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.

Sporotrichosis in Argentina: clinical and epidemiological analysis / Esporotricosis en Argentina: análisis clínico y epidemiológico

Introduction: Sporotrichosis is an implantation mycosis caused by Sporothrix spp. It is distributed worldwide and can be found in vegetation and soil. The most frequent route of infection is by trauma with elements contaminated with fungal propagules. Since domestic cats are the most affected animals and can transmit this infection to humans, sporotrichosis is considered a zoonosis. Clinical presentations include nodular lymphangitis, fixed cutaneous, pulmonary (rare), and disseminated (exceptional). Objectives: To analyze the epidemiology of sporotrichosis in Argentina during 2010 and 2022. To describe the clinical presentation, diagnostic methods, and treatment of cases diagnosed during this period. To know the circulating genotypes and to observe possible associations with the geographic location where the infection was acquired. Materials and methods: Analytical, retrospective, and observational study. We analyzed the medical records of patients with sporotrichosis from 12 health institutions in Argentina, between 2010 and 2022. Results: We present 54 cases in which the most frequent clinical form was nodular lymphangitis, and the treatment of choice was itraconazole. Conventional diagnosis was made in all cases. Culture of clinical samples was more sensitive than direct examination because it allowed the isolation of Sporothrix spp. in all 54 cases. Molecular identification was performed in 22 cases, with Sporothrix schenkii sensu stricto being the most frequently isolated species. Conclusions: This study allowed to know the epidemiology of this mycosis in Argentina, as well as the availability of diagnostic methods and the treatment of choice.

Introducción: La esporotricosis es una micosis de implantación causada por Sporothrix spp. Este se encuentra distribuido mundialmente y se puede encontrar en la vegetación y en el suelo. La ruta más frecuente de adquisición de la infección es por traumatismos con elementos contaminados con propágulos del hongo. Los gatos domésticos son los animales más afectados y pueden transmitirla a los humanos, por lo que es considerada una zoonosis. Las formas clínicas incluyen: la linfangítica nodular, la cutánea fija, la pulmonar (poco habitual) y la diseminada (excepcional). Objetivo: Analizar la epidemiología de la esporotricosis en Argentina entre los años 2010 y 2022. Describir la presentación clínica, los métodos de diagnóstico y el tratamiento de los casos diagnosticados en este período. Conocer los genotipos circulantes y observar su relación con el lugar geográfico de adquisición de la infección. Materiales y métodos. Se llevó a cabo un estudio analítico, retrospectivo y observacional, en el que se analizaron las historias clínicas de los pacientes con esporotricosis de 12 instituciones de salud de Argentina, entre los años 2010 y 2022. Resultados: Se presentan 54 casos en los que la forma clínica más frecuente fue la linfangítica nodular y el tratamiento de elección fue el itraconazol. En todos los casos se realizó diagnóstico convencional. El cultivo de las muestras clínicas resultó más sensible que el examen directo, ya que permitió el desarrollo de Sporothrix spp. en los 54 casos. En 22 casos se hizo identificación molecular y Sporothrix schenkii sensu stricto fue la especie más frecuentemente aislada. Conclusiones: Este estudio permitió conocer la epidemiología de esta micosis en Argentina, así como la disponibilidad de métodos diagnósticos y el tratamiento de elección.

Esporotricosis en Argentina: análisis clínico y epidemiológico / Sporotrichosis in Argentina: clinical and epidemiological analysis

Introducción. La esporotricosis es una micosis de implantación causada por Sporothrix spp. Este se encuentra distribuido mundialmente y se puede encontrar en la vegetación y en el suelo. La ruta más frecuente de adquisición de la infección es por traumatismos con elementos contaminados con propágulos del hongo. Los gatos domésticos son los animales más afectados y pueden transmitirla a los humanos, por lo que es considerada una zoonosis. Las formas clínicas incluyen: la linfangítica nodular, la cutánea fija, la pulmonar (poco habitual) y la diseminada (excepcional). Objetivo. Analizar la epidemiología de la esporotricosis en Argentina entre los años 2010 y 2022. Describir la presentación clínica, los métodos de diagnóstico y el tratamiento de los casos diagnosticados en este período. Conocer los genotipos circulantes y observar su relación con el lugar geográfico de adquisición de la infección. Materiales y métodos. Se llevó a cabo un estudio analítico, retrospectivo y observacional, en el que se analizaron las historias clínicas de los pacientes con esporotricosis de 12 instituciones de salud de Argentina, entre los años 2010 y 2022. Resultados. Se presentan 54 casos en los que la forma clínica más frecuente fue la linfangítica nodular y el tratamiento de elección fue el itraconazol. En todos los casos se realizó diagnóstico convencional. El cultivo de las muestras clínicas resultó más sensible que el examen directo, ya que permitió el desarrollo de Sporothrix spp. en los 54 casos. En 22 casos se hizo identificación molecular y Sporothrix schenkii sensu stricto fue la especie más frecuentemente aislada. Conclusiones. Este estudio permitió conocer la epidemiología de esta micosis en Argentina, así como la disponibilidad de métodos diagnósticos y el tratamiento de elección.

Introduction. Sporotrichosis is an implantation mycosis caused by Sporothrix spp. It is distributed worldwide and can be found in vegetation and soil. The most frequent route of infection is by trauma with elements contaminated with fungal propagules. Since domestic cats are the most affected animals and can transmit this infection to humans, sporotrichosis is considered a zoonosis. Clinical presentations include nodular lymphangitis, fixed cutaneous, pulmonary (rare), and disseminated (exceptional). Objective. To analyze the epidemiology of sporotrichosis in Argentina during 2010 and 2022. To describe the clinical presentation, diagnostic methods, and treatment of cases diagnosed during this period. To know the circulating genotypes and to observe possible associations with the geographic location where the infection was acquired. Materials and methods. Analytical, retrospective, and observational study. We analyzed the medical records of patients with sporotrichosis from 12 health institutions in Argentina, between 2010 and 2022. Results. We present 54 cases in which the most frequent clinical form was nodular lymphangitis, and the treatment of choice was itraconazole. Conventional diagnosis was made in all cases. Culture of clinical samples was more sensitive than direct examination because it allowed the isolation of Sporothrix spp. in all 54 cases. Molecular identification was performed in 22 cases, with Sporothrix schenkii sensu stricto being the most frequently isolated species. Conclusions. This study allowed to know the epidemiology of this mycosis in Argentina, as well as the availability of diagnostic methods and the treatment of choice.

Clinical and Demographic Features of Paracoccidioidomycosis in Argentina: A Multicenter Study Analysis of 466 Cases.

Information on paracoccidioidomycosis (PCM) in Argentina is fragmented and has historically been based on estimates, supported only by a series of a few reported cases. Considering the lack of global information, a national multicentric study in order to carry out a more comprehensive analysis was warranted. We present a data analysis including demographic and clinical aspects of a historical series of 466 cases recorded over 10 years (2012-2021). Patients were aged from 1 to 89 years. The general male: female (M:F) ratio was 9.5:1 with significant variation according to the age group. Interestingly, the age range 21-30 shows an M:F ratio of 2:1. Most of the cases (86%) were registered in northeast Argentina (NEA), showing hyperendemic areas in Chaco province with more than 2 cases per 10,000 inhabitants. The chronic clinical form occurred in 85.6% of cases and the acute/subacute form occurred in 14.4% of cases, but most of these juvenile type cases occurred in northwestern Argentina (NWA). In NEA, the incidence of the chronic form was 90.6%; in NWA, the acute/subacute form exceeded 37%. Diagnosis by microscopy showed 96% positivity but antibody detection displays 17% of false negatives. Tuberculosis was the most frequent comorbidity, but a diverse spectrum of bacterial, fungal, viral, parasitic, and other non-infectious comorbidities was recorded. This national multicenter registry was launched in order to better understand the current status of PCM in Argentina and shows the two endemic zones with a highly diverse epidemiology.

Modulatory effect of Origanum vulgare essential oil and carvacrol on Malassezia spp. virulence factors.

Malassezia sympodialis and Malassezia furfur are recognized as an important part of the human and animals healthy skin microbiota, but also as an opportunistic fungus due to their association with a broad spectrum of skin and systemic infections. Human skin infections associated with Malassezia spp. are often chronic, recurrent, and topical or oral azole treatments are challenging with not always successful outcomes. A wide inter and intraspecies antifungal susceptibility variability that may suggest a combination of wild-type strains and resistant mutants was reported. The essential oils are complex hydrophobic mixtures of different compounds extracted from plants by distillation and have demonstrated antimicrobial activity. In this study, toxicity in the larvae model and the ability of Origanum vulgare essential oil (OVEO) and carvacrol to inhibit virulence factors such as hydrophobicity, adherence, and biofilm formation of M. sympodialis and M. furfur was evaluated. We have demonstrated the antifungal activity of OVEO and carvacrol against both species. Carvacrol was more active and less toxic than OVEO at low concentrations (< 1 mg/ml). Malassezia sympodialis showed a significant decrease in adherence and its ability to form biofilms when OVEO or carvacrol is present. Both Malassezia species showed reduced hydrophobicity in the presence of carvacrol. Further studies will have to be carried out to understand how these substances act, but the inhibition of some virulence factors could represent a new target to overcome the problem of drug resistance.

The work reports the research on the effect of the Origanum vulgare essential oil and carvacrol, against virulence factors such as hydrophobicity, adherence, and biofilm formation of two species of Malassezia. The inhibition of virulence factors could represent a new target to overcome the drug resistance reported.

Biogenic silver nanoparticles as antifungal agents.

In recent years, an increase in multidrug-resistant fungal strains has been observed, which, together with the limited number of clinically available antifungal agents, highlights the need for the development of new antifungal agents. Due to the proven antifungal activity of silver nanoparticles (AgNPs), there is a growing interest in their use in the treatment of fungal infections. Nanoparticles are usually synthesised through a variety of physical and chemical processes that are costly and pollute the environment. For this reason, biogenic synthesis is emerging as an environmentally friendly technology and new strategies are increasingly based on the use of biogenic AgNPs as antifungal agents for clinical use. The aim of this review is to compare the antifungal activity of different biogenic AgNPs and to summarise the current knowledge on the mechanisms of action and resistance of fungi to AgNPs. Finally, a general analysis of the toxicity of biogenic AgNPs in human and veterinary medicine is performed.

Malassezia species: the need to establish epidemiological cutoff values.

Malassezia are common yeasts in human skin microbiome. Under certain conditions these yeasts may cause disease from skin disorders to systemic infections. In the absence of clinical breakpoints, epidemiological cutoff values (ECVs) are useful to differentiate isolates with acquired or mutational resistance. The aim of this work was to propose tentative ECVs of Malassezia furfur, M. sympodialis, M. globosa for fluconazole (FCZ), itraconazole (ITZ), voriconazole (VCZ), ketoconazole (KTZ) and amphotericin B (AMB). A total of 160 isolates (80 M. furfur, 50 M. sympodialis, and 30 M. globosa) were tested. Minimal inhibitory concentrations (MICs) were determined by modified broth microdilution method (CLSI). ECVs were estimated by ECOFFinder software and twofold dilutions beyond the mode. ITZ, KTZ, and VCZ showed the lowest MICs. The highest MIC and widest ranges were for FCZ and AMB. For ITZ, KTZ, and VCZ both ECVs were similar. For FCZ, AMB especially M. furfur, modal ECVs were lower than values obtained by statistical method. When MIC distribution is the only data available, ECV could provide information to help guide therapy decisions. In that drug/species combination in which different peaks in the MIC distribution were observed, difference between both ECV was greater. This is the first study that provides ECV data of 160 Malassezia yeasts. Although ECVs cannot be used as predictors of clinical response, identification of non wild-type isolates suggests that it may be less likely to respond to a given antifungal agent. LAY SUMMARY: Malassezia species causes skin disorders to systemic infections. Epidemiological cutoff value (ECV) allows for differentiation of wild-type and non wild-type isolates. Based on MIC data of 160 isolates we propose tentative ECVs for three Malassezia species. ECVs are useful in surveillance and guide therapy decisions.

Cost-Effective Method to Perform SARS-CoV-2 Variant Surveillance: Detection of Alpha, Gamma, Lambda, Delta, Epsilon, and Zeta in Argentina.

SARS-CoV-2 variants with concerning characteristics have emerged since the end of 2020. Surveillance of SARS-CoV-2 variants was performed on a total of 4,851 samples from the capital city and 10 provinces of Argentina, during 51 epidemiological weeks (EWs) that covered the end of the first wave and the ongoing second wave of the COVID-19 pandemic in the country (EW 44/2020 to EW 41/2021). The surveillance strategy was mainly based on Sanger sequencing of a Spike coding region that allows the identification of signature mutations associated with variants. In addition, whole-genome sequences were obtained from 637 samples. The main variants found were Gamma and Lambda, and to a lesser extent, Alpha, Zeta, and Epsilon, and more recently, Delta. Whereas, Gamma dominated in different regions of the country, both Gamma and Lambda prevailed in the most populated area, the metropolitan region of Buenos Aires. The lineages that circulated on the first wave were replaced by emergent variants in a term of a few weeks. At the end of the ongoing second wave, Delta began to be detected, replacing Gamma and Lambda. This scenario is consistent with the Latin American variant landscape, so far characterized by a concurrent increase in Delta circulation and a stabilization in the number of cases. The cost-effective surveillance protocol presented here allowed for a rapid response in a resource-limited setting, added information on the expansion of Lambda in South America, and contributed to the implementation of public health measures to control the disease spread in Argentina.

Antimicrobial and cytotoxic activity of green synthesis silver nanoparticles targeting skin and soft tissue infectious agents.

Combining traditional medicine with nanotechnology therefore opens the door to innovative strategies for treating skin and soft tissue infections (SSTIs) and also contributes to the fight against the rise of antimicrobial resistance. Acanthospermum australe (Loefl.) Kuntze is a medicinal plant used by indigenous peoples in northeastern Argentina to treat SSTIs. Spherical and stable silver nanoparticles (AgNPs) of 14 ± 2 nm were synthesized from the aqueous extract of A. australe and silver nitrate. The antimicrobial activity against main species causing SSTIs and cytotoxicity on peripheral blood mononuclear cells of AgNP solution and its synthesis components were evaluated. Compared to its synthesis components, AgNP solution showed greater antimicrobial activity and lower cytotoxicity. The antimicrobial activity of AgNPs was due to the silver and not to the metabolites of the aqueous extract present on the surface of the nanoparticles. The plant extract played an important role in the formation of stable AgNPs and acted as a modulator of cytotoxic and immune responses.

Biofilm formation, adherence, and hydrophobicity of M. sympodialis, M. globosa, and M. slooffiae from clinical isolates and normal skinVirulence factors of M. sympodialis, M. globosa and M. slooffiae.

The genus Malassezia comprises a heterogeneous group of species that cause similar pathologies. Malassezia yeasts were considered as the most abundant skin eukaryotes of the total skin mycobiome. The ability of this fungus to colonize or infect is determined by complex interactions between the fungal cell and its virulence factors. This study aims to evaluate in vitro the hydrophobicity levels, the adherence capacity on a polystyrene surface and the ability to form biofilm of 19 isolates, including M. sympodialis, M. globosa, and M. slooffiae, from healthy subjects and from dermatological disorders. Cellular surface hydrophobicity levels were determined by two-phase system. The biofilm formation was determined by tetrazolium salt (XTT) reduction assay and by Scanning Electron Microscopy (SEM). Strain dependence was observed in all virulence factors studied. All isolates of M. sympodialis, M. globosa, and M. slooffiae demonstrated their ability to form biofilm at variable capacities. SEM observations confirmed a variable extracellular matrix after 48 hours of biofilm formation. All isolates of M. globosa were highly adherent and/or hydrophobic as well as biofilm producers. In contrast, M. slooffiae was the least biofilm producer. No significant differences between virulence factors were demonstrated for M. sympodialis, either as clinical isolate or as inhabitant of human microbiota. Results of this work together with the previous M. furfur research confirm that the most frequently Malassezia species isolated from normal subject's skin and patients with dermatosis, form biofilm with different capacities. The study of these virulence factors is important to highlight differences between Malassezia species and to determine their involvement in pathological processes.

Antifungal activity of silver nanoparticles in combination with ketoconazole against Malassezia furfur.

Malassezia furfur is lipophilic and lipid-dependent yeast, inhabitant of human skin microbiota associated with several dermal disorders. In recent years, along with the advances in nanotechnology and the incentive to find new antimicrobial drugs, there has been a growing interest in the utilization of nanoparticles for the treatment of skin microbial infections. This work aimed to study the in vitro inhibitory activity of silver nanoparticles (AgNP) against 41 M. furfur clinical isolates, visualize the interaction between AgNP-Malassezia, evaluate the synergism with ketoconazole (KTZ) and to produce an antimicrobial gel of AgNP-KTZ. The synthesized AgNP were randomly distributed around the yeast surface and showed a fungicidal action with low minimal inhibitory concentration values. AgNP showed no antagonistic effect with KTZ. The broad-spectrum antimicrobial property with fungicidal action of AgNP and its accumulation in affected areas with a sustained release profile, added to the great antifungal activity of KTZ against Malassezia infections and other superficial mycoses, allowed us to obtain a gel based on carbopol formulated with AgNP-KTZ with the potential to improve the topical therapy of superficial malasseziosis, reduce the number of applications and, also, prevent the recurrence.

Emergence of acute/subacute infant-juvenile paracoccidioidomycosis in Northeast Argentina: Effect of climatic and anthropogenic changes?

Argentina has two endemic areas of paracoccidioidomycosis (PCM). Bordering Paraguay and Brazil, Northeast Argentina (NEA) comprises the area with the highest incidence where the chronic adult clinical form has historically been reported. Juvenile form in children and adolescents is rare in this area since only one case was reported in the last 10 years. Despite this, between 2010 and 2012, several cases of acute/subacute clinical forms in children aged 10 to 16 (median 12) were detected. In the last decade, the NEA region has been exposed to ecological variations as consequences of certain climatic and anthropogenic changes, including El Niño-Southern Oscillation phenomenon during 2009, and deforestation. The region has also suffered from the significant ecological effects of the construction of one of the biggest hydroelectric dams of South America. This study aims to describe clinical and epidemiological aspects of acute/subacute PCM cases detected in children from NEA and to discuss climatic and anthropogenic changes as possible contributing factors in the emergence of this disease in children. This acute/subacute PCM cluster was characterized by severe disseminated and aggressive presentations to localized form, with a high spectrum of clinical manifestations uncommonly observed. Due to the lack of experience in acute/subacute PCM in children in the studied area and the atypical clinical manifestations observed, the diagnosis was delayed. In order to avoid misdiagnosis, a higher level of suspicion is now required in NEA and countries bordering the southern part of the endemic area, which are affected by the changes discussed in this article.

Biofilm, adherence, and hydrophobicity as virulence factors in Malassezia furfur.

Malassezia species are natural inhabitants of the healthy skin. However, under certain conditions, they may cause or exacerbate several skin diseases. The ability of this fungus to colonize or infect is determined by complex interactions between the fungal cell and its virulence factors. This study aims to evaluate "in vitro" the hydrophobicity levels, the adherence on a plastic surface and the biofilm formation of 16 clinical isolates of Malassezia furfur. Cellular surface hydrophobicity (CSH) levels were determined by two-phase system. The biofilm formation was determined by tetrazolium salt (XTT) reduction assay and by Scanning Electron Microscopy (SEM). Results showed many isolates were hydrophobic, adherent, and producers of biofilm on abiotic surfaces with different capacity. SEM observations confirmed an abundant extracellular matrix after 48 h of biofilm formation. About 63% of strains with high production of biofilm showed medium to high percentage of hydrophobicity and/or adherence. In addition, it has been demonstrated a correlation between hydrophobicity, adherence, and biofilm formation in about 60% of strains examined. These important virulence factors could be responsible of this yeast changing from a commensal to a pathogenic status.

Protothecosis in a patient with T cell lymphocytic leukemia.

Human protothecosis is a rare infection caused by algae of the genus Prototheca. Prototheca wickerhamii has been recognized as the main species that causes infection in immunocompromised hosts with deficits in innate or cellular immunity. We report a case of persisting subcutaneous protothecosis in a patient with T-cell large granular lymphocyte leukemia, who also presented a history of disseminated histoplasmosis.

In Vivo Paracoccidioides sp. Biofilm on Vascular Prosthesis.

Paracoccidioidomycosis is an endemic mycosis caused by Paracoccidioides species limited to Latin America arising with the chronic form in 90% of cases. The capacity of microorganisms to form biofilms is considered of great importance medical since can contribute to the persistence and to the chronic state of the diseases. The ability of Paracoccidioides to form biofilm has been demonstrated in vitro. In our study, for the first time we have observed this capability in vivo on a vascular prosthesis using scanning electron microscope showing a dense network of Paracoccidioides yeasts covered by an extracellular matrix.