Paracoccidioidomycosis cases by Paracoccidioides lutzii in southeastern Brazil.

BACKGROUND AND OBJECTIVE: Paracoccidioidomycosis (PCM) is a systemic fungal disease caused by the thermodimorphic fungi Paracoccidioides spp. Their distribution is highly variable. Paracoccidioides lutzii is predominantly found in North and Middle-West Brazil and Ecuador. This study evaluated the clinicopathological characteristics of 10 patients diagnosed with PCM caused by P. lutzii in a reference center located in southeastern Brazil. DESIGN: Double immunodiffusion assay (DID) was used to investigate 35 patients' sera with negative serology for P. brasiliensis against a P. lutzii CFA (cell-free antigen). RESULTS: Out of the 35 retested patients, 10 (28.6%) were positive for P. lutzii CFA. Four patients did not report any displacement to P. lutzii endemic areas. Our results reinforce the importance of using different antigens when testing patients with clinical manifestations of PCM and negative serological tests for P. brasiliensis, primarily in cases of the report of displacement to or former residence in P. lutzii endemic regions. CONCLUSIONS: The availability of tests for different Paracoccidioides species antigens is fundamental for reaching an adequate diagnosis, patient follow-up, and definition of prognosis.

Screening paracoccidioidomycosis by double immunodiffusion test in a referred diagnostic center in Brazilian southeastern: an accessible tool.

PURPOSE: This article shows reports of the clinical-epidemiological characteristics and serological screening in patients assisted by a reference center for PCM care, University Hospital Cassiano Antonio Moraes, Federal University of Espirito Santo, Brazil. METHODS: The patient's sera with PCM were analyzed by DID test at the beginning and the end treatment. Clinical and demographic data were also collected to characterize the sample. RESULTS: One hundred patients with a suspected diagnosis of PCM were evaluated. Serology by DID test was used as a screen in all patients. The test was positive for 79 patients (72 for Paracoccidioides brasiliensis and 7 for Paracoccidioides lutzii). Serology was negative in 21 sera, although all of them were diagnosed PCM by histopathologic or direct exam. Serological follow-up was performed during the treatment of all patients. After treatment, 58(58%) had negative serology and 33(33%) low levels of antibodies (≤ 1:16). CONCLUSION: Our results indicate the importance of the DID test for the screening and monitoring of PCM and that the incidence of P. lutzii might be greater than expected in areas where it is not the predominant PCM species. Therefore, this article may contribute to improving the knowledge and clinical management about this disease.

Puzzling paracoccidioidomycosis: Factors associated with the severity of Paracoccidioides lutzii infections.

OBJECTIVES: Historically, the Brazilian Central-West region has had high numbers of paracoccidioidomycosis (PCM) cases caused by the dimorphic fungus Paracoccidioides lutzii. METHODS: This epidemiological, observational, analytical, cross-sectional study was performed to investigate the clinical and laboratory data of 44 PCM patients with a culture-proven P. lutzii infection. All patients were referred to the Systemic Mycosis Center, Júlio Muller University Hospital, Cuiabá, Brazil, during January 2017 to March 2020. The neutrophil to lymphocyte ratio (NLR) was calculated and dichotomized by its median value to include in the identification of factors associated with severity. RESULTS: At admission, 13 (31.7%) patients showed the disseminated multifocal chronic form of PCM and 16 (36.4%) patients met the clinical severity criteria. Treatment prescribed on admission did not follow the recommendations of the Brazilian Guideline for the Clinical Management of Paracoccidioidomycosis in 26% of the severe PCM cases (prevalence ratio 0.26, 95% confidence interval 0.14-0.49; P < 0.0001). Patients with severe PCM had a higher NLR that was greater than the median (≥4.11). CONCLUSIONS: The NLR biomarker complements the criteria for PCM severity. Applying the low-cost NLR test can greatly increase the diagnostic sensitivity when screening patients for PCM and contribute to better control of the disease, management of complications, and therapeutic strategies.

A New Duplex PCR-Assay for the Detection and Identification of Paracoccidioides Species.

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.