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Detection of Paracoccidioides brasiliensis in tissue samples by a nested PCR assay.

ABSTRACT: A nested PCR assay for the detection of Paracoccidioides brasiliensis DNA was evaluated, using a sequence of the immunogenic gp43 gene as a target. This gene encodes an outer membrane protein unique to this dimorphic fungus. DNA from six clinical isolates and the ATCC strain 60885 of P. brasiliensis, as well as DNA from closely related fungi, was examined to determine detection limits and cross-reactions. PCR was done on DNA extracts of lung homogenates from 23 experimentally P. brasiliensis-infected and two uninfected BALB/c mice and from 20 Histoplasma capsulatum-infected ICR mice. The results were compared to quantitative cultures. A detection limit of 0.5 fg of specific DNA was determined using cloned plasmid DNA. In all seven P. brasiliensis isolates, the expected 196-bp nested PCR product was found. Their sequences were 100% identical to the gp43 gene sequence in GenBank. DNA extracts of all other, related fungi were negative. The PCR assay was positive in 21 out of 23 culture-positive lung homogenates with concentrations of 1 x 10(3) to 1.3 x 10(7) CFU of P. brasiliensis per g of lung. Uninfected BALB/c mice and H. capsulatum-infected mice samples gave negative results. The high sensitivity and specificity of this new nested PCR assay for the detection of P. brasiliensis in tissue samples were demonstrated. The assay may be useful for diagnosis in human tissue samples.

SUBMITTER: Bialek R

PROVIDER: S-EPMC87153 | biostudies-literature | 2000 Aug

REPOSITORIES: biostudies-literature

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Detection of Paracoccidioides brasiliensis in tissue samples by a nested PCR assay.

Bialek R R Ibricevic A A Aepinus C C Najvar L K LK Fothergill A W AW Knobloch J J Graybill J R JR

Journal of clinical microbiology 20000801 8

A nested PCR assay for the detection of Paracoccidioides brasiliensis DNA was evaluated, using a sequence of the immunogenic gp43 gene as a target. This gene encodes an outer membrane protein unique to this dimorphic fungus. DNA from six clinical isolates and the ATCC strain 60885 of P. brasiliensis, as well as DNA from closely related fungi, was examined to determine detection limits and cross-reactions. PCR was done on DNA extracts of lung homogenates from 23 experimentally P. brasiliensis-inf ...[more]

PMID: 10921955

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Project description:Paracoccidioidomycosis (PCM) is a life-threatening systemic fungal infection caused by members of the Paracoccidioides brasiliensis complex and P. lutzii. Routine diagnoses of PCM down to the species level using classical mycological approaches are unspecific due to overlapping phenotypes. There is an urgent need for specific, sensitive, and cost-effective molecular tools to diagnose PCM. Variation among the exon-2 of the gp43 gene was exploited to design species-specific primer pairs to discriminate between members of the P. brasiliensis complex and P. lutzii in a duplex PCR assay. Primer-BLAST searches revealed highly species-specific primers, and no significant region of homology was found against DNA databases except for Paracoccidioides species. Primers PbraCx-F and PbraCx-R targeting P. brasiliensis DNA produced an amplicon of 308 bp, while primers Plu-F and Plu-R targeting P. lutzii DNA generated an amplicon of 142 bp. The lower limit of detection for our duplex PCR assay was 1 pg of gDNA. A panel of 62 Paracoccidioides revealed 100% specificity (AUC = 1.000, 95%CI 0.972-1.000, p < 0.0001) without cross-reacting with other medically relevant fungi or human DNA. As a proof of concept, we demonstrated the accurate identification of the P. brasiliensis complex (n = 7) or P. lutzii (n = 6) from a broad range of formalin-fixed, paraffin-embedded (FFPE) tissues of PCM patient's organs. In four cases, FFPE PCR results confirmed, for the first time, co-infection due to P. brasiliensis (S1) and P. lutzii in the same biopsy. Our duplex PCR assay is useful to detect and differentiate members of the P. brasiliensis complex and P. lutzii, providing clinical laboratories with an important tool to be applied routinely, especially in atypical cases such as those featuring negative serology and positive mycological examination of clinical specimens as well as for the investigation of putative co-infection cases. This will likely benefit thousands of infected patients every year in a wide area of the Americas.

Dataset Information

Detection of Paracoccidioides brasiliensis in tissue samples by a nested PCR assay.

Publications

Detection of Paracoccidioides brasiliensis in tissue samples by a nested PCR assay.

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