Epidemiology of paracoccidioidomycosis in Venezuela: a retrospective study from 1954 to 2019

BACKGROUND Paracoccidioidomycosis (PCM) is a systemic mycosis endemic to Latin America. Etiological agents are Paracoccidioides species that diverge phylogenetically throughout South America. OBJECTIVES This study aimed to document the epidemiology of PCM in Venezuela. METHODS We have performed a retrospective cross-sectional descriptive study in 31,081 clinical records of patients from two reference centres during 65 years (1954-2019). FINDINGS PCM diagnosis was confirmed in 745 patients. Chronic PCM was the most prevalent form (90.06% cases); 80.67% were male and the most affected age range was 41-60. Farming and construction were the most prevalent occupation and Miranda State had a higher prevalence. Lung and skin were the most affected organs, followed by oral manifestations. Direct examination, culture and serology showed a high sensibility, and no statistical difference was observed among the diagnostic tools. Out of 17 Paracoccidioides isolates genotyped from Venezuela, one was typed as Paracoccidioides americana and 16 as Paracoccidioides venezuelensis. MAIN CONCLUSIONS Clinical manifestations observed, information about the epidemiology and molecular profile is essential not only for diagnosis but also for understanding therapeutic responses to mycotic drugs and prognosis. Therefore, it is necessary to sequence all positive isolated strains in order to confirm the dominance of P. venezuelensis in Venezuela.

Use of fluorescent oligonucleotide probes for differentiation between Paracoccidioides brasiliensis and Paracoccidioides lutzii in yeast and mycelial phase

BACKGROUND Fluorescence in situ hybridisation (FISH) associated with Tyramide Signal Amplification (TSA) using oligonucleotides labeled with non-radioactive fluorophores is a promising technique for detection and differentiation of fungal species in environmental or clinical samples, being suitable for microorganisms which are difficult or even impossible to culture. OBJECTIVE In this study, we aimed to standardise an in situ hybridisation technique for the differentiation between the pathogenic species Paracoccidioides brasiliensis and Paracoccidioides lutzii, by using species-specific DNA probes targeting the internal transcribed spacer-1 (ITS-1) of the rRNA gene. METHODS Yeast and mycelial phase of each Paracoccidioides species, were tested by two different detection/differentiation techniques: TSA-FISH for P. brasiliensis with HRP (Horseradish Peroxidase) linked to the probe 5’ end; and FISH for P. lutzii with the fluorophore TEXAS RED-X® also linked to the probe 5’ end. After testing different protocols, the optimised procedure for both techniques was accomplished without cross-positivity with other pathogenic fungi. FINDINGS The in silico and in vitro tests show no reaction with controls, like Candida and Cryptococcus (in silico) and Histoplasma capsulatum and Aspergillus spp. (in vitro). For both phases (mycelial and yeast) the in situ hybridisation showed dots of hybridisation, with no cross-reaction between them, with a lower signal for Texas Red probe than HRP-TSA probe. The dots of hybridisation was confirmed with genetic material marked with 4’,6-diamidino-2-phenylindole (DAPI), visualised in a different filter (WU) on fluorescent microscopic. MAIN CONCLUSION Our results indicated that TSA-FISH and/or FISH are suitable for in situ detection and differentiation of Paracoccidioides species. This approach has the potential for future application in clinical samples for the improvement of paracoccidioidomycosis patients prognosis.

Infection of the lymphatic system by Aureobasidium pullulans in a patient with erythema nodosum leprosum

Aureobasidium pullulans is a causal agent of phaeohyphomycosis, occasionally found in men and animals. As an agent of different opportunistic fungal processes, it may cause fungemia, systemic infections and abscesses in different viscera. This paper aims to report a case of a patient with infection of the lymphatic system by A. pullulans. A 23-year-old patient being treated for erythema nodosum leprosum presented a 60-day complaint of daily fever, hoarseness, odynophagia and weight loss. Laboratory tests showed pancytopenia with severe neutropenia, cervical adenomegaly and solid contrast uptake lesion in the oropharyngeal region. Due to neutropenia and sepsis the patient was initially treated with cefepime and vancomycin, but there was no clinical improvement. Lymph node puncture-aspiration showed yeast-form fungus identified as A. pullulans by sequencing ITS region. The patient was treated with amphotericin B deoxycholate, leading to complete recovery of bone marrow function and regression of adenomegaly and the oropharyngeal lesion.