Carbon nanoparticle-based lateral flow assay for the detection of specific double-tagged DNA amplicons of Paracoccidioides spp.

To overcome the limitations of current methods for diagnosing paracoccidioidomycosis (PCM), it is critical to develop novel diagnostic strategies that can be implemented in low-resource settings and dramatically improve turnaround times. This study focused on the development of a portable molecular test to screen for Paracoccidioides spp. The proposed approach integrated double-tagging polymerase chain reaction (PCR) and a paper-based lateral flow assay (LFA) for readout, using carbon nanoparticles as a signal generation system. Primers tagged with biotin and digoxigenin were employed to conduct the double-tagging PCR, which can be conveniently carried out on portable thermocyclers. This method can generate billions of tagged DNA copies from a single target molecule, which can be rapidly detected by the LFA platform, providing results within minutes. Avidin-modified carbon nanoparticles served as a signal generation system, enabling detection in the immunochromatographic assay. The LFA demonstrated the capability to detect double-tagged amplicons as low as 0.21 ng or 0.10 ng, depending on whether the results were assessed visually or with a smartphone equipped with an image processor. These findings suggest that the proposed approach holds great promise as a point-of-care diagnostic tool for the early and accurate detection of PCM in low-resource settings. The diagnostic test is rapid and inexpensive, requires minimal handling and can be easily introduced into the general practitioner's armoury for ambulatory screening of infection. This innovative approach has the potential to make a substantial contribution to PCM diagnosis, ultimately reducing morbidity and mortality associated with this disease.

Paracoccidioidomycosis manifested by sarcoid-like cutaneous lesions with severe systemic disease: a rare and challenging diagnosis.

Cutaneous involvement in paracoccidioidomycosis (PCM) can exhibit a highly polymorphic spectrum. The infiltrative pattern corresponds to up to 26.6% of observed skin lesions, including sarcoid-like plaques, a rare presentation of cutaneous lesions in PCM. This clinical expression is almost exclusively cutaneous, and its histology reveals a tuberculoid granuloma with a scarcity of fungi, leading to misdiagnosis as other granulomatous diseases. Here, we report a rare form of chronic multifocal paracoccidioidomycosis manifesting as sarcoid-like skin lesions misdiagnosed as granulomatous rosacea in a patient with severe systemic disease.

Case Report: Disseminated Paracoccidioidomycosis and Strongyloides Hyperinfection in a Patient with Human T-Lymphotropic Virus Type 1/2 Infection.

Co-occurrence of paracoccidioidomycosis and strongyloidiasis in immunosuppressed patients, particularly those infected with human T-lymphotropic virus type 1/2, is infrequent. We describe the case of a Peruvian farmer from the central jungle with human T-lymphotropic virus type 1/2 infection, with 2 months of illness characterized by respiratory and gastrointestinal symptoms associated with fever, weight loss, and enlarged lymph nodes. Strongyloides stercoralis and Paracoccidioides brasiliensis were isolated in sputum and bronchoalveolar lavage samples, respectively. The clinical evolution was favorable after the patient received ivermectin and amphotericin B. We hypothesize that autoinfestation by S. stercoralis in human T-lymphotropic virus type 1/2-infected patients may contribute to the disseminated presentation of Paracoccidioides spp. Understanding epidemiological context is crucial for suspecting opportunistic regional infections, particularly those that may coexist in immunosuppressed patients.

Can COVID-19 impact the natural history of paracoccidioidomycosis? Insights from an atypical chronic form of the mycosis.

Paracoccidioidomycosis (PCM) is a systemic fungal infection caused by Paracoccidioides spp. It can occur as an acute/subacute form (A/SAF), a chronic form (CF) and rarely as a mixed form combining the features of the two aforementioned forms in an immunocompromised patient. Here, we report a 56-year-old male patient with CF-PCM who presented with atypical manifestations, including the development of an initial esophageal ulcer, followed by central nervous system (CNS) lesions and cervical and abdominal lymphatic involvement concomitant with severe SARS-CoV-2 infection. He was HIV-negative and had no other signs of previous immunodeficiency. Biopsy of the ulcer confirmed its mycotic etiology. He was hospitalized for treatment of COVID-19 and required supplemental oxygen in the intensive unit. The patient recovered without the need for invasive ventilatory support. Investigation of the extent of disease during hospitalization revealed severe lymphatic involvement typical of A/SAF, although the patient`s long history of high-risk exposure to PCM, and lung involvement typical of the CF. Esophageal involvement is rare in non-immunosuppressed PCM patients. CNS involvement is also rare. We suggest that the immunological imbalance caused by the severe COVID-19 infection may have contributed to the patient developing atypical severe CF, which resembles the PCM mixed form of immunosuppressed patients. Severe COVID-19 infection is known to impair the cell-mediated immune response, including the antiviral response, through T-lymphopenia, decreased NK cell counts and T-cell exhaustion. We hypothesize that these alterations would also impair antifungal defenses. Our case highlights the potential influence of COVID-19 on the course of PCM. Fortunately, the patient was timely treated for both diseases, evolving favorably.

Pseudoepitheliomatous hyperplasia: Squamous cell carcinoma versus oral paracoccidioidomycosis, a case from a dermatological perspective / Hiperplasia pseudoepiteliomatosa: carcinoma escamocelular versus paracoccidioidomicosis oral, un caso con mirada dermatológica

Paracoccidioidomycosis is a systemic mycosis endemic in Latin America. The most frequent form involves a chronic compromise of the lungs, skin, and mucosa. The patient started with a single oral lesion that lasted for several years. The absence of other symptoms pointed out a possible malignant neoplasm, specifically a squamous cell carcinoma. Differentiation between both diagnoses ­fungal infection and carcinoma­ depends on the results of the direct examination, the histopathological study, and the initial and subsequent cultures. However, in this case, those findings were not conclusive. The coexistence of both diagnoses is frequent and increases the diagnostic challenge. After several consultations and tests, direct examination, immunodiffusion and real-time PCR findings the multifocal chronic paracoccidioidomycosis diagnosis was confirmed. This case warns about a systematical absence of clinical suspicion of endemic mycoses before the appereance of mucocutaneous lesions, which can be produced by fungi like Paracoccidioides spp, and the importance of considering those mycoses among the differential diagnoses.

La paracoccidioidomicosis es una micosis sistémica endémica en Latinoamérica. La presentación más frecuente compromete crónicamente los pulmones, la piel y las mucosas. Al inicio, este paciente presentó, por varios años, una lesión única en la mucosa oral que, en ausencia de otros síntomas, se relacionó con una neoplasia maligna, específicamente con un carcinoma escamocelular. La diferenciación entre los dos diagnósticos se hace mediante un examen directo, un estudio histopatológico y cultivos iniciales y subsecuentes. Sin embargo, tales estudios no fueron concluyentes. Después de varias consultas y pruebas, con los resultados del examen directo, la inmunodifusión y la PCR en tiempo real se confirmó el diagnóstico de paracoccidioidomicosis crónica multifocal. Este caso alerta sobre la ausencia de sospecha clínica de micosis endémicas, dada la presencia de lesiones mucocutáneas que pueden ser producidas por hongos como Paracoccidioides spp, y la importancia de considerarlas entre los diagnósticos diferenciales.

Fast and cost-effective protocol to produce Paracoccidioides spp. antigens / Protocolo rápido y económico para la producción de antígenos de Paracoccidioides spp

INTRODUCTION: The existing methods for Paracoccidioides spp. antigen production are problematic in terms of standardization, specificity, stability, repeatability, and reproducibility. OBJECTIVE: To optimize the methodology for Paracoccidioides spp. antigen production and evaluate its applicability in paracoccidioidomycosis immunodiagnosis. MATERIALS AND METHODS: The antigens were obtained from Paracoccidioides lutzii isolates (01, 66, and 8334), Paracoccidioides brasiliensis sensu stricto (113), and Paracoccidioides restripiensis (B-339). These fungi were grown at 36 °C ± 1 °C, on modified Fava-Netto agar, according to Freitas et al. (2018). Paracoccidioides lutzii antigens were obtained after , 10, and 20 days of culture, whereas P. brasiliensis and P. restripiensis antigens were obtained after 10 days. Antigens were evaluated in natura, 10 and 20 times concentrated. Antigenic capacity was evaluated using a double immunodiffusion assay against serum samples from patients with paracoccidioidomycosis, histoplasmosis, and aspergillosis, and random blood donors. RESULTS: Cross-reactivity between Paracoccidioides spp. antigens was observed when P. brasiliensis, P. restrepiensis antigens, and P. lutzii antigens were evaluated with the polyclonal antibodies against P. lutzii and P. brasiliensis, respectively. No cross-reactivity was obtained for polyclonal antibodies against Histoplasma capsulatum, Aspergillus fumigatus, and random blood donors. The proposed protocol allowed stable, repeatable, and reproducible genus-specific antigen production at a low cost and in a short cultivation time. CONCLUSION: The proposed protocol allowed us to obtain genus-specific antigens that can be developed and reproduced in all laboratories in Brazil and South America, where paracoccidioidomycosis is a neglected disease, contributing to an early diagnosis, especially in endemic regions, regardless of the species.

Introducción: Los métodos existentes para la producción de los antígenos de Paracoccidioides spp. son problemáticos en su estandarización, especificidad, estabilidad, repetibilidad y reproducibilidad. Objetivo: Optimizar la metodología para la producción de antígenos de Paracoccidioides spp. y evaluar su aplicabilidad en el inmunodiagnóstico de la paracoccidioidomicosis. Materiales y métodos: Los antígenos se obtuvieron de aislamientos de P. lutzii (01, 66 y 8334), P. brasiliensis sensu stricto (113) y P. restripiensis (B-339). Estos hongos se cultivaron a 36 °C ± 1 °C en agar Fava-Netto modificado, según Freitas et al. (2018). Los antígenos de P. lutzii se obtuvieron a los 5, 10 y 20 días de cultivo y los antígenos de P. brasiliensis y P. restripiensis se obtuvieron a los 10 días. Los antígenos se evaluaron in natura, concentrados 10 y 20 veces. La capacidad antigénica se evaluó mediante un ensayo de inmunodifusión doble con muestras de suero de pacientes con paracoccidioidomicosis, histoplasmosis, aspergilosis y donantes de sangre aleatorios. Resultados: Se observó reacción cruzada con Paracoccidioides spp. cuando se evaluaron los antígenos de P. brasiliensis, P. restrepiensis y P. lutzii frente a los anticuerpos policlonales contra P. lutzii y P. brasiliensis, respectivamente. No hubo reactividad cruzada con los anticuerpos policlonales contra Histoplasma capsulatum y Aspergillus fumigatus, ni contra los donantes de sangre aleatorios. El protocolo propuesto permitió la producción estable, repetible y reproducible de antígenos dirigidos de un género específico (Paracoccidiodes) en un tiempo corto de cultivo y a un menor costo. Conclusión: El protocolo propuesto permitió obtener antígenos específicos de un género, que pueden ser desarrollados y reproducidos en todos los laboratorios de Brasil y Surámerica donde la paracoccidioidomicosis es una enfermedad endémica y desatendida. Estos antígenos pueden contribuir al diagnóstico precoz de la infección, independientemente de la especie.

Endemic Systemic Mycoses in Italy: A Systematic Review of Literature and a Practical Update.

Endemic systemic mycoses such as blastomycosis, coccidioidomycosis, histoplasmosis, talaromycosis, paracoccidioidomycosis are emerging as an important cause of morbidity and mortality worldwide. We conducted a systematic review on endemic systemic mycoses reported in Italy from 1914 to nowadays. We found out: 105 cases of histoplasmosis, 15 of paracoccidioidomycosis, 10 of coccidioidomycosis, 10 of blastomycosis and 3 of talaromycosis. Most cases have been reported in returning travelers and expatriates or immigrants. Thirtytwo patients did not have a story of traveling to an endemic area. Fortysix subjects had HIV/AIDS. Immunosuppression was the major risk factor for getting these infections and for severe outcomes. We provided an overview on microbiological characteristics and clinical management principles of systemic endemic mycoses with a focus on the cases reported in Italy.

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.

COVID-19 in patients with paracoccidioidomycosis.

INTRODUCTION: In 2020, we reported the first patient with concomitant COVID-19 and paracoccidioidomycosis (PCM). Since then, no other cases have been recorded in the literature. We aim to update information on the occurrence of COVID-19 in patients with PCM followed at a reference center for infectious diseases at Rio de Janeiro, Brazil. METHODS: We reviewed the medical records from patients diagnosed with PCM who presented with clinical symptoms, radiological findings, and/or laboratory diagnosis of COVID-19 at any time during their acute or follow-up care. The clinical profiles of these patients were described. RESULTS: Between March 2020 and September 2022, we identified six individuals with COVID-19 among the 117 patients with PCM evaluated. The median age was 38 years and the male to female ratio 2:1. Most patients (n = 5) presented for evaluation due to acute PCM. The severity of COVID-19 ranged from mild to severe in acute PCM and only the single patient with chronic PCM died. CONCLUSIONS: There is a range of disease severity in COVID-19 and PCM co-infection and concomitant disease may represent a severe association, especially in the chronic type of the mycosis with pulmonary involvement. As COVID-19 and chronic PCM share similar clinical aspects and PCM is neglected, it is probable that COVID-19 has been hampering simultaneous PCM diagnosis, which can explain the absence of new co-infection reports. With the continued persistence of COVID-19 globally, these findings further suggest that more attention by providers is necessary to identify co-infections with Paracoccidioides.

Oral paracoccidioidomycosis: a retrospective study of 95 cases from a single center and literature review

Background: The ecoepidemiological panorama of paracoccidioidomycosis (PCM) is dynamic and still ongoing in Brazil. In particular, data about the oral lesions of PCM are barely explored. The aim of this study was to report the clinicopathological features of individuals diagnosed with oral PCM lesions at an oral and maxillofacial pathology service in Rio de Janeiro, Brazil, in the light of a literature review. Material and methods: A retrospective study was conducted on oral biopsies obtained from 1958 to 2021. Additionally, electronic searches were conducted in PubMed, Embase, Scopus, Web of Science, Latin American and Caribbean Center on Health Sciences Information, and Brazilian Library of Dentistry to gather information from large case series of oral PCM. Results: Ninety-five cases of oral PCM were surveyed. The manifestations were more frequent among males (n=86/90.5%), middle-aged/older adults (n=54/58.7%), and white individuals (n=40/51.9%). The most commonly affected sites were the gingiva/alveolar ridge (n=40/23.4%) and lip/labial commissure (n=33/19.3%); however, one (n=40/42.1%) or multiple sites (n=55/57.9%) could also be affected. In 90 (94.7%) patients, "mulberry-like" ulcerations/moriform appearance were observed. Data from 21 studies (1,333 cases), mostly Brazilian (90.5%), revealed that men (92.4%; male/female: 11.8:1) and individuals in the fifth and sixth decades of life were the most affected (range: 7-89 years), with the gingiva/alveolar ridge, palate, and lips/labial commissure being the sites most frequently affected. Conclusions: The features of oral PCM lesions are similar to those reported in previous studies from Latin America. Clinicians should be aware of the oral manifestations of PCM, with emphasis on the clinicodemographic aspects and differential diagnoses, especially considering the phenomenon of the emergence of reported cases in rural and/or urban areas of Brazil. (AU)

Oral paracoccidioidomycosis: a retrospective study of 95 cases from a single center and literature review.

BACKGROUND: The ecoepidemiological panorama of paracoccidioidomycosis (PCM) is dynamic and still ongoing in Brazil. In particular, data about the oral lesions of PCM are barely explored. The aim of this study was to report the clinicopathological features of individuals diagnosed with oral PCM lesions at an oral and maxillofacial pathology service in Rio de Janeiro, Brazil, in the light of a literature review. MATERIAL AND METHODS: A retrospective study was conducted on oral biopsies obtained from 1958 to 2021. Additionally, electronic searches were conducted in PubMed, Embase, Scopus, Web of Science, Latin American and Caribbean Center on Health Sciences Information, and Brazilian Library of Dentistry to gather information from large case series of oral PCM. RESULTS: Ninety-five cases of oral PCM were surveyed. The manifestations were more frequent among males (n=86/90.5%), middle-aged/older adults (n=54/58.7%), and white individuals (n=40/51.9%). The most commonly affected sites were the gingiva/alveolar ridge (n=40/23.4%) and lip/labial commissure (n=33/19.3%); however, one (n=40/42.1%) or multiple sites (n=55/57.9%) could also be affected. In 90 (94.7%) patients, "mulberry-like" ulcerations/moriform appearance were observed. Data from 21 studies (1,333 cases), mostly Brazilian (90.5%), revealed that men (92.4%; male/female: 11.8:1) and individuals in the fifth and sixth decades of life were the most affected (range: 7-89 years), with the gingiva/alveolar ridge, palate, and lips/labial commissure being the sites most frequently affected. CONCLUSIONS: The features of oral PCM lesions are similar to those reported in previous studies from Latin America. Clinicians should be aware of the oral manifestations of PCM, with emphasis on the clinicodemographic aspects and differential diagnoses, especially considering the phenomenon of the emergence of reported cases in rural and/or urban areas of Brazil.

Paracoccidioides and Paracoccidioidomycosis in the 21st Century.

Paracoccidioidomycosis (PCM) defines a broad spectrum of human and animal diseases caused by Paracoccidioides species (Onygenales). In the twenty-first century, Paracoccidioides advanced from a monotypic taxon to a genus that harbors seven species, including P. brasiliensis sensu stricto, P. americana, P. restrepiensis, P. venezuelensis, P. lutzii, P. loboi, and P. cetii. Classic PCM, acquired upon inhalation of propagules from P. brasiliensis sensu stricto, P. americana, P. restrepiensis, P. venezuelensis, and P. lutzii, affects the human lungs and may progress to systemic granulomatous disease with tegumentary and visceral involvement. On the other hand, PCM loboi and PCM ceti caused by the unculturable P. loboi and P. cetii are subcutaneous mycoses, typically observed as keloid lesions in humans and dolphins. Such heterogeneity highlights the importance of recognizing species boundaries in Paracoccidioides to gain insights into the ecology, evolution, clinical features, and mitigation strategies to tackle the advance of PCM.

A surprising finding: The curious case of a tongue lesion misdiagnosed as paracoccidioidomycosis.

BACKGROUND: Paracoccidioidomycosis is an endemic mycosis caused by members of the Paracoccidioides genus. Brazil remains the focus area and, to a lesser extent, the disease has been reported from Argentina, Colombia and Venezuela. AIMS: A Venezuelan Paracoccidioides brasiliensis strain, isolated from a patient diagnosed with chronic multifocal paracoccidioidomycosis, was subjected to whole genome sequencing to provide more insight about Paracoccidioides outside the endemic focus area. METHODS: P. brasiliensis strain CBS 118890 was whole genome sequenced using nanopore; library preparation with the 'native barcoding genomic DNA kit' was followed by sequencing on Flongle and MinION flowcells. Batches of strain CBS 118890 were re-identified by sequencing the internal transcribed spacer (ITS) region, and final identification was made based on phylogenetic analysis. RESULTS: Surprisingly, the Venezuelan P. brasiliensis strain CBS 118890 turned out to be a Nannizziopsis species. The batches of this strain were ITS sequenced followed by phylogenetic analysis and resulted in the final identification of Nannizziopsis arthrosporioides. CONCLUSIONS: Nannizziopsis infections are commonly seen in a wide variety of reptiles, but are particularly rare in human infections. This case underlines the need for molecular characterization of cases that clinically mimic paracoccidioidomycosis but that are serologically negative for Paracoccidioides.

Paracoccidioidomycosis and Addison's syndrome - case report.

Paracoccidioidomycosis is caused by a fungus (Paracoccidioides brasiliensis), which is endemic to Brazil. It is most frequently found in the lungs, with haematogenous and lymphatic spread. The condition is more prevalent in men, between 30 and 60 years old, commonly rural workers. It is the third leading cause of death among chronic infectious diseases today. The systemic disease has an insidious and nonspecific course, with adrenal involvement being observed in 5% of cases and requiring the destruction of 80% of the glands for symptoms of adrenal insufficiency to appear. Isolated involvement of this gland is quite rare. In this case report, however, our patient presented wasting and adrenal insufficiency with isolated adrenal involvement by the fungus.

Paracoccidioidomycosis with digestive manifestations in a pediatric patient.

BACKGROUND: Paracoccidioidomycosis is a systemic infection caused by the fungus Paracoccidioides. It may present in two forms: an acute/subacute form, whose most frequent manifestations include weight loss, fever, anemia, and adenopathy, and a chronic condition with mainly respiratory symptoms. Digestive symptoms, although they may occur, are not frequently reported. Paracoccidioidomycosis usually affects adult male agricultural workers; thus, its presentation in children is rare. CASE REPORT: We describe the case of a 9-year-old male patient diagnosed with paracoccidioidomycosis, who showed abdominal pain and diarrhea as initial manifestations of the disease. CONCLUSIONS: This case is reported not only because of the age of presentation but also due to the existence of digestive symptoms from the onset of the disease, both infrequently reported in the literature.

INTRODUCCIÓN: La paracoccidioidomicosis es una infección sistémica producida por el hongo Paracoccidioides. Se puede presentar de dos formasuna forma aguda/subaguda, cuyas manifestaciones más frecuentes incluyen pérdida de peso, fiebre, anemia y adenopatías, y una forma crónica con manifestaciones principalmente respiratorias. Las manifestaciones digestivas, aunque pueden presentarse, no se reportan frecuentemente. La paracoccidioidomicosis afecta usualmente a varones adultos que trabajan en labores agrícolas, por lo que su presentación en niños es poco frecuente. CASO CLÍNICO: Se describe el caso de un paciente de sexo masculino de 9 años de edad con diagnóstico de paracoccidioidomicosis, con dolor abdominal y diarrea como manifestaciones iniciales de la enfermedad. CONCLUSIONES: Se reporta este caso, no solamente por la edad de presentación, sino también por la existencia de síntomas digestivos desde el inicio de la enfermedad, ambos reportados en forma infrecuente en la literatura.

Paracoccidioidomycosis: Current Status and Future Trends.

Paracoccidioidomycosis (PCM), initially reported in 1908 in the city of São Paulo, Brazil, by Adolpho Lutz, is primarily a systemic and neglected tropical mycosis that may affect individuals with certain risk factors around Latin America, especially Brazil. Paracoccidioides brasiliensis sensu stricto, a classical thermodimorphic fungus associated with PCM, was long considered to represent a monotypic taxon. However, advances in molecular taxonomy revealed several cryptic species, including Paracoccidioides americana, P. restrepiensis, P. venezuelensis, and P. lutzii, that show a preference for skin and mucous membranes, lymph nodes, and respiratory organs but can also affect many other organs. The classical diagnosis of PCM benefits from direct microscopy culture-based, biochemical, and immunological assays in a general microbiology laboratory practice providing a generic identification of the agents. However, molecular assays should be employed to identify Paracoccidioides isolates to the species level, data that would be complemented by epidemiological investigations. From a clinical perspective, all probable and confirmed cases should be treated. The choice of treatment and its duration must be considered, along with the affected organs, process severity, history of previous treatment failure, possibility of administering oral medication, associated diseases, pregnancy, and patient compliance with the proposed treatment regimen. Nevertheless, even after appropriate treatment, there may be relapses, which generally occur 5 years after the apparent cure following treatment, and also, the mycosis may be confused with other diseases. This review provides a comprehensive and critical overview of the immunopathology, laboratory diagnosis, clinical aspects, and current treatment of PCM, highlighting current issues in the identification, treatment, and patient follow-up in light of recent Paracoccidioides species taxonomic developments.