MEM INST OSWALDO CRUZ, RIO DE JANEIRO, 101(2) March 2006
PAGES: 219-221 DOI: S0074-02762006000200015 Short communication
Evaluation of polymerase chain reaction for the detection of Paracoccidioides brasiliensis DNA on serum samples from patients with paracoccidioidomycosis

Cecília Eugenia CharbelI,1; José Eduardo LeviII; José Eduardo Costa MartinsIII

IInstituto de Medicina Tropical de São Paulo, Rua Dr. Enéas de Carvalho Aguiar 500, 05403-000 São Paulo, SP, Brasil
IILaboratório de Virologia, LIM-52, Rua Dr. Enéas de Carvalho Aguiar 500, 05403-000 São Paulo, SP, Brasil
IIILaboratório de Micologia Médica, LIM-53, Rua Dr. Enéas de Carvalho Aguiar 500, 05403-000 São Paulo, SP, Brasil

Abstract

The aim of this study was to demonstrate the DNA of Paracoccidioides brasiliensis in human serum samples of patients with paracoccidioidomycosis using the polymerase chain reaction (PCR). The diagnosis of paracoccidioidomycosis (PCM) was defined by microscopic observation of the fungus on direct exam or histopathology, culture, and serological positivity. DNA from serum of 33 patients with PCM was extracted and submitted to nested-PCR using primers from the gp 43 gene. Only one sample was positive on nested-PCR. We conclude that the prevalence of fungemia in patients with different clinical forms of PCM is low, limiting the use of serum DNA detection as an alternative diagnostic tool.

Paracoccidioidomycosis (PCM) is a systemic mycosis with occurrence restricted to South and Central America, being rural residents the largest affected group. The diagnosis of PCM has traditionally relied on the finding of characteristic yeast forms on direct examination, culture, or histopathology (Lacaz 1994). Serological tests such as double immunodifusion and counter-immunoelectrophoresis are also employed for the diagnosis and follow-up of this mycosis (Mendes-Giannini et al. 1994), as well as the detection of a 43-kDa glycoprotein exoantigen (gp43) (Silva et al. 2004).

<font">Studies with blood, cerebrospinal fluid, and sputum specimens from patients with different fungal infections indicated that the polymerase chain reaction (PCR) assay is more sensitive in diagnosing invasive fungal infections than culture methods (Reiss et al. 1998). While several PCR systems have been described, reports on clinical samples from PCM patients are rare. In a study employing sputa specimens, Paracoccidioides brasiliensis DNA was detected by gp43-PCR in 100% (11/11) of patients with X-ray proven, pulmonary infection (Gomes et al. 2000). Goldani and Sugar (1998) used primers from a P. brasiliensis specific sequence spanning a 62 bp fragment, and were able to show its presence in sera of 5/5 experimentally infected mice. Bialek et al. (2000) developed a highly sensitive and specific nested-PCR detecting a fragment from the gp43 gene on lung homogenates from infected animals and in human tissue samples.

<font">Our objective was to evaluate the use of PCR for detection of P. brasiliensis DNA on serasamples from untreated patients with direct evidence of PCM and different clinical forms. This study was approved by the Ethics Committee of the Hospital das Clínicas, Faculdade de Medicina da Universidade de São Paulo.

<font">Thirty-three serum samples from patients with PCM (Table) were submitted to nested-PCR with primers spanning a 196 bp fragment from the gp43 gene as described by Bialek et al. (2000). P. brasiliensis genome contains two copies of the gp43 gene (Cano et al. 1998). Amplification of a fragment of 432 bp from the human growth hormone gene was used as a positive control for the whole process according to Meyer et al. (1999). Among the 33 serum samples analyzed, 25 had the HGH fragment amplified, demonstrated DNA adequacy. When DNAs were submitted to the gp43 nested-PCR assay, only one presented the expected amplified fragment. Moreover, all 33 serum samples, when spiked with P. brasiliensis cells, presented the corresponding gp43 amplicon, discarding major PCR inhibition. Our results suggest that PCR with DNA extracted from serum, is not useful for the diagnosis of PCM, due to the low prevalence of fungemia in patients with different clinical forms of untreated paracoccidiodomy-cosis. Itano et al. (2002) were able to show PCR detection of P. brasiliensis DNA on 5/20 infected patients with DNA isolated from serum. However these results were obtained only after a third-round of PCR and they obtained no amplification on plasma samples from 78 infected mice after two-rounds of amplification (nested-PCR). An interesting observation of this work was a much higher frequency of positive PCR amplification with DNA extracted from clots in comparison to plasma.

<font">In conclusion, our results show a limited value of nested PCR from serum samples, at least on its present form, for the identification of P. brasiliensis DNA. Though disappointing, the low detection rate here reported is corroborated by similar results from heavily infected experimental animals and also from the observation of the rarity of P. brasiliensis fungemia and isolation on blood culture from patients and infected mice (Singer-Vermes et al. 1993). Absence of P. brasiliensis DNA in sera of PCM patients deserves further investigation, as it contrasts to other deep mycosis. One possibility is that fungal DNA and/or cells are rapidly fagocytosed by leukocytes, reinforced by a recent observation showing that P. brasiliensis cells are readily detected and quantified in the bloodstream of experimental animals by staining with a fluorescence dye (Nishikaku & Burger 2003). We plan to carry a parallel comparison between whole blood and plasma/serum in order to clarify this issue.

 

ACKNOWLEDGMENTS

<font">To Dr Raquel dos Anjos Fazioli, Immunology Section of Instituto Adolfo Lutz, São Paulo, Brazil, for providing sera from infected mice and Dr Cláudio Sérgio Pannuti, Virology Laboratory of Instituto de Medicina Tropical, São Paulo, Brazil, for carefully reviewing the manuscript.

 

REFERENCES

<font">Bialek R, Ibricevic A, Aepinus C, Najvar LK, Fothergill AW, Knobloch J, Graybill JR 2000. Detection of Paracoc-cidioides brasiliensis in tissue samples by a nested PCR assay. J Clin Microbiol 38: 2940-2942.

<font">Cano MIN, Cisalpino PS, Galindo I, Ramirez JL, Mortara RA, da Silveira JF 1998. Eletrophortic karyotypes and genome sizing of the pathogenic fungus Paracoccidioides brasiliensis. J Clin Microbiol 36: 742-747.

<font">Goldani LZ, Sugar AM 1998. Short report: Use of the polymerase chain reaction to detect Paracoccidioides brasiliensis in murine paracoccidioidomycosis. Am J Trop Med Hyg 58: 152-153.

<font">Gomes GM, Cisalpino PS, Taborda CP, Camargo ZP 2000. PCR for diagnosis of paracoccidioidomycosis. J Clin Microbiol 38: 3478-3480.

<font">Itano EN, Uno J, Sano A, Yarita K, Kamei K, Miyaji M, Nishimura K, Mikami Y 2002. Detection of the gp 43 gene and (1®3) - b - D glucan of Paracoccidioides brasiliensis in the blood of experimentally infected mice. J Med Mycol 43: 29-35.

<font">Lacaz CS 1994. Paracoccidioides brasiliensis: morphology, evolutionary cycle; maintenance during saprophytic life; biology, virulence, taxonomy. In M Franco, CS Lacaz, A Restrepo-Moreno, A Del Negro (eds), Paracoccidioidomycosis, CRC Press, Boca Raton, FL, p.13-25.

<font">Mendes-Giannini MJS, Del Negro GB, Siqueira AM 1994. Serodiagnosis. In M Franco, CS Lacaz, A Restrepo-Moreno, G Del Negro (eds), Paracoccidioidomycosis, CRC Press, Boca Raton, FL, p. 345-363.

<font">Meyer O, Hildebrandt M, Schulz B, Blasczyk R, Salama A 1999. Simultaneous genotyping of human platelet antigens (HPA)1 through 6 using new sequence-specific primers for HPA-5. Transfusion 39: 1256-1258.

<font">Nishikaku AS, Burger E 2003. Evaluation of fungal burden in experimental paracoccidioidomycosis by using the fluorescent dye Blankophor. J Clin Microbiol 41: 3419-3422.

<font">Reiss E, Tanaka K, Bruker G, Chazalet V, Coleman D, Debeaupuis JP, Hanazawa R, Latgé JP, Lortholary J, Makimura K, Morrison CJ, Murayama SY, Naoe S, Paris S, Sarfati J, Shibuya K, Sullivan D, Uchida K, Yamaguchi H 1998. Molecular diagnosis and epidemiology of fungal infections. Med Mycol 36: 249-257.

<font">Silva SHM, Queiroz-Telles F, Colombo AL, Blotta MHSL, Lopes JD, Camargo ZP 2004. Monitoring gp43 antigenemia in paracoccidioidomycosis patients during therapy. J Clin Microbiol 42: 2419-2424.

<font">Singer-Vermes LM, Caldeira CB, Burger E, Calich VLG 1993. Experimental murine paracoccidioidomycosis: relationship among the dissemination of the infection, humoral and cellular immune responses. Clin Exp Immunol 94: 75-79.

Received 12 September 2005
Accepted 7 March 2006

1 Corresponding author: This e-mail address is being protected from spambots. You need JavaScript enabled to view it.

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