Screening of Candida spp. in wastewater in Brazil during COVID-19 pandemic: workflow for monitoring fungal pathogens.

Fungal diseases are often linked to poverty, which is associated with poor hygiene and sanitation conditions that have been severely worsened by the COVID-19 pandemic. Moreover, COVID-19 patients are treated with Dexamethasone, a corticosteroid that promotes an immunosuppressive profile, making patients more susceptible to opportunistic fungal infections, such as those caused by Candida species. In this study, we analyzed the prevalence of Candida yeasts in wastewater samples collected to track viral genetic material during the COVID-19 pandemic and identified the yeasts using polyphasic taxonomy. Furthermore, we investigated the production of biofilm and hydrolytic enzymes, which are known virulence factors. Our findings revealed that all Candida species could form biofilms and exhibited moderate hydrolytic enzyme activity. We also proposed a workflow for monitoring wastewater using Colony PCR instead of conventional PCR, as this technique is fast, cost-effective, and reliable. This approach enhances the accurate taxonomic identification of yeasts in environmental samples, contributing to environmental monitoring as part of the One Health approach, which preconizes the monitoring of possible emergent pathogenic microorganisms, including fungi.

Severe COVID-19 in HIV/Leishmania infantum coinfected patient: a successfully managed case report.

BACKGROUND: Coronavirus disease 2019 originated in China and swiftly spread worldwide, posing a significant threat to public health. Caused by SARS-CoV-2, it manifests as a flu-like illness that can escalate to Acute Respiratory Distress Syndrome, potentially resulting in fatalities. In countries where HIV/Leishmania infantum is endemic, the occurrence of concurrent SARS-CoV-2/HIV/Leishmania infantum infections is a reality, prompting inquiries into appropriate clinical management. CASE PRESENTATION: We present the case of a 48-year-old woman who was hospitalized for 36 days across three different hospitals in the state of Pernambuco, Brazil. She was diagnosed with SARS-CoV-2/HIV/L. infantum coinfection. The patient exhibited severe COVID-19 symptoms, including fever, productive cough, and dyspnea. Throughout her hospitalization, she experienced oxygen saturation levels of ≤ 93%, along with fluctuations in blood pressure, respiratory rate, and heart rate. Her blood tests revealed lymphopenia, leukopenia, and neutropenia, while laboratory results indicated abnormal levels of d-dimer, AST, ALT, lactate dehydrogenase, ferritin, and C-reactive protein. A computed tomography scan revealed 75% involvement of the lung parenchyma with patchy ground-glass opacities. CONCLUSION: Against all odds, the patient was discharged. The leukopenia associated with HIV/L. infantum may have played a decisive role. Further studies are necessary to better understand diagnostic strategies and clinical management measures for HIV/L. infantum coinfected patients who are susceptible to SARS-CoV-2 infection.

Taxonomy of Candida parapsilosis complex isolated from neonates and the role of Hsp90 inhibitors to enhanced the antifungal activity of micafungin.

Species from Candida parapsilosis complex are frequently found in neonatal candidemia. The antifungal agents to treat this infection are limited and the occurrence of low in vitro susceptibility to echinocandins such as micafungin has been observed. In this context, the chaperone Hsp90 could be a target to reduce resistance. Thus, the objective of this research was to identify isolates from the C. parapsilosis complex and verify the action of Hsp90 inhibitors associated with micafungin. The fungal identification was based on genetic sequencing and mass spectrometry. Minimal inhibitory concentrations were determined by broth microdilution method according to Clinical Laboratory and Standards Institute. The evaluation of the interaction between micafungin with Hsp90 inhibitors was realized using the checkerboard methodology. According to the polyphasic taxonomy, C. parapsilosis sensu stricto was the most frequently identified, followed by C. orthopsilosis and C. metapsilosis, and one isolate of Lodderomyces elongisporus was identified by genetic sequencing. The Hsp90 inhibitor geladanamycin associated with micafungin showed a synergic effect in 31.25% of the isolates, a better result was observed with radicicol, which shows synergic effect in 56.25% tested yeasts. The results obtained demonstrate that blocking Hsp90 could be effective to reduce antifungal resistance to echinocandins.

Virulence factors of Gram-negative bacteria from free-ranging Amazon river dolphins (Inia geoffrensis).

Freshwater cetaceans play a significant role as sentinel animals, providing important data on animal species and aquatic ecosystem health. They also may serve as potential reservoirs of emerging pathogens and host virulence genes in their microbiota. In this study, we evaluated virulence factors produced by Gram-negative bacteria recovered from individuals belonging to two populations of free-ranging Amazon river dolphins (Inia geoffrensis). A total of 132 isolates recovered from the oral cavity, blowhole, genital opening and rectum of 21 river dolphins, 13 from Negro River and 8 from Tapajós River, Brazil, were evaluated for the production of virulence factors, such as biofilms and exoproducts (proteases, hemolysins and siderophores), in planktonic and biofilm forms. In planktonic form, 81.1% (107/132) of the tested bacteria of free-ranging Amazon river dolphins were able to produce virulence factors, with 44/132 (33.4%), 65/132 (49,2%) and 54/132 (40,9%) positive for protease, hemolysin and siderophore production, respectively. Overall, 57/132 (43.2%) of the isolates produced biofilms and, under this form of growth, 66/132 (50%), 88/132 (66.7%) and 80/132 (60.6%) of the isolates were positive for protease, hemolysin and siderophore production. In general, the isolates showed a higher release of exoproducts in biofilm than in planktonic form (P < 0.001). The present findings show that Amazon river dolphins harbor potentially pathogenic bacteria in their microbiota, highlighting the importance of monitoring the micro-organisms from wild animals, as they may emerge as pathogens for humans and other animals.

Proposal for a microcosm biofilm model for the study of vulvovaginal candidiasis.

This study proposes a microcosm biofilm (MiB) model for the study of vulvovaginal candidiasis (VVC). Different conditions that mimic the vaginal environment were tested for MiB formation. The best growth conditions were obtained with samples incubated in vaginal fluid simulator medium pH 4.5 at 35 °C under a microaerophilic atmosphere. MiBs were evaluated for growth kinetics, fluconazole susceptibility and morphology. Samples containing high numbers of bacteria were analyzed for metagenomics. At 48 h, MiBs presented a higher cell density (CFU ml-1), a higher biomass and tolerance to fluconazole than their corresponding monospecies biofilms. Morphological analysis of MiBs revealed blastoconidia preferentially adhered to epithelial cells. Abundant Lactobacillus spp. were detected in two clinical samples; their MiBs showed a lower biomass and a higher fluconazole susceptibility. The proposed model proved to be a useful tool for the study of the complex microbial relationship in the vaginal environment, and may help to find new strategies for VVC control.

Clonal Dissemination of Clinical Isolates of Acinetobacter baumannii Carriers of 16S rRNA Methylase Genes in an Oncological Hospital in Recife, Brazil.

16S rRNA methylases confer high-level resistance to aminoglycosides which are used to treat serious infections caused by gram-negative bacteria, such as Acinetobacter spp. Some genes encoding these enzymes are disseminated worldwide, while others were detected in only some countries. The objective was to characterize the susceptibility profile to aminoglycosides (amikacin and gentamicin) of clinical isolates of Acinetobacter spp. from an oncological hospital in Recife, and given the resistance to both antimicrobials, to characterize minimal inhibitory concentrations (MICs) of amikacin, gentamicin and tobramycin, the occurrence of 16S rRNA methylase genes (armA, rmtB, rmtC and rmtD) and of ß-lactamase gene (blaKPC) and the clonal profile. Isolates resistant to both antimicrobials, amikacin and gentamicin, were selected by disk diffusion technique in Mueller-Hinton agar and identified. Broth microdilution was conducted to determine MICs of amikacin, gentamicin, and tobramycin. These isolates were subjected to polymerase chain reaction and pulsed-field gel electrophoresis. Among 23 analyzed isolates, 12 (52.2%) were resistant to gentamicin and amikacin and identified as Acinetobacter baumannii. Among these, 11 (91.7%), 12 (100%), and 9 (75%) isolates showed respectively MICs > 256 µg/mL of amikacin, > 64 µg/mL of gentamicin, and > 64 µg/mL of tobramycin. The armA gene was found in 12 (100%) isolates and 6 (50%) showed coexistence of armA, rmtB, and rmtC genes. The rmtD and blaKPC genes were not detected. These isolates showed high genetic similarity (92%) and were classified as clone A. Elaboration and fulfillment of measures are thus essential to prevent the spread of this resistance mechanism.

High-Virulence Cat-Transmitted Ocular Sporotrichosis.

The article describes an atypical case of cat-transmitted ocular sporotrichosis due Sporothrix brasiliensis in a young patient. This report contributes significantly to knowledge of sporotrichosis as an important neglected tropical disease. In addition, this condition is an important public health problem in ascending incidence.

In vitro effects of promethazine on cell morphology and structure and mitochondrial activity of azole-resistant Candida tropicalis.

The aim of this study was to evaluate the effect of promethazine on the antifungal minimum inhibitory concentrations against planktonic cells and mature biofilms of Candida tropicalis, as well as investigate its potential mechanisms of cell damage against this yeast species. Three C. tropicalis isolates (two azole-resistant and one azole-susceptible) were evaluated for their planktonic and biofilm susceptibility to promethazine alone and in combination with itraconazole, fluconazole, voriconazole, amphotericin B, and caspofungin. The antifungal activity of promethazine against C. tropicalis was investigated by performing time-kill curve assays and assessing rhodamine 6G efflux, cell size/granularity, membrane integrity, and mitochondrial transmembrane potential, through flow cytometry. Promethazine showed antifungal activity against planktonic cells and biofilms at concentrations of 64 and 128 µg/ml, respectively. The addition of two subinhibitory concentrations of promethazine reduced the antifungal MICs for all tested azole drugs against planktonic growth, reversing the resistance phenotype to all azoles. Promethazine decreased the efflux of rhodamine 6G in an azole-resistant strain. Moreover, promethazine decreased cell size/granularity and caused membrane damage, and mitochondrial membrane depolarization. In conclusion, promethazine presented synergy with azole antifungals against resistant C. tropicalis and exhibited in vitro cytotoxicity against C. tropicalis, altering cell size/granularity, membrane integrity, and mitochondrial function, demonstrating potential mechanisms of cell damage against this yeast species.

Targeting the Cryptococcus neoformans var. grubii cell wall using lectins: study of the carbohydrate-binding domain.

Cryptococcus neoformans var. grubii is considered to be the major cause of cryptococcosis in immunosuppressed patients. Understanding cell wall glycoproteins using lectins is of medical interest and can contribute to specific therapy. The aim of this study was to evaluate the carbohydrates on the cell wall of Cryptococcus neoformans var. grubii clinical isolates, using a fluorescein isothiocyanate-lectin binding protocol. Thirty yeast strains stocked in the culture collection were cultivated for 2 days at 30 °C with shaking. Cells were obtained by centrifugation, washed in phosphate-buffered saline, and a suspension of 107 cells/mL was obtained. To determine the binding profile of lectins, concanavalin A (Con A), wheat germ agglutinin (WGA), Ulex europaeus agglutinin I (UEA-I), and peanut agglutinin (PNA) conjugated to fluorescein were used. All the tested clinical isolates of Cryptococcus neoformans var. grubii were intensely stained by WGA, moderately stained by Con A, and weakly stained by PNA and UEA-I. Thus, Cryptococcus can be detected in clinical specimens such as blood and cerebrospinal fluid using the fluorescent lectin WGA, which may be considered as an option for detection in cases of suspected cryptococcosis with low laboratory sensitivity. Future applications may be developed using this basic tool.

Assessing antimicrobial stewardship governance in Northeast Brazilian hospitals: a survey-based analysis.

Background: Effective governance of antimicrobial stewardship (AMS) and infection prevention control (IPC) in healthcare facilities is crucial for safeguarding patients against healthcare-associated infections and enhancing patient outcomes by optimizing antibiotic use and curbing the spread of antimicrobial-resistant (AMR) pathogens. Objectives: To assess the current AMS governance in two public hospitals in Northeast of Brazil, specifically focusing on identifying institutional antibiotic policies and operational practices. Methods: A survey was conducted by team leaders of both hospitals from 2020 to 2022 using a questionnaire adapted from the Pan American Health Organization (PAHO) recommendations for implementing AMS programmes (ASP) in Latin America and the Caribbean, alongside criteria from the National Health Surveillance Agency (ANVISA) and CDC. Results: Fifty leaders, from senior management to coordinators, answered the questionnaire. Results indicate a lack of AMS process measures, specialist support, systematic antimicrobial utilization analysis and structured IPC programmes, especially in one hospital where patient records remain in paper format. Conclusions: The empirical use of antimicrobials without local epidemiological or susceptibility data underscores the absence of logistical support for microbiological cultures in the region. These findings emphasize the urgent need for systematic AMS processes and multiprofessional teams to drive AMS and IPC practices, essential for patient care and safety.

Levels of soluble TNF receptors (sTNFR1 and sTNFR2) increase with clinical worsening of patients and are related to COVID-19 mortality.

The present study aimed to inspect the serum levels of the soluble receptors, sTNFR1 and sTNFR2, in patients with COVID-19. The large production of inflammatory cytokines is an essential process in the pathogenesis of COVID-19. TNF is a multifaceted proinflammatory cytokine which has soluble and membrane receptors. Thus, knowing the role of these receptors will help better understand this disease's immunopathogenesis. We included 131 patients confirmed for SARS-CoV-2, separated into three groups: ward patients without O2 support, group A (n = 14); ward patients with O2 support, group B (n = 85), and patients in an intensive care unit (ICU), group C (n = 32), making up the receptors dosed by flow cytometry. The results showed that sTNFR1 and sTNFR2 are associated with disease severity, being higher in group C when compared to group A. As for the levels of receptors and their relationship with the degree of lung involvement, we found higher values of sTNFR1 in patients in group 1 (pulmonary involvement < 25%), suggesting that inflammatory processes related to TNF are not necessarily associated with the primary site of infection. When we analysed the patients who passed away compared to those who recovered, both receptors significantly increased the mortality numbers. These findings suggest a relevant influence of soluble receptors in the inflammatory processes involved in the pathogenesis of COVID-19. Wherefore, we suggest using these receptors as biomarkers of severity and mortality of the disease.

Colonized patients by Candida auris: Third and largest outbreak in Brazil and impact of biofilm formation.

Objective: To describe the clinical-epidemiological features of patients colonized by Candida auris in the largest outbreak in Brazil and to show the biofilm formation capacity of yeast strains. Methods: Clinical yeasts suspected of C. auris isolated from urine and surveillance samples were seeded on chromogenic media at 30°C and Sabouraud agar at 42°C. matrix-assisted laser desorption/ionization-time of flight mass spectometry was used for reliable identification. After proteomic confirmation, the genomic approach and culture on Chromagar Candida Plus media were carried out. Biofilm formation was investigated based on metabolic activity, and the clinical-epidemiological profile of patients was described. Results: A total of 11 C. auris clinical yeasts from nine patients were identified between the end of December 2021 and March 2022. Two clinical yeasts were isolates from urine and nine clinical yeasts were isolates from axillary and inguinal surveillance swabs. No case is related to previous Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. However, all the yeasts showed a high ability of biofilm formation. Conclusion: C. auris requires great vigilance as its high capacity to colonize and form biofilms contributes to its dissemination. The rapid and precise identification of this species is essential for the management, control, and prevention of infections.

Clinical and Epidemiological Characteristics of Neurocryptococcosis Associated with HIV in Northeastern Brazil.

Cryptococcal meningitis is a serious infection of the central nervous system that is predominant in developing countries, caused by fungi of the genus Cryptococcus, and which affects immunosuppressed patients, especially those with HIV. Here, we aim to diagnose and characterize the clinical-epidemiological profile of cryptococcosis in patients admitted to two tertiary public hospitals in northeastern Brazil. The study is divided into three moments: (1) the isolation of fungus and diagnosis from biological samples collected between 2017 and 2019, (2) a description of the clinical and epidemiological characteristics of the patients, and (3) the experimental tests related to an in vitro susceptibility antifungal profile. The species were identified by MALDI-TOF/MS. Among the 100 patients evaluated, 24 (24.5%) were diagnosed with cryptococcosis based on positive culture. Clinical-epidemiological analysis showed a slightly higher prevalence in men between 30 and 39 years. When comparing the date of HIV diagnosis and the development of cryptococcosis, it was observed that 50% received the diagnosis of infection by cryptococcosis after or equal to a period of 12 months from being diagnosed with HIV; the other 50% received it within the first 30 days of the HIV diagnosis. Neurocryptococcosis was the most prevalent clinical form, and, at the time of hospital admission, the most common clinical signs were high fever (75%), intense headache (62.50%), and neck stiffness (33.33%). The cerebrospinal fluid showed 100% sensitivity and positivity for direct examination by India ink, and fungal culture. The mortality rate in this study was 46% (11/24), a lower rate than in the other literature. An antifungigram showed that 20 (83.33%) isolates were susceptible to amphotericin B and 15 (62.5%) to fluconazole. Mass spectrometry identified 100% of the isolates as Cryptococcus neoformans. In Brazil, this infection is not mandatory notifiable. Therefore, although there is little information on the subject, it is obsolete and does not express the reality of the facts, mainly in the northeast region, where this information is insufficient. The data obtained in this research contribute to the epidemiological knowledge of this mycosis in Brazil and will serve as a basis for future globally comparative epidemiological studies.

Efficacy and non-toxicity of ciclopirox olamine-loaded liposomes against Cryptococcus neoformans clinical isolates.

The aim of this study was to evaluate the efficacy and non-toxicity of ciclopirox olamine-loaded liposomes against Cryptococcus neoformans clinical isolates. Initially, 24-1 fractional experimental design was carried out to obtain an optimized formulation of liposomes containing CPO (CPO-LipoC), which were then used to prepare stealth liposomes (CPO-LipoS). Liposomal formulations were characterized by their mean size diameter, polydispersity index (PDI), and drug encapsulation efficiency (EE%). Immunosuppressed mice were exposed to CPO-LipoS at 0.5 mg/kg/day for 14 days to verify possible histopathological alterations in the liver and kidneys. Immunosuppressed mice infected with C. neoformans were treated with CPO-LipoS at 0.5 mg/kg/day for 14 days to quantify the fungal burden in spleen, liver, lungs, and brain. CPO-LipoS presented a mean size diameter, PDI, and EE% of 101.4 ± 0.7 nm, 0.307, and 96.4 ± 0.9%, respectively. CPO-LipoS was non-toxic for the liver and kidneys of immunosuppressed mice. At the survival curve, all infected animals submitted to treatment with CPO-LipoS survived until the end of the experiment. Treatment with CPO-LipoS reduced C. neoformans cells in the spleen (59.3 ± 3.4%), liver (75.0 ± 3.6%), lungs (75.7 ± 6.7%), and brain (54.2 ± 3.2%). CPO-LipoS exhibit antifungal activity against C. neoformans, and the encapsulation of CPO into stealth liposomes allows its use as a systemic drug for treating cryptococcosis.

Malassezia spp. and Candida spp. from patients with psoriasis exhibit reduced susceptibility to antifungals.

INTRODUCTION: Psoriasis is a chronic inflammatory disease that affects over 125 million people worldwide. Many studies have shown the importance of the microbiome for psoriasis exacerbation. AIM: Explore the fungal load and species composition of cultivable yeasts on the skin of psoriatic patients (PP) and healthy volunteers living in a tropical area and evaluate the susceptibility to antifungals. METHODOLOGY: A cross-sectional study with 61 participants (35 patients and 26 healthy controls) was performed during August 2018 and May 2019. Clinical data were collected from patient interviewing and/or medical records review. Samples were collected by swabbing in up to five anatomic sites. Suggestive yeast colonies were counted and further identified by phenotypical tests, PCR-REA, and/or MALDI-TOF. Susceptibility of Malassezia spp. and Candida spp. to azoles, terbinafine, and amphotericin B was evaluated by broth microdilution. RESULTS: Nearly 50% of the patients had moderate to severe psoriasis, and plaque-type psoriasis was the most common clinical form. Yeast colonies count was significantly more abundant among PP than healthy controls. Malassezia and Candida were the most abundant genus detected in all participants. Higher MIC values for ketoconazole and terbinafine were observed in Malassezia strains obtained from PP. Approximately 42% of Candida isolates from PP showed resistance to itraconazole in contrast to 12.5% of isolates from healthy controls. MIC values for fluconazole and amphotericin B were significantly different among Candida isolates from PP and healthy individuals. CONCLUSION: This study showed that Malassezia and Candida strains from PP presented higher MIC values to widespread antifungal drugs than healthy individuals.

Fungal esophagitis associated with tuberculous pericarditis in an human immunodeficiency virus-positive patient: a case report.

BACKGROUND: Opportunistic infections are frequent in people living with the human immunodeficiency virus who either do not have access to antiretroviral therapy (ART) or use it irregularly. Tuberculosis is the most frequent infectious disease in PLHIV and can predispose patients to severe fungal infections with dire consequences. CASE PRESENTATION: We describe the case of a 35-year-old Brazilian man living with human immunodeficiency virus (HIV) for 10 years. He reported no adherence to ART and a history of histoplasmosis with hospitalization for 1 month in a public hospital in Natal, Brazil. The diagnosis was disseminated Mycobacterium tuberculosis infection. He was transferred to the health service in Recife, Brazil, with a worsening condition characterized by daily fevers, dyspnea, pain in the upper and lower limbs, cough, dysphagia, and painful oral lesions suggestive of candidiasis. Lymphocytopenia and high viral loads were found. After screening for infections, the patient was diagnosed with tuberculous pericarditis and esophageal candidiasis caused by Candida tropicalis. The isolated yeasts were identified using the VITEK 2 automated system and matrix-assisted laser desorption/ionization time-of-flight-mass spectrometry. Antifungal microdilution broth tests showed sensitivity to fluconazole, voriconazole, anidulafungin, caspofungin, micafungin, and amphotericin B, with resistance to fluconazole and voriconazole. The patient was treated with COXCIP-4 and amphotericin deoxycholate. At 12 days after admission, the patient developed sepsis of a pulmonary focus with worsening of his respiratory status. Combined therapy with meropenem, vancomycin, and itraconazole was started, with fever recurrence, and he changed to ART and tuberculostatic therapy. The patient remained clinically stable and was discharged with clinical improvement after 30 days of hospitalization. CONCLUSION: Fungal infections should be considered in patients with acquired immunodeficiency syndrome as they contribute to worsening health status. When mycoses are diagnosed early and treated with the appropriate drugs, favorable therapeutic outcomes can be achieved.

Carbohydrate profiling of fungal cell wall surface glycoconjugates of Trichophyton tonsurans and other keratinophilic filamentous fungi using lectins.

Various researchers have concluded that lectins are useful reagents for the study of fungal cell wall surface glycoconjugates. In this study, we evaluated the expression of N-acetyl-D-glucosamine, L-fucose, D-galactose and glucose/mannose on the cell wall surface of Trichophyton tonsurans and other keratinophilic filamentous fungi, using a simple lectin-binding protocol. The fungal cultures used were isolated from soils obtained from public parks by the hair-bait technique. The lectin assays used concanavalin A (Con A), wheat germ agglutinin (WGA), Ulex europeus agglutinin I (UEA-I) and peanut agglutinin (PNA), all conjugated with horseradish peroxidase. Adhesive tape was placed sticky-side down over the fungal colony, gently pressed and then removed. The fungal-tape samples were incubated with the lectin for 1 h at 4 °C. Lectin binding was visualised using 3,3-diaminobendizine (DAB) and hydrogen peroxidase. There was a high expression of N-acetyl-D-glucosamine on the cell wall surface of all fungi species tested, whereas the expression of L-fucose, D-galactose and glucose/mannose demonstrated inter-specific variations. The lectin-binding assay presented in this article eliminates many of the laborious steps involved in other protocols. The amount and quality of the mycelium and spores immobilised by the adhesive tapes were suitable for obtaining the carbohydrate profile in glycoconjugates of the cell wall surface of filamentous fungi.