Validation and clinical application of a nested PCR for paracoccidioidomycosis diagnosis in clinical samples from Colombian patients

Abstract

Paracoccidioidomycosis is a systemic and endemic mycosis, restricted to tropical and subtropical areas of Latin America. The infection is caused by the thermal dimorphic fungus Paracoccidioides brasiliensis and Paracoccidioides lutzii. The diagnosis of paracoccidioidomycosis is usually performed by microscopic examination, culture and immunodiagnostic tests to respiratory specimens, body fluids and/or biopsies; however these methods require laboratory personnel with experience and several days to produce a result. In the present study, we have validated and evaluated a nested PCR assay targeting the gene encoding the Paracoccidioides gp43 membrane protein in 191 clinical samples: 115 samples from patients with proven infections other than paracoccidioidomycosis, 51 samples as negative controls, and 25 samples from patients diagnosed with paracoccidioidomycosis. Additionally, the specificity of the nested PCR assay was also evaluated using purified DNA isolated from cultures of different microorganisms (n=35) previously identified by culture and/or sequencing. The results showed that in our hands, this nested PCR assay for gp43 protein showed specificity and sensitivity rates of 100%. The optimized nested PCR conditions in our laboratory allowed detection down to 1fg of P. brasiliensis DNA.

Brazilian guidelines for the clinical management of paracoccidioidomycosis

“…For instance, the north and midwest regions have been associated with PCM infections caused by P. lutzii because of molecular identification of isolates in these areas 1,5,9 and higher sera reactivity of patients with proven disease in these areas to P. lutzii antigen 63,64 . Although clinical data and therapeutic response are similar for both species (data from Mato Grosso do Sul), little is known about the isolation and molecular identification of the species in patients 8 because mycological and histopathological examinations cannot distinguish these species and molecular tests are not available in routine laboratories 66 .…”

“…Additional resources and techniques for PCM diagnosis have been developed, but they are not available for PCM routine assessment. These examinations include the immunoblot technique, the triage ELISA test, the detection of specific antigens and PCR 65,66 .…”

II Consenso Brasileiro em Paracoccidioidomicose - 2017*

“…Entre esses exames, podem--se citar a técnica de imunoblot, o teste de ELISA para triagem, a pesquisa de antígeno específico e técnicas de PCR. 65,66 Até o momento, não se dispõe de técnica sorológica validada para o diagnóstico acurado de infecção por P. lutzii. Infelizmente, não há nenhum sistema comercial disponível para o diagnóstico de paracoccidioidomicose, sendo todos os testes disponíveis baseados em sistemas desenvolvidos in house.…”

“…63,64 Embora os dados clínicos e a resposta à terapêutica sejam aparentemente similares para ambas as espécies (dados do Mato Grosso do Sul), pouco se conhece com base em dados de isolamento e identificação molecular em pacientes, 8 uma vez que os exames micológico e histopatológico não são capazes de distinguir essas espécies, e as provas moleculares não estão disponíveis em laboratórios de rotina. 66 Permanecem como questões a serem respondidas: o conhecimento do perfil epidemiológico-molecular de isolados de Paracoccidioides no país, visando fundamentar o diagnóstico clínico-epidemiológico; a caracterização do perfil antigênico dos isolados e sua correlação com os dados clínico-laboratoriais das doenças causadas por ambas as espécies, 1,7 incluindo sequelas; e a resposta aos diferentes medicamentos, bem como a análise da susceptibilidade às drogas. 112,113 Da maior importância para a saúde pública é a implantação de métodos simples, como o exame micológico, como método de primeira escolha no diagnóstico em regiões carentes, particularmente quando o diagnóstico de tuberculose for excluído.…”

Biosensors and Diagnostics for Fungal Detection

“…Modified "nested PCR" protocols have been established for improved specificity and sensitivity. This is achieved by subjecting samples to two consecutive PCR reactions using two sets of primers, thus allowing for the detection of fungal DNA as low as 1 fg with 100% specificity; however, this is dependent on sample type and concentration and is extremely vulnerable to contamination [86]. Endoscopic sinus surgery specimens were found to be particularly unsuitable for this technique perhaps due to contamination with high levels of environmental fungi in the nasal cavity [87].…”