Etiologic Agent Research Articles

First report of Colletotrichum nymphaeae causing anthracnose on Hass avocados in Brazil.

The Hass cultivar is one of the most cultivated and distributed avocado varieties throughout the world, having high productivity, nutritional quality, market acceptance and adaptation to different climatic zones (Dreher and Davenport 2013). Anthracnose affects avocado production in tropical and subtropical regions, causing economic losses, especially post-harvest (Fuentes-Aragón et al. 2020). Correct identification of Colletotrichum species causing anthracnose is essential, as different species vary in important phenotypes such as virulence and sensitivity to fungicides (Chen et al. 2016). Twelve samples from batches of avocados with anthracnose were collected in Minas Gerais State, Brazil, in September 2023. The observed symptoms were brown to black depressed circular spots, ranging from a few millimeters to 3 cm in diameter on the epicarp of the fruits, covered in center by mucilaginous layers of pathogen sporulation. Isolation was performed directly from the spore masses and monoconidial isolates were cultured in PDA at 25°C for 7 days for morphological characterization and preserved in sterile water at 4°C. One of the morphotypes commonly recovered from lesions, represented by isolate UCBV 362 (Culture Collection COAD 3843), formed fast-growing colonies having white aerial mycelium and intense salmon-colored sporulation. The cylindrical conidia were 13 to 17.5 μm long and 4.5 to 7 μm wide (average 14.5 x 5.7 μm, N=100), produced on conidiophores dispersed in the aerial mycelium or aggregated on melanized conidiomata formed on the agar. The partial sequence of the second largest subunit of the RNA polymerase II gene (RPB2) from isolate UCBV 362 (GenBank: PQ034617, 1116 nt) showed 99% of coverage and 99.37% of nucleotide identity with the RPB2 sequence of the ex-epitype strain of Colletotrichum nymphaeae ICMP 17918 (=CBS 515.78) (GenBank: JN985506). In a Maximum Likelihood phylogenetic tree composed with RBP2 sequences from reference strains of the Colletotrichum acutatum species complex, the isolate UCBV 362 formed a highly supported clade with the ex-epitype and other reference strains of Colletotrichum nymphaeae, occupying the Clade 2 of the species complex together with C. scovillae and C. simmondsii (Damm et al. 2012). This result shows the reliability of RPB2 for phylogeny and species delimitation within Colletotrichum. To confirm pathogenicity, 10-mm discs from a 7-day-old colony were inoculated at 3 different points on healthy-looking avocado fruits and incubated at 28°C. Uninoculated fruits served as controls. The first symptoms appeared 5 days after inoculation and were similar to those observed in the original samples, while the fruits in the control group remained asymptomatic. The pathogen was reisolated from the lesions and identified morphologically, fulfilling Koch's postulates. Colletotrichum nymphaeae has been associated with avocado anthracnose in Mexico (Fuentes-Aragón et al. 2020). In Brazil, a study based on molecular phylogeny identified Colletotrichum siamense and C. karsti as etiological agents of this disease (Soares et al. 2021). This report expands the range of species that cause avocado anthracnose in Brazil and provides etiological information for the implementation and monitoring of control methods, especially chemical control.

Etiological Agents of Superficial Surgical Site Infections among Post Operative Patients Attending a Teaching Hospital in Southeastern Nigeria

Surgical site infections (SSI) are infections at or around the site of incision where surgical procedure is conducted. It is a major cause of hospital morbidity, resulting in prolonged hospital stay, increased treatment costs, higher rates of re-operation, and even increased mortality rates. The etiological agents of superficial surgical site infections among post-operative patients attending a Teaching Hospital in Southeastern Nigeria were evaluated using standard microbiological methods. Seventy superficial surgical wound swab specimens were properly and aseptically obtained from post-operative patients after ethical clearance was duly obtained from the hospital’s ethical committee. Pure microbial cultures were obtained after 24 - 48 hours incubation, aerobically at 25oC, on blood, nutrient and Sabouraud’ Dextrose agar plates. The isolates were identified based on their morphological, physiological, biochemical and molecular characteristics. Out of the 70 surgical swab specimens collected, 81% were bacterial isolates which included Staphylococcus aureus 41%, Escherichia coli 16%, Pseudomonas aeruginosa 14% and Staphylococcus xylosus 10%. The fungi isolates (19%) were Candida tropicalis 8% and Trichosporon asahii 11%. These isolates were probably endogenous and exogenous flora from the patients’ and hospital’ environments respectively; thereby necessitating the need for adequate patient care before, during and after surgical procedures.

Gut bacteria: an etiological agent in human pathological conditions.

Through complex interactions with the host's immune and physiological systems, gut bacteria play a critical role as etiological agents in a variety of human diseases, having an impact that extends beyond their mere presence and affects the onset, progression, and severity of the disease. Gaining a comprehensive understanding of these microbial interactions is crucial to improving our understanding of disease pathogenesis and creating tailored treatment methods. Correcting microbial imbalances may open new avenues for disease prevention and treatment approaches, according to preliminary data. The gut microbiota exerts an integral part in the pathogenesis of numerous health conditions, including metabolic, neurological, renal, cardiovascular, and gastrointestinal problems as well as COVID-19, according to recent studies. The crucial significance of the microbiome in disease pathogenesis is highlighted by this role, which is comparable to that of hereditary variables. This review investigates the etiological contributions of the gut microbiome to human diseases, its interactions with the host, and the development of prospective therapeutic approaches. To fully harness the benefits of gut microbiome dynamics for improving human health, future research should address existing methodological challenges and deepen our knowledge of microbial interactions.

Distribuição espacial e características clínico epidemiológicas dos casos de síndrome respiratória aguda grave por covid-19 no Brasil: estudo das duas primeiras ondas

The new coronavirus (SARS-CoV-2), the COVID-19 etiological agent, has been established as a challenge for health systems and can present as Flu Syndrome (FS) or Severe Acute Respiratory Syndrome (SARS). This work was aimed at describing the clinical epidemiological profile of SARS cases due to COVID-19 and the spatial distribution of the incidence among the Federative Units of Brazil (FU) in the first two waves. This is a descriptive and ecological study, with the FU as settings. The data collected are related to SARS cases due to COVID-19, with a time frame between March 2020 and June 2021. The sociodemographic and clinical-epidemiological variables were selected with their respective absolute and relative values. The spatial distribution of incidence was determined to identify the presence of spatial autocorrelation in the first two waves. A total of 613,324 SARS cases due to COVID-19 were reported, most were related to males, aged>60 years, typified as non-nosocomial and with a healing outcome; signs and symptoms were: cough,fever, dyspnea, and at least presence of one comorbidity. The average incidence in the countrywas 286.85 cases/100,000 inhabitants, while the Moran Index showed statistical significance I=0.329 (p value<0.001), with global spatial autocorrelation of the incidence in the second wave. The distribution was heterogeneous. These results constitute subsidies for management in health systems and services regarding the strategic implementation for the control and clinical management of this disease.

Respiratory Isolation for Tuberculosis: A Historical Perspective.

Although tuberculosis is an ancient disease, recognition of its airborne route of transmission, with implications for respiratory isolation, is only relatively recent. Since the time of Hippocrates, the dogma among health practitioners was that the disease was hereditary or that it could be contracted by inhaling "miasma", or corrupted air. Consequently, isolation of patients was not routine practice, and, in fact, patients with scrofula (morbus regius, or "king's evil) sought to be cured by the "royal touch" throughout the middle ages. The sanatorium, which emerged in the mid-19th century, initially served as a place of healing, where patients could receive the appropriate diet, rest therapy, graduated exercise, and abundant fresh air. Major scientific breakthroughs, including Robert Koch's 1882 discovery of the tubercle bacillus as the disease's etiological agent and early 20th century experimental evidence that the organism could be transmitted via expectorated droplet nuclei, helped to reinforce the important public health role of sanatoria and tuberculosis hospitals in preventing disease transmission through isolation. The advent of highly efficacious and oral antitubercular regimens in the mid-20th century and the concurrent declining incidence of the disease contributed to the closure of tuberculosis sanatoria and hospitals in the US and western Europe. Over the past several decades, tuberculosis treatment in the US has been conducted in the outpatient setting under the supervision of local public health departments. Patients receiving treatment are required to remain in respiratory isolation in the home until they are deemed noninfectious based on multiple sputum samples. This historical review demonstrates that despite changing medical knowledge, drug therapies, and social conditions over time, the role of isolation remains an important topic of debate in the treatment of patients with pulmonary tuberculosis.

Commercial bacteriophage preparations for the control of Listeria monocytogenes and Shiga toxin-producing Escherichia coli in raw and pasteurized milk

Listeria monocytogenes was the etiologic agent in nearly all recent outbreaks in North America attributed to pasteurized dairy products, whereas Escherichia coli O157 infections were responsible for most of the rare, yet serious complications from outbreaks involving unpasteurized dairy. This study determined the susceptibility of selected strains of L. monocytogenes and Shiga toxin-producing E. coli (STEC) to commercial phage preparations and their ability to control these pathogens in pasteurized and raw milk during 7-day storage at 7 °C. Both phage products demonstrated high lytic efficiency against 17 strains of L. monocytogenes whereas the efficiency of E. coli phages was more variable against 11 strains of O157 and non-O157 STEC. Broth microdilution assays identified effective endpoint multiplicities of infection (MOI) ranging from log 2.53 to 5.13, which differed between strains of L. monocytogenes and phage products. Mean log MOIs of E. coli phages against STEC also varied within and between products from 0.43 to 7.05. Despite these observations, the change in counts over time of three L. monocytogenes strains exposed to phage in pasteurized milk (log MOI 6) was similar with counts ∼ 4 log CFU/mL lower than control at day 7. Results for STEC O157 varied by strain but counts were lower than control in all cases by 72 h thorough day 7. Titers on isolates of both pathogens isolated from pasteurized milk indicated that the surviving populations were less susceptible to phage. The addition of a phage preparation to raw milk did not reduce populations of either pathogen or affect the change in counts of any strain over time when compared to control. Reduced efficacy in raw milk may be attributed to reduced phage binding as titers in raw milk decreased steadily (∼2 log PFU/mL) during storage. Commercial phage products may be a promising pathogen control intervention for pasteurized dairy products, warranting further investigation.

Tylvalosin tartrate inhibits the replication stage of the porcine reproductive and respiratory syndrome virus life cycle

The aetiological agent of porcine reproductive and respiratory syndrome, a deadly disease that affects pigs and seriously jeopardises the global swine industry, is a porcine reproductive and respiratory syndrome virus (PRRSV). Tylvalosin tartrate, which is a macrolide antibiotic, is the active ingredient in Aivlosin. In recent years, tylvalosin tartrate has widely been used to control porcine reproductive and respiratory syndrome in swine herds in China. However, whether tylvalosin tartrate has exerts anti-PRRSV effects remains controversial. In the present study, tylvalosin tartrate exhibited no effect on PRRSV susceptibility but suppressed the replication of PRRSV and the activity of infecting Marc-145 cells. Next, the relationship between the replication cycle of PRRSV and the activity of tylvalosin tartrate was further assessed. Tylvalosin tartrate did not affect the attachment and release stages of PRRSV or act during the internalisation stage of the virus in HuN4; however, contrasting effects were noted for strains CH-1a and SDVD-HN21. Tylvalosin tartrate acted on the replication stage of PRRSV and was not strain-specific in the replication stage of the PRRSV life cycle. The study findings provide an initial clarification of the inhibitory effects of tylvalosin tartrate on PRRSV, providing new insights into the treatment of PRRS.

Microsporum audouinii: Emergence of an etiological agent of tinea capitis in Rio de Janeiro, Brazil (2012-2019).

The etiology of tinea capitis changes over time, mainly due to trends in migration. We report 19 cases of tinea capitis caused by Microsporum audouinii, an uncommon agent in South America, all of them confirmed by molecular methods. All patients were male. The average age was 6.1 years. Fifteen patients were residents of Rio de Janeiro city and four were from neighboring cities. Among the patients submitted to follow-up, griseofulvin was prescribed for eight of them. Due to medication shortages, terbinafine was prescribed for five patients, needing to be switched in three cases, with a bigger total average time until clinical improvement. The study reaffirms the emergence of a new etiological agent in Rio de Janeiro, Brazil.

Site-specific sulfations regulate the physicochemical properties of papillomavirus-heparan sulfate interactions for entry.

Certain human papillomaviruses (HPVs) are etiological agents for several anogenital and oropharyngeal cancers. During initial infection, HPV16, the most prevalent cancer-causing type, specifically interacts with heparan sulfates (HSs), not only enabling initial cell attachment but also triggering a crucial conformational change in viral capsids termed structural activation. It is unknown, whether these HPV16-HS interactions depend on HS sulfation patterns. Thus, we probed potential roles of HS sulfations using cell-based functional and physicochemical assays, including single-molecule force spectroscopy. Our results demonstrate that N-sulfation of HS is crucial for virus binding and structural activation by providing high-affinity sites, and that additional 6O-sulfation is required to mechanically stabilize the interaction, whereas 2O-sulfation and 3O-sulfation are mostly dispensable. Together, our findings identify the contribution of HS sulfation patterns to HPV16 binding and structural activation and reveal how distinct sulfation groups of HS synergize to facilitate HPV16 entry, which, in turn, likely influences the tropism of HPVs.

Evaluation of the Safety and Immunogenicity of a Multiple Epitope Polypeptide from Canine Distemper Virus (CDV) in Mice.

Morbillivirus canis is the etiological agent of a highly contagious disease that affects diverse domestic and wild animals. Vaccination is considered the most suitable strategy for controlling CDV dissemination, transmission, and distemper disease. However, the emergence of new CDV strains has led to the need to update the current vaccine strategies employed to prevent CDV infection in domestic and wild animals. Currently, there is a lack of effective alternatives for wild animals. Diverse computational tools, especially peptide-based therapies, enable the development of new universal vaccines. The aim of this study was to evaluate the safety and humoral and cellular immune response of a new generation of vaccines based on CDV peptides as single-peptide mixtures or multiepitope CDV polypeptides in mice. Twenty-four BALB/c mice were subjected to a three-dose regimen for 28 days. Seroconversion was evaluated via ELISA, and cellular immune responses were evaluated via flow cytometry through activation-induced markers (AIMs). Compared with the placebo, the peptide mixture and multiepitope CDV polypeptide were safe, and seroconversion was statistically significant in the multiepitope CDV polypeptide and commercial vaccine (CV) groups. The numbers of antigen-specific CD4+CD134+ and IFN-γ+ T cells, CD8+ T cells and TNF-α- and IL-6-producing cells were greater in the mice immunized with the multiepitope CDV polypeptide than in the control mice. This combined approach represents a potential step forward in developing new immunization candidates or enhancing current commercial vaccines to control CDV disease in domestic dogs and wild animals.

Spatio-temporal distribution and international context of bovine viral diarrhoea virus genetic diversity in France

Bovine viral diarrhoea (BVD) is one of the most economically damaging livestock enzootic diseases in the world. BVD aetiological agents are three pestiviruses (BVDV-1, -2 and HoBi-like pestivirus), which exhibit high genetic diversity and complex transmission cycles. This considerably hampers the management of the disease, which is why eradication plans have been implemented in several countries. In France, a national plan has been in place since 2019. Our understanding of its impact on the distribution of BVDV genotypes is limited by the availability of French genetic data. Here, we conducted a molecular epidemiology study to refine our knowledge of BVDV genetic diversity in France, characterise its international relationships, and analyse national spatio-temporal genotypic distribution. We collated 1037 BVDV-positive samples throughout France between 2011 and 2023, with a greater sampling effort in two major cattle production areas. We developed a high-throughput sequencing protocol which we used to complete the 5’UTR genotyping of this collection. We show that two main BVDV-1 genotypes, 1e and 1b, account for 88% of genotyped sequences. We also identified seven other BVDV-1 genotypes occurring at low frequencies and three BVDV-2 samples (genotype 2c). Phylogenetic analyses indicate different worldwide distribution patterns between the two main BVDV-1 genotypes. Their relative frequencies present no major changes in France since the 1990s and few variations at the national scale. We also found some degree of local spatial structuring in western France. Overall, our results demonstrate the potential of large-scale sequence-based surveillance to monitor changes in the epidemiological situation of enzootic diseases.

Fusarium as potential pathogenic fungus of Ginger (Zingiber officinale Roscoe) wilt disease

The wilt disease of ginger, caused by various Fusarium species, imperils the cultivation of this valuable crop. However, the pathogenic mechanisms and epidemiology of ginger wilt remain elusive. Here, we investigate the association between ginger rhizome health and the prevalence of Fusarium conidia, as well as examine fungal community composition in symptomatic and asymptomatic ginger tissues. Our findings show that diseased rhizomes have reduced tissue firmness, correlating negatively with Fusarium conidia counts. Pathogenicity assays confirmed that both Fusarium oxysporum and Fusarium solani are capable of inducing wilt symptoms in rhizomes and sterile seedlings. Furthermore, Fungal community profiling revealed Fusarium to be the dominant taxon across all samples, yet its relative abundance was significantly different between symptomatic and asymptomatic tissues. Specifically, there is a higher incidence of Fusarium amplicon sequence variants (ASVs) in symptomatic above-ground parts. Our results unequivocally implicate F. oxysporum or F. solani as the etiological agents responsible for ginger wilt and demonstrate that Fusarium is the principal fungal pathogen associated with this disease. These findings provide critical insights for efficacious disease management practices within the ginger industry.

Avian malaria in a feral-pet pigeon: a case report

BackgroundAvian malaria is caused by diverse parasite species of the genus Plasmodium, and it affects various bird species. The occurrence of this disease in some wild bird species is sparsely documented due to the scarce availability of samples. Hence the pathogenicity in some hosts is not completely known. In addition, feral birds may act as reservoirs bridging the transmission cycle from wild migratory birds to domestic and zoo-kept bird species.Case presentationAn owner of pigeons adopted a feral pigeon (Columba livia forma domestica) and housed it together with his other pet-pigeons. The bird died unexpectedly a few weeks after a surgical procedure and necropsy revealed a severely anaemic carcass, with pale organs and hydropericardium. Histopathologic analysis revealed inflammatory infiltrates in the lung and liver, and monocytes and Kupffer cells contained haemozoin pigment indicative of phagocytosis of Plasmodium-infected erythrocytes. A high erythrocytic infection rate of 18% was evident in tissues and blood vessels in various organs. Furthermore, the thyroid had masses classified as thyroid carcinomas. Immunohistochemistry with anti- Plasmodium falciparum HSP70 antibody revealed positive signals in erythrocytes and intravascular leucocytes. Further microscopy analysis using a Hemacolor-stained impression smear revealed a high parasitaemia with an asynchronous infection showing all erythrocytic stages. Molecular diagnosis by PCR identified Plasmodium relictum, lineage GRW11 as the aetiological agent. The bird presented died most likely due to an acute infection as evidenced by the high blood parasitaemia, leading to major erythrocyte destruction. Further analyses of feral pigeons (n = 22) did not reveal any additional cases of Plasmodium infections.ConclusionThis study reports the first mortality associated with P. relictum lineage GRW11. The study supports previous studies, suggesting that Plasmodium infections are not frequent in pigeons. Host conditions like immunosuppression due to the tumour may have influenced the infection outcome in this fatal case. Use of anti-P. falciparum HSP70 antibody for detection of P. relictum antigens for immune assays in blood and tissue samples will be a useful tool for future studies.

Ergosterol Biosynthesis and Regulation Impact the Antifungal Resistance and Virulence of Candida spp.

Ergosterol is a key fungal sterol that is mainly found in the plasma membrane and is responsible for the proper membrane structure, rigidity, permeability and activity of membrane proteins. Ergosterol plays a crucial role in the ability of fungi to adapt to environmental stresses. The biosynthesis of ergosterol is also intimately connected with the antifungal resistance and virulence of pathogenic fungi. The most common etiological agents of life-threatening fungal infections are yeasts belonging to the genus Candida. The antifungal agents mostly used to treat Candida spp. infections are azoles, which act as competitive inhibitors of sterol demethylase, a key enzyme in the fungal ergosterol biosynthetic pathway. Although most studies on ergosterol biosynthesis, its regulation and the uptake of sterols are from the baker’s yeast Saccharomyces cerevisiae, the study of ergosterol biosynthesis and its relationship to antifungal drug resistance and virulence in pathogenic fungi is of utmost importance. The increasing antifungal drug resistance of Candida spp. and the limited armamentarium of antimycotics pose a challenge in the development of new therapeutic approaches. This review summarizes the available data on ergosterol biosynthesis and related phenomena in Candida albicans and non-albicans Candida species (Candida glabrata, Candida parapsilosis, Candida tropicalis and Candida auris) with special emphasis on C. albicans and C. glabrata as the most common etiological agents of systemic candidiasis.

Fungal endometritis associated with embryonic loss in a maiden mare

SummaryFungal endometritis and placentitis have been reported as a rare but important cause of infertility in the mare. There are no reported clinical cases of fungal endometritis causing embryonic loss in the mare. This case report aims to highlight the potential of fungal endometritis as an aetiological agent in embryonic loss in the mare; an incidental diagnosis of a case of fungal endometritis and embryonic loss while undertaking routine pregnancy assessments in a Standardbred mare. After a positive pregnancy diagnosis after 13 days post‐ovulation, a mare was diagnosed with endometritis and pregnancy loss associated with the growth of an unusual fungus, likely of the Acremonium spp., 28 days post‐ovulation. While fungal embryonic loss is rare, it should be considered a potential reason for pregnancy wastage in the mare.

Prevalence, aetiology and factors associated with onychomycosis among patients with diabetes at a Tertiary Care Centre in Galle, Sri Lanka

Introduction: Epidemiological data of onychomycosis, a common fungal nail infection found in diabetics, is scarce in Sri Lanka. Objective: To determine the prevalence, aetiological agents and associated factors of onychomycosis among patients with clinically suspected diabetics in southern Sri Lanka. Method: A cross-sectional study was done using a sample of 97 diabetic patients suspected of having onychomycosis in a tertiary diabetes care centre in Galle. Nail clippings were collected, and microscopic and cultural examinations were done. Both microscopic and culture positive cases were considered as having onychomycosis. Results: The prevalence of onychomycosis was 18.6% (95% CI: 11.38%, 27.73%). Non-dermatophyte moulds (NDMs) (55.7%) and yeast (30.9%) were the dominant aetiological agents. The leading pathogens found among NDMs were Aspergillus sp. (35.1%) and Fusarium sp. (1.4%) and Candida albicans (8.2%). Aspergillus niger (20.6%) was the most frequently isolated Aspergillus sp. in the NDMs. Frequent use of detergents and walking bare foot were the significant risk factors of onychomycosis. Only 15.5% of the participants were aware of onychomycosis. Conclusion: Onychomycosis is prevalent to a considerable level among diabetics. Regular dermatological investigations of nails and health education are needed to combat the disease. Large scale research is warranted to confirm these assertions.

Elucidation of the mechanisms of fluconazole resistance and repurposing treatment options against urinary Candida spp. isolated from hospitalized patients in Alexandria, Egypt

BackgroundThe incidence of fungal urinary tract infections (UTIs) has dramatically increased in the past decades, with Candida arising as the predominant etiological agent. Managing these infections poses a serious challenge to clinicians, especially with the emergence of fluconazole-resistant (FLC-R) Candida species. In this study, we aimed to determine the mechanisms of fluconazole resistance in urinary Candida spp. isolated from hospitalized patients in Alexandria, Egypt, assess the correlation between fluconazole resistance and virulence, and explore potential treatment options for UTIs caused by FLC-R Candida strains.ResultsFluconazole susceptibility testing of 34 urinary Candida isolates indicated that 76.5% were FLC-R, with a higher prevalence of resistance recorded in non-albicans Candida spp. (88.9%) than in Candida albicans (62.5%). The calculated Spearman’s correlation coefficients implied significant positive correlations between fluconazole minimum inhibitory concentrations and both biofilm formation and phospholipase production. Real-time PCR results revealed that most FLC-R isolates (60%) significantly overexpressed at least one efflux pump gene, while 42.3% significantly upregulated the ERG11 gene. The most prevalent mutation detected upon ERG11 sequencing was G464S, which is conclusively linked to fluconazole resistance. The five repurposed agents: amikacin, colistin, dexamethasone, ketorolac, and sulfamethoxazole demonstrated variable fluconazole-sensitizing activities in vitro, with amikacin, dexamethasone, and colistin being the most effective. However, the fluconazole/colistin combination produced a notable reduction (49.1%) in bladder bioburden, a 50% decrease in the inflammatory response, and tripled the median survival span relative to the untreated murine models.ConclusionsThe fluconazole/colistin combination offers a promising treatment option for UTIs caused by FLC-R Candida, providing an alternative to the high-cost, tedious process of novel antifungal drug discovery in the battle against antifungal resistance.

Analysis of virulence factors in extracellular vesicles secreted by Naegleria fowleri

Naegleria fowleri is the etiological agent of primary amebic meningoencephalitis (PAM), a rapidly progressive acute and fulminant infection that affects the central nervous system, particularly of children and young adults, which has a mortality rate greater than 95%, and its symptomatologic similarity with other meningitis caused by virus or bacteria makes it difficult to make a quick and timely diagnosis that prevents the progression of the infection. It is necessary to know the antigenic determinants as well as the pathogenicity mechanisms of this amoeba to implement strategies that allow for better antiamoebic therapeutic and diagnostic targets that directly impact the health sector. Therefore, the aim of this work was to analyze some virulence factors as part of extracellular vesicle (EV) cargo secreted by N. fowleri. The EV secretion to the extracellular medium was evaluated in trophozoites fixed and incubated with anti-N. fowleri antibody while molecular identification of EV cargo was performed by SDS-PAGE, Western blot, and RT-PCR. Our results showed that N. fowleri secretes a wide variety of vesicle sizes ranging from 0.2 to > 2 μm, and these EVs were recognized by antibodies anti-Naegleropore B, anti-19 kDa polypeptide band, anti-membrane protein Mp2CL5, anti-protease cathepsin B, and anti-actin. Furthermore, these vesicles were localized in the trophozoites cytoplasm or secreted into the extracellular medium. Specifically in relation to small vesicles, our purified exosomes were recognized by CD63 and Hsp70 markers, along with the previously mentioned proteins. RT-PCR analysis was made through the isolation of EVs from N. fowleri trophozoite culture by concentration, filtration, and ultracentrifugation. Interestingly, we obtained PCR products for Nfa1, NPB, Mp2CL5, and CatB genes as part of exosomes cargo. This suggests that the molecules identified in this work could play an important role in communication as well as in infectious processes caused by this amoeba. Therefore, the study and characterization of the pathogenicity mechanisms, as well as the virulence factors released by N. fowleri remains a key point to provide valuable information for the development of therapeutic treatments, vaccine design, or biomarkers for a timely diagnosis against infections caused by protozoa.

Trypanosoma cruzi P21 protein exacerbates Leishmania (L.) amazonensis infection

The protozoan parasite Trypanosoma cruzi, the etiological agent of Chagas disease, affects millions of people worldwide. Current treatments rely on drugs effective only in the acute phase, making the search for new therapeutic targets a priority. While a recombinant protein based on T. cruzi P21 (rP21) exhibits immunomodulatory properties and contributes to controlling parasitism and inflammation during T. cruzi infection, its efficacy against other trypanosomatids remains unexplored. This study investigated the impact of rP21 on Leishmania (L.) amazonensis infection in a murine model. Contrary to our expectations, treatment with rP21 did not ameliorate L. (L.) amazonensis infection. Instead, rP21 treatment resulted in increased parasite load in the paws of infected BALB/c mice, evidenced by larger lesion sizes and higher parasite burdens, accompanied by an intensified inflammatory infiltrate in the paw tissue. These findings suggest that despite its promising effects in the context of T. cruzi infection, rP21 may not be a suitable therapeutic candidate for L. amazonensis infection and might even exacerbate disease.

Exploration of Novel “Ferroxazide/Ferrazone” Derivatives as Antitrypanosomatid Agents: Design, Synthesis, and Biological Efficacy

ABSTRACTTrypanosomatids are the etiologic agents of numerous parasitic diseases that cause significant morbidity and mortality in millions of people and animals around the world. Approved antitrypanosomatid agents are limited by several drawbacks, such as severe toxicity, lengthy treatment, need for hospitalization, and susceptibility to drug resistance. Consequently, parasitic diseases remain a substantial public health problem, and new drugs are required, especially drugs suitable for rural health systems that have limited resources. In an attempt to find antitrypanosomatid agents to address this problem, we report here on the synthesis and biological efficacy of ferrocene derivatives of nifuroxazide and nitrofurazone, which were designed by replacing the nitrofuran scaffold within their structures with the ferrocene moiety. The 1,2‐disubstituted ferrocene intermediates 8 and 9, featuring amine and carboxaldehyde groups, exhibited the best in vitro antiamastigote activity against Leishmania major strain NIH S and Leishmania donovani strain 9515, respectively. Ferroxazide derivative 15 was revealed as a mammalian cell nontoxic hit compound against Trypanosoma congolense strain IL3000 trypomastigotes; however, no in vivo treatment efficacy was observed against T. congolense strain IL3000‐infected BALB/c mice during a preliminary animal study. The synthesized ferrocene derivatives were poorly soluble in the in vitro and in vivo testing media, hindering uniform sampling and dosing. This study's outcome indicates that replacing the 5‐nitrofuran moiety with ferrocene did not increase antitrypanosomatid activity compared to the nitrofuran parent drugs nifuroxazide and nitrofurazone.