A semi-nested PCR assay for molecular detection of Paracoccidioides brasiliensis in tissue samples Semi-nested PCR para a detecção molecular de Paracoccidioides brasiliensis em amostras de tecido

Introduction: Paracoccidioidomycosis is a systemic infection caused by Paracoccidioides brasiliensis. Methods: In this study, a semi-nested PCR for paracoccidioidomycosis diagnosis was developed. The primers ITS1 and ITS4 were used in the first reaction, while the primers MJ03 and ITS1 primer were used in the second reaction. The semi-nested PCR was used to investigate biopsies of five patients with oral lesions that resembled paracoccidioidomycosis. Results: The semi-nested PCR was positive for four samples and negative for a sample from a patient later diagnosed with leishmaniasis. Conclusions: The new semi-nested PCR describe is useful for paracoccidioidomycosis diagnosis. Key-words: Paracoccidioides brasiliensis. Semi-nested PCR. Molecular detection. RESUMO Introdução: A paracoccidioidomicose é uma infecção sistêmica causada pelo Paracoccidioides brasiliensis. Métodos: Neste estudo, uma semi-nested PCR foi desenvolvida para o diagnóstico da paracoccidioidomicose. Os oligonucleotídeos iniciadores ITS1 e ITS4 foram usados na primeira reação, enquanto os oligonucleotídeos iniciadores MJ03 e ITS1 foram usados na segunda reação. A semi-nested PCR foi usada para investigar biopsias de cinco pacientes com lesões orais que se assemelhavam a paracoccidioidomicose. Resultados: A semi-nested PCR foi positiva para quatro amostras e negativa para a amostra de um paciente, posteriormente diagnosticado com leishmaniose. Conclusões: A semi-nested PCR descrita aqui é útil para o diagnóstico da paracoccidioidomicose. Palavras-chaves: Paracoccidioides brasiliensis. Semi-nested PCR. Detecção molecular. The thermal dimorphic fungus Paracoccidioides brasiliensis is the etiologic agent of paracoccidioidomycosis (PCM), a systemic mycosis endemic in Latin America1. PCM diagnosis can be determined by direct observation or the culture of clinical samples, histological analyses and serological methods. However, these techniques each have concerns: using direct observation, P. brasiliensis yeast cells may not be observed or could be mistaken for other dimorphic fungi1; sample culture is slow and frequently negative; in histological analysis, the pseudoepitheliomatous hyperplasia typical of PCM resembles squamous cell carcinoma so closely that it is possible to mistake one for the other; finally, serological assays are highly sensitivity, but not totally specific. Alternative diagnostic methods for PCM have been developed, including polymerase chain reactions (PCR). The most frequently used target sequences for molecular detection of P. brasiliensis by PCR are the gp432-4 and ribosomal DNA genes5-8. PCR assays have been used experimentally to detect P. brasiliensis in the serum and tissues of infected mice2,4, in artificially contaminated soil and in environmental samples8. In PCM patients, PCR has been used on sputa, cerebrospinal fluid and paraffin-embedded tissues3,9,10. However, molecular diagnosis of PCM is not used in clinical routine. Here, we report a semi-nested PCR (snPCR) assay for the molecular detection of P. brasiliensis using two universal primers for fungi and a specific primer designed to detect a specific DNA ribosomal sequence of P. brasiliensis. Two strains of P. brasiliensis (LDR1 and Pb18) and one isolate of each of the following fungi, Candida albicans (CR15), Histoplasma capsulatum, Sporothrix schenckii, Cryptococcus sp and Tricophyton rubrum were used. All fungi isolates were maintained on Sabouraud dextrose agar at room temperature, with the exception of P. brasiliensis and Cryptococcus sp, which were maintained at 35oC. DNA from fungal cells and tissues from mice or patients were extracted by maceration in liquid nitrogen followed by phenolchloroform-isoamyl alcohol treatment and sodium acetate-ethanol precipitation. DNA concentration and purity were determined by spectrophotometry. Swiss male mice (n= 5) were injected with 1.5 x 106 yeast cells of P. brasiliensis Pb18 via the tail vein. The mice were killed 16h after fungal inoculation and the lungs were removed under aseptic conditions, weighed and manually homogenized in PBS (100μL to 20mg of tissue). Lung samples were cultured on BHI agar plates supplemented with 4% horse serum, 5% growth factor11 and 1% penicillin and streptomycin solution and the number of CFU/g of tissue was calculated. For histological analysis, the lungs were routinely processed for the preparation of paraffin-embedded tissue sections and stained with hematoxylin-eosin (HE). The following primers ITS1 (5’-TCCGTAGGTGAACCTGCGG-3’) and ITS4 (5’-TCCTCCGCTTATTGATATGC-3’), described elsewhere12, were used in the snPCR . The primer MJ03 (5’-GTCTCAGACGTCA A AGCTCC-3’) was designed by

The thermal dimorphic fungus Paracoccidioides brasiliensis is the etiologic agent of paracoccidioidomycosis (PCM), a systemic mycosis endemic in Latin America 1 .
PCM diagnosis can be determined by direct observation or the culture of clinical samples, histological analyses and serological methods.However, these techniques each have concerns: using direct observation, P. brasiliensis yeast cells may not be observed or could be mistaken for other dimorphic fungi 1 ; sample culture is slow and frequently negative; in histological analysis, the pseudoepitheliomatous hyperplasia typical of PCM resembles squamous cell carcinoma so closely that it is possible to mistake one for the other; finally, serological assays are highly sensitivity, but not totally specific.
Alternative diagnostic methods for PCM have been developed, including polymerase chain reactions (PCR).The most frequently used target sequences for molecular detection of P. brasiliensis by PCR are the gp43 2-4 and ribosomal DNA genes [5][6][7][8] .PCR assays have been used experimentally to detect P. brasiliensis in the serum and tissues of infected mice 2,4 , in artificially contaminated soil and in environmental samples 8 .In PCM patients, PCR has been used on sputa, cerebrospinal fluid and paraffin-embedded tissues 3,9,10 .However, molecular diagnosis of PCM is not used in clinical routine.
Here, we report a semi-nested PCR (snPCR) assay for the molecular detection of P. brasiliensis using two universal primers for fungi and a specific primer designed to detect a specific DNA ribosomal sequence of P. brasiliensis.
Two strains of P. brasiliensis (LDR1 and Pb18) and one isolate of each of the following fungi, Candida albicans (CR15), Histoplasma capsulatum, Sporothrix schenckii, Cryptococcus sp and Tricophyton rubrum were used.All fungi isolates were maintained on Sabouraud dextrose agar at room temperature, with the exception of P. brasiliensis and Cryptococcus sp, which were maintained at 35ºC.DNA from fungal cells and tissues from mice or patients were extracted by maceration in liquid nitrogen followed by phenolchloroform-isoamyl alcohol treatment and sodium acetate-ethanol precipitation.DNA concentration and purity were determined by spectrophotometry.
Swiss male mice (n= 5) were injected with 1.5 x 10 6 yeast cells of P. brasiliensis Pb18 via the tail vein.The mice were killed 16h after fungal inoculation and the lungs were removed under aseptic conditions, weighed and manually homogenized in PBS (100μL to 20mg of tissue).Lung samples were cultured on BHI agar plates supplemented with 4% horse serum, 5% growth factor 11 and 1% penicillin and streptomycin solution and the number of CFU/g of tissue was calculated.For histological analysis, the lungs were routinely processed for the preparation of paraffin-embedded tissue sections and stained with hematoxylin-eosin (HE).
The following primers ITS1 (5' -TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'), described elsewhere 12 , were used in the snPCR .The primer MJ03 (5'-GTCTCAGACGTCAAAGCTCC-3') was designed by  As expected, in the first reaction, amplicons of different sizes were generated with DNA of all the fungi tested (Figure 1A).The second round PCR only amplified a fragment of 212bp with DNA of P. brasiliensis, thus showing the specificity of this reaction (Figure 1B).In relation to sensitivity, the test was able to detect 0.25pg of P. brasiliensis DNA (Figure 1C) and in mice lungs samples spiked with yeast cells from P. brasiliensis, it detected as few as 10 yeast cells (Figure 1D).
The histopathological analysis and snPCR were able to detect P. brasiliensis in all the samples tested, while culture was positive in three out of five mice samples (Table 1).
In clinical samples from patients with an initial clinical diagnose of PCM, the snPCR for P. brasiliensis was positive in four out of five patients.The negative sample was from a patient later diagnosed with leishmaniasis (Figure 1E and Table 2).
Molecular biological methods for detection and characterization of microorganisms have revolutionized diagnostic microbiology and the PCR technique is of great importance in this respect.The snPCR described here uses the universal to fungi ITS1 and ITS4 primers and the MJ03 primer that is specific for P. brasiliensis.This specific primer was design comparing sequences from GenBank database and anneals to the internal transcribed spacer 1 (ITS1) variable region.In a preview study using the ITS1-5.8S-ITS2region as target, an unexpected cross-reaction with H. capsulatum was observed 6 .In this study, the snPCR was specific for P. brasiliensis and no amplification product was observed with DNA from the other tested fungi; moreover, it showed greater sensitivity than some previous reports 10 and similar to a nested PCR assay for S. schenckii 13 .The high sensitivity in the present test is probably the result of choosing the high copy number ITS1-5.8S-ITS2 region 14 in association with snPCR, recognized as having a 1,000-fold greater sensitivity than conventional PCR.Furthermore, detection of amplicons by polyacrylamide gels/silver nitrate methodology is recognized as more sensitive than agarose and ethidium bromide detection.
To show the potential application of this snPCR for the molecular detection of P. brasiliensis in clinical samples, mouse lung samples spiked with yeast cells from P. brasiliensis and positive results were obtained with as few as 10 yeast cells.Similar results were observed with a PCR assay for the detection of P. brasiliensis in spiked sputum samples 3 .
Furthermore, the comparative test among culture, histopathological analysis and snPCR performed using experimentally infected mice, showed that histopathological analysis and snPCR were slightly more sensitive than culture.
In clinical samples from patients with an initial clinical diagnose of PCM, the snPCR for P. brasiliensis was positive for four patients, and negative for a sample, later diagnosed as leishmaniasis, a disease with lesions that can resemble oral PCM lesions.The diagnoses were later confirmed by histopathological analysis.
The semi-nested PCR described in this study is a rapid (approximately 12 hours), specific and sensitive method and was useful for detecting the presence of P. brasiliensis DNA in culture and tissue.

TABLE 2 -Patient clinical data.
P. brasiliensis and genetically close species, B. dermatitidis, C. immitis, H. capsulatum and S. schenckii.The MJ03 primer was used with ITS1 primer in the second round PCR to generate a fragment of 212bp.The first round PCR consisted of 5µl of DNA sample in a total volume of 25µl, with 20mM Tris-HCl, (pH To evaluate snPCR with clinical biopsy, samples of lesions from 5 patients with an initial clinical diagnose of PCM from University Dentistry Center at the State University of Londrina, Brazil were used.The samples were stored at -20ºC until DNA extraction.This study was approved by the Ethics Committee of the Londrina State University, Brazil.