L19 284H3/04 – L20 509H2/05 – L21 34H6/01 – L16 832H5/03 – L18 68H5/99 – L25 808H8/06 – L26 245H8/01 – L24 56H5/99 – L17 521H2/98 – L27 559H6/02 – L14 44H10/98 – L15

FIGURE 1 - (A) An example of the random amplifi cation of polymorphic DNA patterns obtained with primer OPG-17 5´d [ACGACCGACA] - 3´ using Candida albicans

isolates. Line Y: 100-pb ladder (Invitrogen); Line 1: Positive control (ATCC) 10231; and L2-L27, samples isolated from bloodstream infections. (B) Dendrograms showing the

genotypic profi les of the Candida albicans isolates. Clinical samples were obtained from patients at the University Hospital in Campo Grande, State of Mato Grosso do Sul, Brazil.

A

B

A

_B

The authors thank Fundect-MS, Embrapa Beef Cattle, and

the UFMS Dean for Research for their fi nancial support.

ACKNOWLEDGMENTS 2. Pinto PM, Resende M, Koga-Ito CY, Tendler M. Genetic variability analysis among clinical Candida spp. Isolates using randon amplifi ed polymorphic DNA. Mem Inst Oswaldo Cruz 2004; 99:147-152.

3. Valério HM, Weikert-Oliveira RCB, Resende MA. Differentiation of

Candida species obtained from nosocomial candidemia using RAPD-PCR technique.Rev Soc Bras Med Trop2006; 39:174-178.

4. Resende PCJ, Franco GR, Rosa CA, Hahn RC, Hamdan JS. Phenotypic

and genotypic identifi cation of Candida spp. isolated from hospitalized patients. Rev Iberoam Micol 2004; 21:24-28.