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We aimed to develop and validate a Loop-mediated Isothermal Amplification (LAMP) assay to Sporothrix brasiliensis. LAMP reaction was developed using six primers designed based on calmodulin gene. In the LAMP reaction, we tested twenty isolates of S. brasiliensis from animals and humans, along with ten tissue samples extracted from the left footpad of mice that had been experimentally infected with S. brasiliensis. In addition, it included DNA samples from various other fungal species for specificity evaluation. All S. brasiliensis isolates yielded positive results in the LAMP, and the limit of DNA detection was 1 ng/µL. All murine samples were positive in the test while DNA from other fungal species were all negative, resulting in 100% of sensitivity and specificity of primers. LAMP diagnosis technique is a promising alternative to sporotrichosis diagnosis, in a simple and cost-effective way. Further studies are warranted to validate this technique using animal model samples obtained from both humans and animals.
Assuntos
Primers do DNA , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico , Sensibilidade e Especificidade , Sporothrix , Esporotricose , Sporothrix/genética , Sporothrix/isolamento & purificação , Sporothrix/classificação , Esporotricose/diagnóstico , Esporotricose/microbiologia , Esporotricose/veterinária , Animais , Técnicas de Amplificação de Ácido Nucleico/métodos , Técnicas de Diagnóstico Molecular/métodos , Camundongos , Humanos , Primers do DNA/genética , Modelos Animais de Doenças , Calmodulina/genéticaRESUMO
Zoonotic sporotrichosis caused by Sporothrix brasiliensis is an emerging mycosis in Latin America. One of the problems to quickly treat infected animals and break the transmission chain is associated with the time-consuming gold-standard diagnosis method (culture). We aimed to evaluate a species-specific polymerase chain reaction (PCR) for the diagnosis of sporotrichosis caused by S. brasiliensis using non-invasive samples. We performed a retrospective cross-sectional study using samples collected with swabs from humans and cats with clinical suspicion of sporotrichosis. Deoxyribonucleic acid (DNA) was extracted using a commercial kit, and a species-specific PCR for S. brasiliensis detection was performed. One hundred ten samples were included. PCR showed a good concordance with culture (86% of agreement) for human and cat samples (Kappa coefficient = 0.722, and 0.727, respectively). In conclusion, our data shows that this adapted PCR using non-invasive samples can be applied to sporotrichosis diagnosis, being a good alternative mainly in regions with a lack of mycologists to identify the fungus in culture, contributing to the control of this emergent zoonosis.
We aimed to evaluate a molecular method for diagnosing sporotrichosis caused by Sporothrix brasiliensis in humans and cats. We observed that the technique is in good agreement with the classic method and is a good alternative for assisting in the diagnosis and consequent control of this zoonosis.
Assuntos
Doenças do Gato , Reação em Cadeia da Polimerase , Sporothrix , Esporotricose , Esporotricose/diagnóstico , Esporotricose/microbiologia , Esporotricose/veterinária , Gatos , Sporothrix/genética , Sporothrix/isolamento & purificação , Sporothrix/classificação , Humanos , Animais , Reação em Cadeia da Polimerase/métodos , Doenças do Gato/diagnóstico , Doenças do Gato/microbiologia , Estudos Retrospectivos , Estudos Transversais , Zoonoses/diagnóstico , Zoonoses/microbiologia , DNA Fúngico/genética , Técnicas de Diagnóstico Molecular/métodos , Sensibilidade e EspecificidadeRESUMO
The impact of invasive pulmonary aspergillosis (IPA) on non-neutropenic critically ill patients in intensive care units (ICU) has been demonstrated in recent decades. Furthermore, after the start of the COVID-19 pandemic, COVID-19 associated with pulmonary aspergillosis (CAPA) has become a major concern in ICUs. However, epidemiological data from different regions are scarce. We evaluated the prevalence and clinical-epidemiological data of IPA in patients with COVID-19 requiring mechanical ventilation (MV) in the ICU ("severe COVID-19") and non-COVID ICU patients in MV of a tertiary hospital in the southern region of Brazil. Eighty-seven patients admitted between June 2020 and August 2022 were included; 31 with severe COVID-19. For the diagnosis of IPA or CAPA, algorithms including host factors and mycological criteria (positive culture for Aspergillus spp., immunoassay for galactomannan detection, and/or qPCR) were utilized. The overall incidence of IPA and CAPA in our ICU was 73 cases/1000 ICU hospitalizations. Aspergillosis occurred in 13% (4/31) of the COVID-19 patients, and in 16% (9/56) of the critically ill patients without COVID-19, with mortality rates of 75% (3/4) and 67% (6/9), respectively. Our results highlight the need for physicians enrolled in ICU care to be aware of aspergillosis and for more access of the patients to sensitive and robust diagnostic tests by biomarkers detection.
Assuntos
COVID-19 , Estado Terminal , Unidades de Terapia Intensiva , Aspergilose Pulmonar Invasiva , Centros de Atenção Terciária , Humanos , COVID-19/complicações , COVID-19/epidemiologia , Brasil/epidemiologia , Centros de Atenção Terciária/estatística & dados numéricos , Masculino , Feminino , Pessoa de Meia-Idade , Idoso , Aspergilose Pulmonar Invasiva/epidemiologia , Aspergilose Pulmonar Invasiva/diagnóstico , Adulto , SARS-CoV-2/isolamento & purificação , Respiração Artificial , Prevalência , Incidência , Idoso de 80 Anos ou maisRESUMO
Vertebrate animal models are essential in research; however, efforts need to be made to decrease animal suffering as much as possible. It could be useful to determine humane endpoints that could serve as surrogates for a fatal outcome. We address this issue with respect to infectious diseases. We propose a humane endpoint for studies of Sporothrix brasiliensis infection. BALB/c mice were inoculated subcutaneously in the footpad. To define a humane endpoint, we considered two groups: animals who died during the experiment, and those euthanized at the end of the experiment. The groups were compared for colony-forming units (CFU) in internal organs, clinical condition, and body weight. Thirteen (22%) animals died before the end of the experiment due to the progression of local infection to severe and disseminated sporotrichosis. Analyzing data of the groups, we propose the following future criteria for euthanasia as a humane endpoint: neurological impairment OR necrosis of the footpad OR loss of ≥ 20% body weight AND moderate to severe dehydration. In view of the current global epidemiological impact of zoonotic sporotrichosis caused by S. brasiliensis, our data could facilitate the utility of models used to study the disease, particularly therapy.
Assuntos
Modelos Animais de Doenças , Camundongos Endogâmicos BALB C , Sporothrix , Esporotricose , Animais , Esporotricose/microbiologia , Camundongos , Peso CorporalRESUMO
Healthcare-associated infections (HAI) are illnesses acquired during healthcare and are often the most important adverse event during healthcare. With the aim of increasing the effectiveness of disinfection/decontamination processes in the health service with safe and not promote microbial resistance, we propose the development of portable equipment associated with type C ultraviolet light (UVC). The efficiency of the irradiance emitted by the equipment (at dosages 3.5, 5.0, and 60 mJ/cm2) was determined by the action exerted after exposure against four different bacterial (Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus) and three different fungi (Candida albicans, C. parapsilosis, and Aspergillus section Fumigati). It was possible to observe that all treatments were capable of inactivating the bacterial species evaluated (p < 0.05), causing the irreversible death of these microorganisms. The most effective elimination of fungal agents was at a dose of 60 mJ/cm2 of UVC radiation, with a decrease in the fungal inoculum varying between 94% and 100% in relation to the control without exposure. Thus, our study showed that the application of the portable prototype with UVC light (254 nm) at a distance of 48 mm, allowed an average irradiance of 3.5 mW/cm2, with doses of 3.5 ≈ 60 mJ/cm2 (from 1 to 60 s of exposure), which can promote the total reduction of the bacteria evaluated and significantly reduce fungal growth. Therefore, this prototype could be used safely and effectively in the hospital environment, considerably reducing contamination and contributing to the reduction of healthcare-associated infection risk.
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Sporothrix brasiliensis is recognized as an emergent fungal pathogen and the high amount of fungal propagules in the lesions of infected cats allows the contamination of surfaces by direct contact. Given that the environment can play a role in the transmission of this fungus, effective methods to eliminate this pathogen from contaminated surfaces are necessary. Physical methods, such as ultraviolet light C (UVC), are broad used for surfaces disinfection, however, non-data about its activity against S. brasiliensis is reported. Therefore, we aimed to evaluate an easy handled prototype of a UVC device, in the inhibition of S. brasiliensis. Three doses and times of exposure of irradiance were tested: 3.5 mJ/cm2 (1 s), 5.25 mJ/cm2 (1.5 s) and 329 mJ/cm2 (94 s) against a standardized inoculum of yeast and mold phase of S. brasiliensis. A decrease in CFU was shown in all doses of irradiance in both phases of S. brasiliensis, the average reduction ranged from 78 to 100% among doses, being a complete fungicidal activity achieved against the yeast phase after the 94 s exposure (329 mJ/cm2). Our data shows that UVC is a potential physical method for disinfection of surfaces contaminated with S. brasiliensis, and the prototype device developed provides an easy handling, and quickly results.
Assuntos
Desinfecção , Sporothrix , Raios Ultravioleta , Sporothrix/efeitos da radiação , Desinfecção/métodos , Desinfecção/instrumentação , Animais , GatosRESUMO
Zoonotic sporotrichosis, a subcutaneous mycosis caused by Sporothrix brasiliensis, has become hyperendemic and a serious public health issue in Brazil and an emerging disease throughout the world. Typical sporotrichosis is defined as fixed or lymphocutaneous lesion development, however, reports of atypical presentations have been described in hyperendemic areas, which may result in a worse prognosis. Thus, considering an increase in atypical cases and in more severe extracutaneous cases and hospitalizations reported in Brazil, we aimed to perform a systematic review to search for hypersensitivity reactions (HRs) and extracutaneous presentations associated with zoonotic sporotrichosis. A systematic review was performed, following the PRISMA guidelines to search for atypical/extracutaneous cases (mucosal, osteoarthritis, HRs, pulmonary, meningeal) of zoonotic sporotrichosis. A total of 791 published cases over 26 years (1998-2023) in eleven Brazilian states were reviewed. Most cases corresponded to a HR (47%; n = 370), followed by mucosal (32%; n = 256), multifocal (8%; n = 60), osteoarthritis (7%; n = 59), meningeal (4%; n = 32), and pulmonary (2%; n = 14) infections. When available (n = 607), the outcome was death in 7% (n = 43) of cases. Here, we show a frequent and worrisome scenario of zoonotic sporotrichosis in Brazil, with a high and dispersed incidence of atypical/extracutaneous cases throughout the Brazilian territory. Therefore, educational measures are necessary to make health professionals and the overall population aware of this fungal pathogen in Brazil as well as in other countries in the Americas.
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The drugs available to treat sporotrichosis, an important yet neglected fungal infection, are limited. Some Sporothrix spp. strains present reduced susceptibility to these antifungals. Furthermore, some patients may not be indicated to use these drugs, while others may not respond to the therapy. The anthelmintic drug niclosamide is fungicidal against the Sporothrix brasiliensis type strain. This study aimed to evaluate whether niclosamide also has antifungal activity against Sporothrix globosa, Sporothrix schenckii and other S. brasiliensis strains with distinct genotypes and antifungal susceptibility status. Minimal inhibitory and fungicidal concentrations (MIC and MFC, respectively) were determined using the microdilution method according to the CLSI protocol. The checkerboard method was employed to evaluate niclosamide synergism with drugs used in sporotrichosis treatment. Metabolic activity of the strains under niclosamide treatment was evaluated using the resazurin dye. Niclosamide was active against all S. brasiliensis strains (n = 17), but it was ineffective (MIC > 20 µM) for some strains (n = 4) of other pathogenic Sporothrix species. Niclosamide MIC values for Sporothrix spp. were similar for mycelial and yeast-like forms of the strains (P = 0.6604). Niclosamide was fungicidal (MFC/MIC ratio ≤ 2) for most strains studied (89%). Niclosamide activity against S. brasiliensis is independent of the fungal genotype or non-wild-type phenotypes for amphotericin B, itraconazole, or terbinafine. These antifungal drugs presented indifferent interactions with niclosamide. Niclosamide has demonstrated potential for repurposing as a treatment for sporotrichosis, particularly in S. brasiliensis cases, instigating in vivo studies to validate the in vitro findings.
Assuntos
Anti-Helmínticos , Antifúngicos , Testes de Sensibilidade Microbiana , Niclosamida , Sporothrix , Sporothrix/efeitos dos fármacos , Sporothrix/genética , Sporothrix/classificação , Niclosamida/farmacologia , Antifúngicos/farmacologia , Anti-Helmínticos/farmacologia , Esporotricose/microbiologia , Esporotricose/tratamento farmacológico , Genótipo , Humanos , Farmacorresistência Fúngica , Sinergismo FarmacológicoRESUMO
Sporotrichosis is a subcutaneous mycosis caused by pathogenic Sporothrix species. Among them, Sporothrix brasiliensis is the main species associated with endemic regions in South America, especially Brazil. It is highly virulent and can be spread through zoonotic transmission. Molecular epidemiological surveys are needed to determine the extent of genetic variation, to investigate outbreaks, and to identify genotypes associated with antifungal resistance and susceptibility. This study investigated the sequence variation of different constitutive genes and established a novel multilocus sequence typing (MLST) scheme for S. brasiliensis. Specific primers were designed for 16 genes using Primer-BLAST software based on the genome sequences of three S. brasiliensis strains (ATCC MYA-4823, A001 and A005). Ninety-one human, animal, and environmental S. brasiliensis isolates from different Brazilian geographic regions (South, Southeast, Midwest and Northeast) andtwo isolates from Paraguay were sequenced. The loci that presented the highest nucleotide diversity (π) were selected for the MLST scheme. Among the 16 studied genetic loci, four presented increased π value and were able to distinguish all S. brasiliensis isolates into seven distinct haplotypes. The PCR conditions were standardized for four loci. Some of the obtained haplotypes were associated with the geographic origin of the strains. This study presents an important advance in the understanding of this important agent of sporotrichosis in Brazil. It significantly increased the discriminatory power for genotyping of S. brasiliensis isolates, and enabled new contributions to the epidemiological studies of this human and animal pathogen in Brazil and in other countries.
Assuntos
Sporothrix , Esporotricose , Animais , Humanos , Esporotricose/epidemiologia , Esporotricose/microbiologia , Tipagem de Sequências Multilocus , Genótipo , Brasil/epidemiologiaRESUMO
Background: Sporotrichosis caused by Sporothrix brasiliensis is a globally emerging infectious disease with limited therapeutic options. Thus, we aimed to evaluate the in vitro activity of amlodipine (AML) and lufenuron (LUF) alone and their interaction with itraconazole (ITZ), the first-choice drug against S. brasiliensis. Methods: Twenty clinical isolates of S. brasiliensis from two hyperendemic regions were tested through a microdilution assay to evaluate the minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) of AML and LUF. Checkerboard assay was performed with 10 isolates for both drug interactions with ITZ. Results: AML showed inhibitory and fungicidal activity against all isolates included, with MIC values ranging from 32 to 256 µg/mL, and MFC from 64 to 256 µg/mL. However, none of the S. brasiliensis isolates were inhibited by the highest soluble concentration of LUF (MIC >64 µg/mL for all strains). Synergic interaction of AML and LUF with ITZ occurred in 50% and 40% of the isolates tested, without any antagonistic effects. Conclusion: Both repurposing drugs evaluated in our study showed a promising in vitro activity, especially in synergy with ITZ against S. brasiliensis, warranting future in vivo investigations regarding its activity.