Combination Therapy Using Benznidazole and Aspirin during the Acute Phase of Experimental Chagas Disease Prevents Cardiovascular Dysfunction and Decreases Typical Cardiac Lesions in the Chronic Phase.

Chagas disease, caused by the protozoan Trypanosoma cruzi, is one of the main causes of death due to cardiomyopathy and heart failure in Latin American countries. The treatment of Chagas disease is directed at eliminating the parasite, decreasing the probability of cardiomyopathy and disrupting the disease transmission cycle. Benznidazole (BZ) and nifurtimox (Nfx) are recognized as effective drugs for the treatment of Chagas disease by the World Health Organization, but both have high toxicity and limited efficacy, especially in the chronic disease phase. At low doses, aspirin (ASA) has been reported to protect against T. cruzi infection. We evaluated the effectiveness of BZ in combination with ASA at low doses during the acute disease phase and evaluated cardiovascular aspects and cardiac lesions in the chronic phase. ASA treatment prevented the cardiovascular dysfunction (hypertension and tachycardia) and typical cardiac lesions. Moreover, BZ+ASA-treated mice had a smaller cardiac fibrotic area than BZ-treated mice. These results were associated with an increase in numbers of eosinophils and reticulocytes and levels of nitric oxide in the plasma and cardiac tissue of ASA-treated mice relative to respective controls. These effects of ASA and BZ+ASA in chronically infected mice were inhibited by pretreatment with the lipoxin A4 (LXA4) receptor antagonist Boc-2, indicating that the protective effects of ASA are mediated by ASA-triggered lipoxin. These results emphasize the importance of exploring new drug combinations for treatments of the acute phase of Chagas disease that are beneficial for patients with chronic disease.

Fatal sepsis caused by mecA-positive oxacillin-susceptible Staphylococcus aureus: First report in a tertiary hospital of southern Brazil.

mecA-positive oxacillin phenotypically susceptible Staphylococcus aureus (OS-MRSA) is increasingly reported worldwide. This bacterium poses a therapeutic threat, as it can be misidentified as an oxacillin-susceptible organism by phenotypic methods that are routinely used in the majority of clinical microbiology laboratories. Herein, we report the first case of fatal sepsis in a 43-year-old female patient caused by an OS-MRSA SCCmec type IVa/ST1/CC1 in a tertiary hospital in southern Brazil, which highlights the difficulties involved in diagnosing this bacterium. Blood cultures and phenotypic susceptibility tests on admission yielded a penicillin-resistant S. aureus. Although vancomycin therapy was initiated, this antibacterial was replaced by oxacillin, based on the susceptibility result. However, the clinical conditions of the patient deteriorated rapidly evolving to fatal septic shock. Clinical microbiology laboratories should consider the use of additional tests to accurately distinguish between various antimicrobial phenotypes of S. aureus.

Differences in cNOS/iNOS Activity during Resistance to Trypanosoma cruzi Infection in 5-Lipoxygenase Knockout Mice.

Infection with the protozoan Trypanosoma cruzi causes Chagas disease and consequently leads to severe inflammatory heart condition; however, the mechanisms driving this inflammatory response have not been completely elucidated. Nitric oxide (NO) is a key mediator of parasite killing in T. cruzi-infected mice, and previous studies have suggested that leukotrienes (LTs) essentially regulate the NO activity in the heart. We used infected 5-lipoxygenase-deficient mice (5-LO-/-) to explore the participation of nitric oxide synthase isoforms, inducible (iNOS) and constitutive (cNOS), in heart injury, cytokine profile, and oxidative stress during the early stage of T. cruzi infection. Our evidence suggests that the cNOS of the host is involved in the resistance of 5-LO-/- mice during T. cruzi infection. iNOS inhibition generated a remarkable increase in T. cruzi infection in the blood and heart of mice, whereas cNOS inhibition reduced cardiac parasitism (amastigote nests). Furthermore, this inhibition associates with a higher IFN-γ production and lower lipid peroxidation status. These data provide a better understanding about the influence of NO-interfering therapies for the inflammatory response toward T. cruzi infection.

Development of a melting-curve based multiplex real-time PCR assay for simultaneous detection of Streptococcus agalactiae and genes encoding resistance to macrolides and lincosamides.

BACKGROUND: Streptococcus agalactiae or Group B Streptococcus (GBS) remains the leading cause of infections in newborns worldwilde. Prenatal GBS screening of pregnant women for vaginal-rectal colonization is recommended in many countries to manage appropriate intrapartum antimicrobial prophylaxis for those identified as carriers. In this study, a novel melting-curve based multiplex real-time PCR assay for the simultaneous detection of GBS and macrolide and lincosamide resistance markers was developed. The usefulness of the assay was evaluated for rapid and accurate prenatal GBS screening. METHODS: One hundred two pregnant women who were at 35-37 weeks of gestation were enrolled in this study. The analytical performance of the multiplex real-time PCR was first tested using a panel of reference and clinical bacterial and fungal strains. To test the clinical performance, vaginal-rectal swabs were obtained from pregnant women who were seen at the teaching hospital for regular prenatal care. The results of real-time were compared with those obtained from microbiological analyses. RESULTS: The real-time PCR assay showed 100% specificity and a limit of detection of 104 colony forming units equivalent per reaction. The prevalence of GBS colonization among the population studied was 15.7% (16/102) based on a positive culture and the real-time PCR results. Agreement between the two assays was found for 11 (68.75%) GBS colonized women. Using the culture-based results as a reference, the multiplex real-time PCR had a sensitivity of 91.7% (11/12, CI 59.7-99.6%), a specificity of 95.5% (86/90, CI 89.8-98.7%), a positive predictive value of 73.3% (11/15, CI 44.8-91.1%) and a negative predictive value of 98.9% (86/87, CI 92.9-99.9%). CONCLUSION: The multiplex real-time PCR is a rapid, affordable and sensitive assay for direct detection of GBS in vaginal-rectal swabs.

Concanavalin-A displays leishmanicidal activity by inducing ROS production in human peripheral blood mononuclear cells.

The context of the article: Leishmania amazonensis has a wide geographical distribution throughout South American countries and can cause self-healing to severe cases as mucocutaneous or visceral forms. Leishmaniasis presents a balance of inflammatory and anti-inflammatory cytokines which is responsible for promoting the activation of phagocytes, essential to control the infection and lead to tissue repair/resolution of the disease, respectively. Results and discussion: Our model revealed that the treatment with Con-A was capable to stimulate human PBMC cells by increasing the phagocytic capacity and promoting parasite elimination. The pretreatment with Con-A promoted inflammatory (IFN-γ, TNF-α, IL-2 and IL-6) and anti-inflammatory (IL-4 and IL-10) cytokines production, increased the reactive oxygen species (ROS) sinthesys as well as the expression and presence of iNOS enzyme, but not nitric oxide production. Conclusion: Based on the data obtained, it was possible to infer that Con-A induces the ROS production, responsible for eliminating parasites in addition to regulatory cytokines synthesis which are important for disease resolution.

Trypanosoma cruzi: Inhibition of infection of human monocytes by aspirin.

Cell invasion by Trypanosoma cruzi and its intracellular replication are essential for progression of the parasite life cycle and development of Chagas disease. Prostaglandin E2 (PGE2) and other eicosanoids potently modulate host response and contribute to Chagas disease progression. In this study, we evaluated the effect of aspirin (ASA), a non-selective cyclooxygenase (COX) inhibitor on the T. cruzi invasion and its influence on nitric oxide and cytokine production in human monocytes. The pretreatment of monocytes with ASA or SQ 22536 (adenylate-cyclase inhibitor) induced a marked inhibition of T. cruzi infection. On the other hand, the treatment of monocytes with SQ 22536 after ASA restored the invasiveness of T. cruzi. This reestablishment was associated with a decrease in nitric oxide and PGE2 production, and also an increase of interleukin-10 and interleukin-12 by cells pre-treated with ASA. Altogether, these results reinforce the idea that the cyclooxygenase pathway plays a fundamental role in the process of parasite invasion in an in vitro model of T. cruzi infection.

Accurate and sensitive real-time PCR assays using intergenic spacer 1 region to differentiate Cryptococcus gattii sensu lato and Cryptococcus neoformans sensu lato.

In this work, two accurate and sensitive real-time polymerase chain reaction (PCR) assays to differentiate pathogenic Cryptococcus gattii sensu lato (s.l.) and C. neoformans sensu lato (s.l.) targeting the intergenic spacer 1 (IGS1) region from rDNA locus were developed. Specific primers were designed based on their IGS1 sequence analyses and the optimal real-time PCR assays showed that the dissociation curves generated two different melting peaks, at 82.8 and 84.2ºC for C. gattii s.l. and C. neoformans s.l., respectively. No amplifications were observed in the negative template control. The minimum limit of detection of both primers was 100 plasmid copies per reaction, and they were highly specific when tested with a range of fungal DNAs. Overall, the results showed that the designed primers completely differentiated C. gattii s.l. and C. neoformans s.l. from clinical and environmental sources with great accuracy when compared to phenotypic identification, with no cross-reactivity to other fungal DNA.

Combination of fluconazole with silver nanoparticles produced by Fusarium oxysporum improves antifungal effect against planktonic cells and biofilm of drug-resistant Candida albicans.

Silver nanoparticles (AgNPs) have been extensively studied because of their anti-microbial potential. Here, we evaluated the effect of biologically synthesized silver nanoparticles (AgNPbio) alone and in combination with fluconazole (FLC) against planktonic cells and biofilms of FLC-resistant Candida albicans AgNPbio exhibited a fungicidal effect, with a minimal inhibitory concentration (MIC) and fungicidal concentration ranging from 2.17 to 4.35 µg/ml. The combination of AgNPbio and FLC reduced the MIC of FLC around 16 to 64 times against planktonic cells of allC. albicans There was no significant inhibitory effect of AgNPbio on biofilm cells. However, FLC combined with AgNPbio caused a significant dose-dependent decrease in the viability of both initial and mature biofilm. All concentrations of AgNPbio, alone or in combination with FLC, were not cytotoxic to mammalian cells.The results highlight the effectiveness of the combination of AgNPbio with FLC against FLC-resistant C. albicans.

Aspirin treatment exacerbates oral infections by Trypanosoma cruzi.

Oral transmission of the protozoan parasite Trypanosoma cruzi, the etiological agent of Chagas disease, has been documented in Latin American countries. The reported cases of infection were due to the ingestion of contaminated fresh fruit, juices, or sugar cane juice. There have been few studies on the physiopathology of the disease in oral transmission cases. Gastritis is a common ailment that can be caused by poor dietary habits, intake of alcohol or other gastric irritants, bacterial infection, or by the widespread use of non-steroidal anti-inflammatory drugs (NSAIDs). This study investigated in a mouse model whether gastric mucosal injury, induced by aspirin, would affect the course of disease in animals infected with T. cruzi by the oral route. The CL14 and G strains of T. cruzi, both of low infectivity, were used. To this end, groups of BALB/c mice were treated during 5 days with aspirin (100 mg kg(-1)) before oral infection with T. cruzi metacyclic forms (4 × 10(5) or 5 × 10(7) parasites/mouse). Histological analysis and determination of nitric oxide and TNF-α were performed in gastric samples obtained 5 days after infection. Parasitemia was monitored from the thirteenth day after infection. The results indicate that aspirin treatment of mice injured their gastric mucosa and facilitated invasion by both CL14 and G strains of T. cruzi. Strain CL14 caused more severe infection compared to the G strain, as larger numbers of amastigote nests were found in the stomach and parasitemia levels were higher. Our study is novel in that it shows that gastric mucosal damage caused by aspirin, a commonly used NSAID, facilitates T. cruzi infection by the oral route.

Molecular Characterization of Trypanosoma cruzi Tc8.2 Gene Indicates Two Differential Locations for the Encoded Protein in Epimastigote and Trypomastigote Forms.

This report describes the molecular characterization of the Tc8.2 gene of Trypanosoma cruzi. Both the Tc8.2 gene and its encoded protein were analyzed by bioinformatics, while Northern blot and RT-PCR were used for the transcripts. Besides, immunolocalization of recombinant protein was done by immunofluorescence and electron microscopy. Analysis indicated the presence of a single copy of Tc8.2 in the T. cruzi genome and 2-different sized transcripts in epimastigotes/amastigotes and trypomastigotes. Immunoblotting showed 70 and 80 kDa polypeptides in epimastigotes and trypomastigotes, respectively, and a differential pattern of immunolocalization. Overall, the results suggest that Tc8.2 is differentially expressed during the T. cruzi life cycle.

Aspirin modulates innate inflammatory response and inhibits the entry of Trypanosoma cruzi in mouse peritoneal macrophages.

The intracellular protozoan parasite Trypanosoma cruzi causes Chagas disease, a serious disorder that affects millions of people in Latin America. Cell invasion by T. cruzi and its intracellular replication are essential to the parasite's life cycle and for the development of Chagas disease. Here, we present evidence suggesting the involvement of the host's cyclooxygenase (COX) enzyme during T. cruzi invasion. Pharmacological antagonist for COX-1, aspirin (ASA), caused marked inhibition of T. cruzi infection when peritoneal macrophages were pretreated with ASA for 30 min at 37°C before inoculation. This inhibition was associated with increased production of IL-1ß and nitric oxide (NO(∙)) by macrophages. The treatment of macrophages with either NOS inhibitors or prostaglandin E2 (PGE2) restored the invasive action of T. cruzi in macrophages previously treated with ASA. Lipoxin ALX-receptor antagonist Boc2 reversed the inhibitory effect of ASA on trypomastigote invasion. Our results indicate that PGE2, NO(∙), and lipoxins are involved in the regulation of anti-T. cruzi activity by macrophages, providing a better understanding of the role of prostaglandins in innate inflammatory response to T. cruzi infection as well as adding a new perspective to specific immune interventions.

Commensal Streptococcus agalactiae isolated from patients seen at University Hospital of Londrina, Paraná, Brazil: capsular types, genotyping, antimicrobial susceptibility and virulence determinants.

BACKGROUND: Streptococcus agalactiae or Group B Streptococci (GBS) have the ability to access various host sites, which reflects its adaptability to different environments during the course of infection. This adaptation is due to the expression of virulence factors that are involved with survival, invasion and bacterial persistence in the host. This study aimed to characterize GBS isolates from women of reproductive age seen at University Hospital of Londrina, according to capsular typing, genetic relatedness, antimicrobial susceptibility profile and occurrence of virulence determinants. RESULTS: A total of 83 GBS isolates were enrolled in this study. Capsular types Ia (42.2%), II (10.8%), III (14.5%) and V (30.1%) were identified in most GBS. One isolate each was classified as type IX and non-typeable.A total of 15 multiple locus variable number of tandem repeat analysis (MLVA) types were identified among the isolates, seven were singletons and eight were represented by more than four isolates. All isolates were susceptible to penicillin, ampicillin, cefepime, cefotaxime, chloramphenicol, levofloxacin and vancomycin. Resistance to erythromycin and clindamycin was observed in 19.3 and 13.3% of isolates, respectively. All isolates resistant to clindamycin were simultaneously resistant to erythromycin and were distributed in the capsular types III and V. One isolate showed the constitutive macrolide-lincosamide-streptogramin B (cMLS(B)) phenotype and ten showed the inducible MLS(B) (iMLS(B)) phenotype. The mechanism of resistance to erythromycin and clindamycin more prevalent among these isolates was mediated by the gene ermA, alone or in combination with the gene ermB. The isolates displaying resistance only to erythromycin belonged to capsular type Ia, and showed the M phenotype, which was mediated by the mefA/E gene. All isolates harbored the gene hylB and at least one pilus variant, PI-1, PI-2a or PI-2b. Although cylE was observed in all GBS, four isolates were classified as gamma-hemolytic and carotenoid pigment non-producers. CONCLUSIONS: Our results indicate the potential virulence of commensal GBS isolates, reinforcing the need for continued screening for this bacterium to prevent infections. The distribution of capsular and pili antigens, and MLVA profiles was also identified, which may contribute to the development of new strategies for the prevention and treatment of GBS infection.

Dermatological Manifestations in COVID-19: A Case Study of SARS-CoV-2 Infection in a Genetic Thrombophilic Patient with Mthfr Mutation.

The present case study describes the dermatological manifestations of COVID-19 in a patient with genetic thrombophilia (MTHFR-C677T mutation) and the identification of a SARS-CoV-2 variant of interest (VOI). A female patient, 47 years old, unvaccinated, with thrombophilia, was diagnosed with COVID-19. She presented with urticarial and maculopapular eruptions from the seventh day of symptoms, which progressed to multiple lesions with dark centers (D-dimer value > 1450 ng/mL). The dermatological manifestations disappeared after 30 days, corroborating the reduction in D-dimer levels. Viral genome sequencing revealed infection by the VOI Zeta (P.2). Antibody testing, performed 30 days after the onset of symptoms, detected only IgG. The virus neutralization test showed the highest neutralizing titer for a P.2 strain, validating the genotypic identification. Lesions were suggested to be due to infection in skin cells causing a direct cytopathic effect or release of pro-inflammatory cytokines triggering erythematous and urticarial eruptions. In addition, vascular complications are also proposed to be due to the MTHFR mutation and increased D-dimer values. This case report is an alert about COVID-19 in patients with pre-existing vascular diseases, especially in unvaccinated patients, by VOI.

The Antibacterial and Wound Healing Properties of Natural Products: A Review on Plant Species with Therapeutic Potential against Staphylococcus aureus Wound Infections.

Wounds of an acute or chronic etiology affect millions of people worldwide, with increasing prevalence every year. Microbial infections are one of the main causes that impair the wound healing process, and Staphylococcus aureus, a commensal member of the skin microbiota, is one of the main causative agents of wound infections. Crucially, a high proportion of these infections are caused by methicillin-resistant Staphylococcus aureus, which, in addition to ß-lactams, has acquired resistance to almost all the antibacterial agents used to treat it, limiting therapeutic options. Studies on the antimicrobial and healing activities of extracts, essential oils, or metabolites obtained from native plants have been reported in many countries that have a diverse flora and traditions with the use of medicinal plants for the treatment of wound infections. Due to their great chemical diversity, plants have proven to be promising sources of bioactive molecules for the discovery and development of new drugs or strategies for the treatment of wounds. This review highlights the main herbal preparations that have antimicrobial and healing activities with potential for the treatment of wound infections caused by Staphylococcus aureus.

High Prevalence of Panton-valentine Leukocidin-encoding Genes in Methicillin-resistant Staphylococcus aureus Isolated from Inpatients with Invasive Infections at a University Hospital in Southern Brazil.

BACKGROUND: Staphylococcus aureus is a major cause of a wide diversity of infections in humans, and the expression of Panton-Valentine Leukocidin (PVL) has been associated with severe clinical syndromes. OBJECTIVES: The present study aimed to investigate the prevalence of PVL-encoding genes in S. aureus isolated from clinical samples of inpatients with invasive infections in a teaching hospital in Southern Brazil. Furthermore, phenotypic and genotypic characteristics of bacterial isolates were analyzed. METHODS: A total of 98 S. aureus isolates recovered from different body sites were characterized according to their antimicrobial susceptibility profile, methicillin-resistance and SCCmec typing, genetic relatedness and occurrence of virulence-encoding genes, such as icaA, lukS-PV/lukF-PV, and tst. RESULTS: Sixty-eight (69.4%) isolates were classified as methicillin-resistant, and among them, four (5.9%) did not harbor the mecA gene. The mecA-harboring methicillin-resistant S. aureus (MRSA) isolates were grouped into SCCmec types I (6.3%), II (64.1%), III (6.3%), IV (15.6%), V (4.7%), and VI (1.6%). One isolate (1.6%) was classified as non-typeable (NT). Seventy isolates (71.4%) were classified as multidrug-resistant. The overall prevalence of virulence-encoding genes was as follows: icaA, 99.0%; tst, 27.5%; and lukS-PV/lukF-PV, 50.0%. The presence of tst gene was significantly higher (p < 0.001) in methicillin-susceptible S. aureus (MSSA) compared to MRSA isolates. CONCLUSION: The present study reports a high prevalence of multidrug-resistant S. aureus harboring lukS-PV/lukF-PV and tst genes in invasive infections. The continuous monitoring of the antimicrobial susceptibility profile and virulence of S. aureus is an important measure for the control of infections caused by this bacterium.

The Therapeutic Potential of Angeli's Salt in Mitigating Acute Trypanosoma cruzi Infection in Mice.

Chagas disease (CD), caused by Trypanosoma cruzi, is a neglected tropical disease prevalent in Latin America. Infected patients are treated to eliminate the parasite, reduce the cardiomyopathy risk, and interrupt the disease transmission cycle. The World Health Organization recognizes benznidazole (BZ) and nifurtimox as effective drugs for CD treatment. In the chronic phase, both drugs have low cure rates and serious side effects. T. cruzi infection causes intense tissue inflammation that controls parasite proliferation and CD evolution. Compounds that liberate nitric oxide (NO) (NO donors) have been used as anti-T. cruzi therapeutics. Currently, there is no evidence that nitroxyl (HNO) affects T. cruzi infection outcomes. This study investigated the effects of the HNO donor Angeli's salt (AS) on C57BL/6 mice infected with T. cruzi (Y strain, 5 × 103 trypomastigotes, intraperitoneally). AS reduced the number of parasites in the bloodstream and heart nests and increased the protective antioxidant capacity of erythrocytes in infected animals, reducing disease severity. Furthermore, in vitro experiments showed that AS treatment reduced parasite uptake and trypomastigote release by macrophages. Taken together, these findings from the murine model and in vitro testing suggest that AS could be a promising therapy for CD.

Synergistic Antifungal Interaction between Pseudomonas aeruginosa LV Strain Metabolites and Biogenic Silver Nanoparticles against Candida auris.

Candida auris has been found to be a persistent colonizer of human skin and a successful pathogen capable of causing potentially fatal infection, especially in immunocompromised individuals. This fungal species is usually resistant to most antifungal agents and has the ability to form biofilms on different surfaces, representing a significant therapeutic challenge. Herein, the effect of metabolites of Pseudomonas aeruginosa LV strain, alone and combined with biologically synthesized silver nanoparticles (bioAgNP), was evaluated in planktonic and sessile (biofilm) cells of C. auris. First, the minimal inhibitory and fungicidal concentration values of 3.12 and 6.25 µg/mL, respectively, were determined for F4a, a semi-purified bacterial fraction. Fluopsin C and indolin-3-one seem to be the active components of F4a. Like the semi-purified fraction, they showed a time- and dose-dependent fungicidal activity. F4a and bioAgNP caused severe changes in the morphology and ultrastructure of fungal cells. F4a and indolin-3-one combined with bioAgNP exhibited synergistic fungicidal activity against planktonic cells. F4a, alone or combined with bioAgNP, also caused a significant decrease in the number of viable cells within the biofilms. No cytotoxicity to mammalian cells was detected for bacterial metabolites combined with bioAgNP at synergistic concentrations that presented antifungal activity. These results indicate the potential of F4a combined with bioAgNP as a new strategy for controlling C. auris infections.

Antiprotozoal Activity of Benzoylthiourea Derivatives against Trypanosoma cruzi: Insights into Mechanism of Action.

For decades, only two nitroheterocyclic drugs have been used as therapeutic agents for Chagas disease. However, these drugs present limited effectiveness during the chronic phase, possess unfavorable pharmacokinetic properties, and induce severe adverse effects, resulting in low treatment adherence. A previous study reported that N-(cyclohexylcarbamothioyl) benzamide (BTU-1), N-(tert-butylcarbamothioyl) benzamide (BTU-2), and (4-bromo-N-(3-nitrophenyl) carbamothioyl benzamide (BTU-3) present selective antiprotozoal activity against all developmental forms of Trypanosoma cruzi Y strain. In this study, we investigated the mechanism of action of these compounds through microscopy and biochemical analyses. Transmission electron microscopy analysis showed nuclear disorganization, changes in the plasma membrane with the appearance of blebs and extracellular arrangements, intense vacuolization, mitochondrial swelling, and formation of myelin-like structures. Biochemical results showed changes in the mitochondrial membrane potential, reactive oxygen species content, lipid peroxidation, and plasma membrane fluidity. In addition, the formation of autophagic vacuoles was observed. These findings indicate that BTU-1, BTU-2, and BTU-3 induced profound morphological, ultrastructural, and biochemical alterations in epimastigote forms, triggering an autophagic-dependent cell death pathway.

Synergistic antifungal interaction of N-(butylcarbamothioyl) benzamide and amphotericin B against Cryptococcus neoformans.

Introduction: Cryptococcus neoformans is one of the leading causes of invasive fungal infections worldwide. Cryptococcal meningoencephalitis is the main challenge of antifungal therapy due to high morbidity and mortality rates, especially in low- and middle-income countries. This can be partly attributed to the lack of specific diagnosis difficulty accessing treatment, antifungal resistance and antifungal toxicity. Methods: In the present study, the effect of the synthetic thiourea derivative N-(butylcarbamothioyl) benzamide (BTU-01), alone and combined with amphotericin B (AmB), was evaluated in planktonic and sessile (biofilm) cells of C. neoformans. Results: BTU-01 alone exhibited a fungistatic activity with minimal inhibitory concentrations (MICs) ranging from 31.25 to 62.5 µg/mL for planktonic cells; and sessile MICs ranging from 125.0 to 1000.0 µg/mL. BTU-01 caused a concentration-dependent inhibitory activity on cryptococcal urease and did not interfere with plasma membrane fluidity. Molecular docking was performed on Canavalia ensiformis urease, and BTU-01 showed relevant interactions with the enzyme. The combination of BTU-01 and AmB exhibited synergistic fungicidal activity against planktonic and sessile cells of C. neoformans. Microscopic analysis of C. neoformans treated with BTU-01, alone or combined with AmB, revealed a reduction in cell and capsule sizes, changes in the morphology of planktonic cells; a significant decrease in the number of cells within the biofilm; and absence of exopolymeric matrix surrounding the sessile cells. Neither hemolytic activity nor cytotoxicity to mammalian cells was detected for BTU-01, alone or combined with AmB, at concentrations that exhibited antifungal activity. BTU-01 also displayed drug-likeness properties. Conclusion: These results indicate the potential of BTU-01, for the development of new strategies for controlling C. neoformans infections.

Development of a Melting-Curve-Based Multiplex Real-Time PCR Assay for the Simultaneous Detection of Viruses Causing Respiratory Infection.

The prompt and accurate identification of the etiological agents of viral respiratory infections is a critical measure in mitigating outbreaks. In this study, we developed and clinically evaluated a novel melting-curve-based multiplex real-time PCR (M-m-qPCR) assay targeting the RNA-dependent RNA polymerase (RdRp) and nucleocapsid phosphoprotein N of SARS-CoV-2, the Matrix protein 2 of the Influenza A virus, the RdRp domain of the L protein from the Human Respiratory Syncytial Virus, and the polyprotein from Rhinovirus B genes. The analytical performance of the M-m-qPCR underwent assessment using in silico analysis and a panel of reference and clinical strains, encompassing viral, bacterial, and fungal pathogens, exhibiting 100% specificity. Moreover, the assay showed a detection limit of 10 copies per reaction for all targeted pathogens using the positive controls. To validate its applicability, the assay was further tested in simulated nasal fluid spiked with the viruses mentioned above, followed by validation on nasopharyngeal swabs collected from 811 individuals. Among them, 13.4% (109/811) tested positive for SARS-CoV-2, and 1.1% (9/811) tested positive for Influenza A. Notably, these results showed 100% concordance with those obtained using a commercial kit. Therefore, the M-m-qPCR exhibits great potential for the routine screening of these respiratory viral pathogens.