Real Time Polymerase Chain Reaction (rt-PCR): A New Patent to Diagnostic Purposes for Paracoccidioidomycosis

Rocha-Silva, Fabiana; Gomes, Luciana I.; Gracielle-Melo, Cidiane; Góes, Alfredo M.; Caligiorne, Rachel Basques

doi:10.2174/1872214810666160905150958

Cited by 9 publications

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“…A great performance of our test was observed with clinical samples, reducing the need for isolation of Paracoccidioides , thus saving critical time. Overall, the application of PCR-based diagnostics shows that the test might work better on sputum than in blood samples [ 50 ], whereas the tissue detection would have no problem if the available Paracoccidioides species’ DNA is greater than 10 fg [ 67 ], which is in agreement with our results. Therefore, we recommend detecting Paracoccidioides DNA in clinical routine and emphasize that the major feature of maintaining diagnostic accuracy is acquiring an excellent clinical specimen free of PCR inhibitors [ 49 ].…”

Section: Discussionsupporting

confidence: 89%

Development of a Multiplex qPCR Assay for Fast Detection and Differentiation of Paracoccidioidomycosis Agents

Pinheiro

Pôssa

Ricci³

et al. 2023

JoF

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Classic paracoccidioidomycosis (PCM) is a potentially deadly neglected tropical systemic mycosis caused by members of the Paracoccidioides brasiliensis complex (P. brasiliensis s. str., P. americana, P. restrepiensis, and P. venezuelensis) and P. lutzii. The laboratorial diagnosis of PCM relies on observing pathognomonic structures such as the “steering wheel” or “Mickey Mouse” shape in the direct mycological examination, fresh biopsied tissue in 10% KOH, histopathological analysis, and/or the isolation of the fungus in culture. However, these procedures are time-consuming and do not allow for the speciation of Paracoccidioides due to overlapping morphologies. Here, we propose a new one-tube multiplex probe-based qPCR assay to detect and recognize agents of the P. brasiliensis complex and P. lutzii. Primers (Paracoco-F and Paracoco-R) and TaqMan probes (PbraCx-Fam, Plu-Ned, and Paracoco-Vic) were developed to target the rDNA (ITS2/28S) in the Paracoccidioides genome. A panel of 77 Paracoccidioides isolates revealed a 100% specificity (AUC = 1.0, 95% CI 0.964–1.000, p < 0.0001) without cross-reacting with other medically relevant fungi or human and murine DNA. The lower limit of detection was 10 fg of gDNA and three copies of the partial rDNA amplicon. Speciation using qPCR was in perfect agreement with AFLP and TUB1-RFLP markers (kappa = 1.0). As a proof of concept, we assessed a panel of 16 formalin-fixed and paraffin-embedded specimens from histopathologically confirmed PCM patients to reveal a significant sensitivity of 81.25% and specificity of 100% (AUC = 0.906 ± 0.05, 95% CI = 0.756–0.979, p < 0.0001, Youden index J = 0.8125). Our assay achieved maximum sensitivity (100%) and specificity (100%) using fresh clinical samples (n = 9) such as sputum, bronchoalveolar lavage, and tissue fragments from PCM patients (AUC = 1.0, 95% CI 0.872–1.000, p < 0.0001, Youden index J = 1.0). Overall, our qPCR assay simplifies the molecular diagnosis of PCM and can be easily implemented in any routine laboratory, decreasing a critical bottleneck for the early treatment of PCM patients across a vast area of the Americas.

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Section: Discussionsupporting

confidence: 89%

Development of a Multiplex qPCR Assay for Fast Detection and Differentiation of Paracoccidioidomycosis Agents

Pinheiro

Pôssa

Ricci³

et al. 2023

JoF

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“…In this case, it was decisive, leading to rapid and safe diagnosis, essential for the prompt treatment of the patient. This case study confirms the standardization of the rt-PCR technique for molecular diagnosis of PCM, which was patented last year and has already been published [8] . Without a doubt, this technique will contribute a lot to help, especially in cases of challenging diagnosis.…”

Section: Discussionsupporting

confidence: 80%

“…The serology was performed by double radial immunodiffusion (ID) for detecting antibodies against gp43 antigens, specific for the genus Paracoccidioides , using the kit produced in Federal University of Paraná, Brazil. Molecular diagnosis in tissue and peripheral blood DNA through real-time PCR (rtPCR) was performed using TaqMan system, using primers and probe for detecting the gene for Pb27 protein, specific for Paracoccidioides genus, according standardized by Rocha-Silva et al, 2016 [8] . Although serology ( Fig.…”

Section: Casementioning

confidence: 99%

Disseminated paracoccidioidomycosis prediagnosticated as neoplasm: An important challenge in diagnosis using rt-PCR

Rocha-Silva

Guimarães

Júnior

et al. 2018

Medical Mycology Case Reports

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This paper presents a case of disseminated paracoccidioidomycosis in a 62-year-old male patient, who lives in Belo Horizonte, MG, Brazil. The patient was hospitalized with icteric syndrome of cholestatic pattern and weight loss, with loss 30 kg in 5 months. The imaging of the abdomen showed lesion of infiltrative pattern, affecting gallbladder and intrahepatic bile ducts, suggesting neoplasia of malignant behavior, besides to presenting the yellow nail syndrome. Dermatological examination presented erythematous-infiltrated plaques in the occipital region. Also, the patient presented tegumentary lesions on the scalp and lumbar region from which the histopathological examination was carried out, which evidenced yeasts cells. The drug of choice for therapy was Liposomal Amphotericin-B. At the end of the antifungal treatment, liver enzyme dosages were normalized and there was improvement of the general condition of the patient, as well as the skin lesions. Here, we demonstrate the importance of molecular biology to confirm the diagnosis. Especially in cases of difficult diagnosis.

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“…for genotypic studies and clinical diagnosis performed directly from samples of patients with PCM [5,11,12,16,17]. Some methodologies such as PCR-Nested [18], conventional PCR [16], qualitative PCR real time [19,20], microsatellites [8,21], mitochondrial markers [22], Multilocus Sequence Typing (MLST) [5] and whole genomic sequencing [23] have been used for the purpose of phylogenetic understanding and for answering important questions that relate genotypic data to epidemiological and serological data. A technique that identifies the species (not variety) of the Paracoccidioides spp.…”

Section: Introductionmentioning

confidence: 99%

An update on the occurrence of Paracoccidioides species in the Midwest region, Brazil: Molecular epidemiology, clinical aspects and serological profile of patients from Mato Grosso do Sul State

et al. 2021

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Background Paracoccidioidomycosis (PCM) is a systemic and endemic fungal infection in Latin American, mainly in Brazil. The majority of PCM cases occur in large areas in Brazil, comprising the South, Southeast and Midwest regions, with the latter demonstrating a higher incidence of the species Paracoccidioides lutzii. Methodology and main findings This study presents clinical, molecular and serological data of thirteen new PCM cases during 2016 to 2019 from the state of Mato Grosso do Sul, located in the Midwest region, Brazil. From these thirteen cases, sixteen clinical isolates were obtained and their genomic DNAs were subjected to genotyping by tub1 -PCR-RFLP. Results showed Paracoccidioides brasiliensis sensu stricto (S1) (11/16; 68.8%), Paracoccidioides restrepiensis (PS3) (4/16; 25.0%) and P. lutzii (1/16; 6.2%) as Paracoccidiodes species. Therefore, in order to understand whether the type of phylogenetic species that are circulating in the state influence the reactivity profile of serological tests, we performed double agar gel immunodiffusion (DID), using exoantigens from genotyped strains found in this series of PCM cases. Overall, our DID tests have been false negative in about 30% of confirmed PCM cases. All patients were male, most with current or previous rural activity, with ages ranging from 17 to 59 years, with 11 patients (84.6%) over 40 years of age. No clinical or epidemiological differences were found between Paracoccidioides species. However, it is important to note that the only case of P. lutzii died as an outcome. Conclusions This study suggests P. brasiliensis sensu stricto (S1) as the predominant species, showing its wide geographic distribution in Brazil. Furthermore, our findings revealed, for the first time, the occurrence of P. restrepiensis (PS3) in the state of Mato Grosso do Sul, Brazil. Despite our setbacks, it would be interesting to provide the complete sequencing of these clinical isolates to complement the molecular information presented.

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Real Time Polymerase Chain Reaction (rt-PCR): A New Patent to Diagnostic Purposes for Paracoccidioidomycosis

Cited by 9 publications

References 0 publications

Development of a Multiplex qPCR Assay for Fast Detection and Differentiation of Paracoccidioidomycosis Agents

Development of a Multiplex qPCR Assay for Fast Detection and Differentiation of Paracoccidioidomycosis Agents

Disseminated paracoccidioidomycosis prediagnosticated as neoplasm: An important challenge in diagnosis using rt-PCR

An update on the occurrence of Paracoccidioides species in the Midwest region, Brazil: Molecular epidemiology, clinical aspects and serological profile of patients from Mato Grosso do Sul State

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