Role of Brazilian bats in the epidemiological cycle of potentially zoonotic pathogens.

Bats (Chiroptera) are flying mammals of great biodiversity and habits. These characteristics contribute for them being natural reservoirs and part of the epidemiological cycle of several potentially zoonotic pathogens, such as viruses, protozoa, fungi and bacteria. Brazil hosts approximately 15% of the world's bat diversity, with 181 distinct species, 68 genera and 9 families. About 60% of infectious diseases in humans are of zoonotic origin and, in the last decades, the detection of zoonotic pathogens in bats and their environment has been reported, such as Rabies virus (RABV) and Histoplasma capsulatum. Thus, the aim of this work was to review the reports of zoonotic pathogens associated with bats in Brazil in the past ten years. We reviewed the main pathogenic microorganisms described and the species of bats most frequently involved in the epidemiological cycles of these zoonotic agents. The obtained data show an upward trend in the detection of zoonotic pathogens in Brazilian bats, such as RABV, Bartonella sp., Histoplasma capsulatum and Leishmania spp., with emphasis on the bat species Artibeus lituratus, Carollia perspicillata, Desmodus rotundus and Molossus molossus. These findings highlight the importance of monitoring bat-associated microrganisms to early identify pathogens that may threaten bat populations, including potentially zoonotic microrganisms, emphasizing the importance of the One Health approach to prevent and mitigate the risks of the emergence of zoonotic diseases.

Ex situ model of biofilm-associated wounds: providing a host-like environment for the study of Staphylococcus aureus and Pseudomonas aeruginosa biofilms.

AIM: This study aimed to assess an ex situ model of biofilm-associated wounds on porcine skin for the study of Staphylococcus aureus and Pseudomonas aeruginosa biofilms in a host-like environment, after 48 to 120 h of incubation. MATERIAL AND RESULTS: Ex situ and in vitro biofilms were comparatively analysed. Overall, CFU-counts and matrix quantification yielded significantly (P < 0·05) higher results for ex situ than in vitro biofilms. Confocal microscopy revealed greater (P < 0·05) biomass and thickness at 48-72 h and greater (P < 0·05) robustness at 72 h of growth. S. aureus ex situ biofilms produced less (P < 0·05) siderophore and proteases than in vitro biofilms, while P. aeruginosa ex situ biofilms produced more (P < 0·05) siderophores and less proteases than in vitro biofilms. CONCLUSIONS: Biofilms grown ex situ present a greater amount of bacterial cells and polymeric matrix than their in vitro counterparts, reaching maturity at 72 h of growth. Moreover the production of virulence factors differs between ex situ and in vitro biofilms. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings emphasize the importance of using ex situ biofilm models, once they mimic in vivo conditions. The use of these models brings perspectives for the pursuit of therapeutic alternatives, as tests may be performed in a host-like environment.

The global epidemiology of emerging Histoplasma species in recent years.

Histoplasmosis is a serious infectious disease in humans caused by Histoplasma spp. (Onygenales), whose natural reservoirs are thought to be soil enriched with bird and bat guano. The true global burden of histoplasmosis is underestimated and frequently the pulmonary manifestations are misdiagnosed as tuberculosis. Molecular data on epidemiology of Histoplasma are still scarce, even though there is increasing recognition of histoplasmosis in recent years in areas distant from the traditional endemic regions in the Americas. We used multi-locus sequence data from protein coding loci (ADP-ribosylation factor, H antigen precursor, and delta-9 fatty acid desaturase), DNA barcoding (ITS1/2+5.8s), AFLP markers and mating type analysis to determine the genetic diversity, population structure and recognise the existence of different phylogenetic species among 436 isolates of Histoplasma obtained globally. Our study describes new phylogenetic species and the molecular characteristics of Histoplasma lineages causing outbreaks with a high number of severe outcomes in Northeast Brazil between 2011 and 2015. Genetic diversity levels provide evidence for recombination, common ancestry and clustering of Brazilian isolates at different geographic scales with the emergence of LAm C, a new genotype assigned to a separate population cluster in Northeast Brazil that exhibited low diversity indicative of isolation. The global survey revealed that the high genetic variability among Brazilian isolates along with the presence of divergent cryptic species and/or genotypes may support the hypothesis of Brazil being the center of dispersion of Histoplasma in South America, possibly with the contribution of migratory hosts such as birds and bats. Outside Brazil, the predominant species depends on the region. We confirm that histoplasmosis has significantly broadened its area of occurrence, an important feature of emerging pathogens. From a practical point of view, our data point to the emergence of histoplasmosis caused by a plethora of genotypes, and will enable epidemiological analysis focused on understanding the processes that lead to histoplasmosis. Further, the description of this diversity opens avenues for comparative genomic studies, which will allow progress toward a consensus taxonomy, improve understanding of the presence of hybrids in natural populations of medically relevant fungi, test reproductive barriers and to explore the significance of this variation.

The Importance of Wild Canids in the Epidemiology of Rabies in Northeast Brazil: A Retrospective Study.

Rabies is an endemic disease in Brazil, where it is considered a serious public health problem. Although the number of human and dog-transmitted cases has declined in recent decades, rabies in wildlife has emerged considerably. Among the sylvatic animals, wild canids have been considered important hosts of the rabies virus. We performed a retrospective study of reported cases of rabies in wild canids and human victims in Ceará state (Northeast Brazil) during 2003 to 2013. Information was provided by governmental laboratories involved in rabies detection and by the Ministry of Health. From January 2003 to December 2013, a total of 11 931 animal samples were examined for rabies. Positivity were detected in 438 samples (3.67%), of which 229 (52.28%) were domestic animals, 105 (23.97%) wild canids and 104 (23.74%) other wild animals (bats, marmosets and raccoons). Approximately 33% of wild canids surveyed (n = 317) were positive for rabies. During the studied period, a total of 1923 attacks on humans by wild canids were registered. Males (n = 1405) were more affected than females (n = 520; 72.98% versus 27.01%), and the median age of all cases was 36.5 years. Injuries to individuals up to 19 years old corresponded to approximately 30% (n = 565) of all cases. Most of the victims lived in rural areas (72.46%; n = 1395), and the majority showed bites (81.13%; n = 1677) or scratches (12.23%; n = 253). Injuries were considered profound (52.1%; n = 1003), superficial (40.91; n = 788) or multiple with severe laceration (6.98%; n = 134). Only 1300 (67.53%) victims were enrolled for the complete rabies post-exposure prophylaxis scheme. Data from the present study confirm that wild canids are important hosts of rabies virus in northeastern Brazil and jeopardize rabies control in this area. Local authorities should focus their efforts in education of health professionals. In addition, strategies should be formulated to preserve wildlife.

Farnesol increases the susceptibility of Burkholderia pseudomallei biofilm to antimicrobials used to treat melioidosis.

AIMS: The aim of this study was to analyse the in vitro activity of farnesol alone and combined with the antibacterial drugs amoxicillin, doxycycline, ceftazidime and sulfamethoxazole-trimethoprim against Burkholderia pseudomallei biofilms. METHODS AND RESULTS: Susceptibility was assessed by the broth microdilution test and cell viability was read with the oxidation-reduction indicator dye resazurin. The biofilms were evaluated through three microscopic techniques (optical, confocal and electronic microscopy). The minimum biofilm erradication concentration (MBEC) for farnesol was 75-2400 mmol l(-1). In addition, farnesol significantly reduced the MBEC values for ceftazidime, amoxicillin, doxycycline and sulfamethoxazole-trimethoprim by 256, 16, 4 and 4 times respectively (P < 0·05). Optical, confocal and electronic microscopic analyses of farnesol-treated B. pseudomallei biofilms demonstrated that this compound damages biofilm matrix, probably facilitating antimicrobial penetration in the biofilm structure. CONCLUSIONS: This study demonstrated the effectiveness of farnesol against B. pseudomallei biofilms and its potentiating effect on the activity of antibacterial drugs, in particular ceftazidime, amoxicillin, doxycycline and sulfamethoxazole-trimethoprim. SIGNIFICANCE AND IMPACT OF THE STUDY: The intrinsic antimicrobial resistance of B. pseudomallei is a serious challenge for the treatment of melioidosis. Thus, this paper reports the inhibitory potential of farnesol against B. pseudomallei biofilms, as well as highlights the favourable pharmacological interaction of farnesol with antibiotics tested, not only on cell viability, but also in the structural morphology of biofilms.

In vitro antifungal activity of miltefosine and levamisole: their impact on ergosterol biosynthesis and cell permeability of dimorphic fungi.

AIMS: This study aimed to evaluate the in vitro activity of miltefosine and levamisole against strains of Coccidioides posadasii in the filamentous phase and strains of Histoplasma capsulatum in filamentous and yeast phases. METHODS AND RESULTS: Strains of C. posadasii in the filamentous phase (n = 22) and strains of H. capsulatum in filamentous (n = 40) and yeast phases (n = 13) were, respectively, submitted to broth macrodilution and broth microdilution methods, as described by the Clinical and Laboratory Standards Institute, to determine the minimum inhibitory concentration (MIC) and the minimum fungicidal concentration (MFC) of miltefosine and levamisole. The effect of the drugs on cell membrane permeability under osmotic stress conditions and total ergosterol production were also assessed, along with quantification of extravasated molecules. The results show the inhibitory effect of levamisole and miltefosine against C. posadasii and H. capsulatum and the effect of these drugs on ergosterol synthesis and the permeability of the plasma membrane using subinhibitory concentrations against strains subjected to osmotic stress. Levamisole was also able to cause the release of nucleic acids. CONCLUSIONS: Miltefosine and levamisole are capable of inhibiting the in vitro growth of C. posadasii and H. capsulatum, probably by altering the permeability of the cellular membrane. SIGNIFICANCE AND IMPACT OF THE STUDY: This work presents alternatives for the treatment of histoplasmosis and coccidioidomycosis, raising the possibility of the use of miltefosine and levamisole as adjuvants in antifungal therapy, providing perspectives for the design of in vivo studies.

Emergence of azole-resistant Candida albicans in small ruminants.

Small ruminant production is a common agricultural activity worldwide. However, studies on the fungal microbiota of these animals are scarce. Therefore, this study aimed at isolating yeasts from goats and sheep and evaluating the antifungal susceptibility of the recovered Candida albicans. A total of 120 animals from farms in Ceará State, Brazil, were assessed in this study. The samples were collected from nasal, oral and rectal cavities with sterile swabs. Candida spp., Trichosporon spp. and Rhodotorula spp. were isolated from small ruminants. Resistance to three azole drugs was observed in C. albicans. In summary, Candida spp. were predominantly observed as part of the microbiota of the nasal, oral and rectal cavities of small ruminants, including azole-resistant strains of C. albicans.

Azole-resistant Candida albicans from a wild Brazilian porcupine (Coendou prehensilis): a sign of an environmental imbalance?

This study aimed at evaluating the in vitro antifungal susceptibility of Candida albicans isolates obtained during necropsy of a wild Brazilian porcupine and the mechanism of azole resistance. Initially, we investigated the in vitro susceptibility of the three isolates to amphotericin B, caspofungin, fluconazole, itraconazole, ketoconazole and voriconazole. Afterwards, three sub-inhibitory concentrations (47, 21 and 12 mg/l) of promethazine, an efflux pump inhibitor, were tested in combination with the antifungal drugs in order to evaluate the role of these pumps in the development of antifungal resistance. In addition, the three isolates were submitted to RAPD-PCR and M13-fingerprinting analyses. The minimum inhibitory concentrations (MICs) obtained with the isolates were 1, 0.03125, 250, 125, 8 and 250 mg/l for amphotericin B, caspofungin, fluconazole, itraconazole, ketoconazole and voriconazole, respectively, and the isolates were found to be resistant to all tested azoles. The addition of the three subinhibitory concentrations of promethazine resulted in statistically significant (P < 0.05) reductions in the MICs for all tested drugs, with decreases to azoles being statistically greater than those for amphotericin B and caspofungin (P < 0.05). The molecular analyses showed a genetic similarity among the three tested isolates, suggesting the occurrence of candidemia in the studied animal. These findings highlight the importance of monitoring antifungal susceptibility of Candida spp. from veterinary sources, especially as they may indicate the occurrence of primary azole resistance even in wild animals.

Antifolates inhibit Cryptococcus biofilms and enhance susceptibility of planktonic cells to amphotericin B.

The Cryptococcus neoformans species complex contains the most important agents of fungal meningoencephalitis. Therapeutic choices are limited and issues related to toxicity and resistance to antifungals have been described. The present study evaluated the inhibitory effect of the antifolate combinations sulfamethoxazole-trimethoprim (SMX/TMP) and sulfadiazine-pyrimethamine (SDZ/PYR) against planktonic cells and biofilms of C. neoformans and C. gattii. The influence of the antifolate combinations on the amphotericin minimum inhibitory concentration (MIC) of planktonic cells was also investigated. In addition, the effect of these combinations on the cellular ergosterol content of planktonic cells was studied. Strains of C. neoformans (n = 15) and C. gattii (n = 15) obtained from environmental or clinical sources were evaluated by the broth microdilution method. SMX/TMP and SDZ/PYR showed antifungal activity against free living cells and sessile cells of Cryptococcus spp. Moreover, planktonic cells showed increased susceptibility to amphotericin B after pre-incubation with sub-inhibitory concentrations of SMX/TMP or SDZ/PYR. The drug combinations SMX/TMP and SDZ/PYR were able to prevent the biofilm formation and showed inhibitory effect against mature biofilms of both species. Additionally, the study showed that antifolate drugs reduced the ergosterol content in C. neoformans and C. gattii planktonic cells. Our results highlight the antifungal potential of antifolate drugs.

Ciprofloxacin shows synergism with classical antifungals against Histoplasma capsulatum var. capsulatum and Coccidioides posadasii.

This study evaluated the in vitro interaction between ciprofloxacin (CIP) and classical antifungals against Histoplasma capsulatum var. capsulatum in mycelial (n = 16) and yeast-like forms (n = 9) and Coccidioides posadasii in mycelial form (n = 16). This research was conducted through broth microdilution and macrodilution, according to Clinical Laboratory Standards Institute. Inocula were prepared to obtain from 0.5 × 10(3) to 2.5 × 10(4) cfu ml(-1) for H. capsulatum and from 10(3) to 5 × 10(3) cfu ml(-1) for C. posadasii. Initially, minimum inhibitory concentration (MIC) for each drug alone was determined. Then, these MICs were used as the highest concentration for each drug during combination assays. The procedures were performed in duplicate. For all combination assays, MICs were defined as the lowest concentration capable of inhibiting 80% of visible fungal growth, when compared to the drug-free control. Drug interaction was evaluated by paired sample t-Student test. The obtained data showed a significant MIC reduction for most tested combinations of CIP with antifungals, except for that of CIP and voriconazole against yeast-like H. capsulatum. This study brings potential alternatives for the treatment of histoplasmosis and coccidioidomycosis, raising the possibility of using CIP as an adjuvant antifungal therapy, providing perspectives to delineate in vivo studies.

Viral protease inhibitors affect the production of virulence factors in Cryptococcus neoformans.

The effects of the protease inhibitors saquinavir, darunavir, ritonavir, and indinavir on growth inhibition, protease and phospholipase activities, as well as capsule thickness of Cryptococcus neoformans were investigated. Viral protease inhibitors did not reduce fungal growth when tested in concentrations ranging from 0.001 to 1.000 mg/L. A tendency toward increasing phospholipase activity was observed with the highest tested drug concentration in a strain-specific pattern. However, these drugs reduced protease activity as well as capsule production. Our results confirm a previous finding that antiretroviral drugs affect the production of important virulence factors of C. neoformans.

Sesquiterpene farnesol contributes to increased susceptibility to ß-lactams in strains of Burkholderia pseudomallei.

This study aimed to evaluate the in vitro combination of farnesol and ß-lactams against Burkholderia pseudomallei. A total of 12 ß-lactamase-positive strains were tested according to CLSI standards. All strains were inhibited by farnesol, with MICs ranging from 75 to 150 µM. The combination of this compound with ß-lactams resulted in statistically significant ß-lactam MIC reduction (P ≤ 0.05). This study provides new perspectives for the use of farnesol combined with ß-lactam antibiotics against strains of B. pseudomallei.

Central nervous system involvement in dengue: a study in fatal cases from a dengue endemic area.

OBJECTIVE: This study aimed to determine the frequency of CNS infection by dengue virus (DENV) in individuals with fatal outcomes. METHODS: Samples of 150 individuals suspect of an infection disease and with fatal outcomes were investigated for evidence of the presence of DENV. The sampling was made up of 150 CSF, 120 tissue samples, and 109 blood specimens. The tests used were viral isolation, reverse transcriptase PCR, immunohistochemistry, nonstructural 1 antigen, and immunoglobulin M detection. RESULTS: Out of 150 studied patients, 84 were dengue positive. Evidence of the presence of DENV was found in 41 CSF, showing the following neurologic diagnosis: 46.3% encephalitis, 34.1% meningoencephalitis, and 19.5% meningitis, giving a frequency of 48.8% of the 84 dengue-positive cases. The major clinical manifestations observed on these individuals were fever, headache, mental irritability, breathless, vomiting, muscle pain, tiredness, abdominal pain, somnolence, restlessness, dizziness, cough, seizure, coma, and neck stiffness. CONCLUSION: Clinical manifestations and laboratory-positive results in CSF that may indicate the presence of DENV led to consider the invasion of CNS by DENV in these fatal cases studied, and showed that neurologic pathology was an important fatal complication in dengue cases.

Detection of the dengue non-structural 1 antigen in cerebral spinal fluid samples using a commercially available enzyme-linked immunosorbent assay.

The involvement of the central nervous system in dengue infections has been reported in countries where the disease in endemic. The purpose of this study was to determine whether an enzyme-linked immunosorbent assay kit designed to detect the dengue NS1 antigen in serum was able to detect this antigen in cerebral spinal fluid (CSF) samples from patients with fatal outcomes. To evaluate the sensitivity of the kit, 26 dengue-positive CSF samples were used. The Pan-E Dengue Early kit was able to detect the NS1 antigen in 13 of 26 dengue-positive CSF samples, resulting in a sensitivity of 50% (95% confidence interval, 29.9-70.1%) and specificity of 100% (95% confidence interval, 75.3-100%). The kit was able to detect the NS1 antigen in CSF of individuals who had died of dengue. When used in combination with IgM, the detection rate rose to 92.3%. This study reports a method for rapidly detecting the dengue virus in CSF, thereby increasing the diagnosis of dengue fever cases with unusual neurological manifestations.

Serological evidence of Histoplasma capsulatum infection among dogs with leishmaniasis in Brazil.

Histoplasmosis is a systemic infection caused by the fungus Histoplasma capsulatum. Environmental sources of infection for humans and animals in certain regions and the prevalence of infection in animals are frequently unknown. Because of the clinical and epidemiological similarities between histoplasmosis and leishmaniasis in northeastern Brazil, we decided to investigate the serologic evidence of H. capsulatum in dogs, considering that these animals can act as sentinels for histoplasmosis. A total of 224 serum samples from dogs were tested for antibodies against H. capsulatum through immunodiffusion. A total of 128 (57.14%) samples were positive for leishmaniasis by indirect immunofluorescence assay and four (1.78%) samples were positive for antibodies against H. capsulatum. Immunological evidence of the co-existence of histoplasmosis and leishmaniasis in dogs living in urban areas was observed. Diagnosis and clinical management of these diseases in endemic areas should be improved by veterinarians.

Candida parapsilosis meningitis as the first manifestation of AIDS: case report.

Candida meningitis is a rare condition that occurs more frequently in premature infants, immunocompromised patients or patients after neurosurgery. We describe a case of a previously healthy 41-year-old man with Candida parapsilosis meningitis associated with oropharyngeal candidiasis as the first manifestation of AIDS.

Mycotic aneurysm caused by Burkholderia pseudomallei: report of a Brazilian strain genetically related to Thai strains.

Melioidosis, a severe infectious disease caused by Burkholderia pseudomallei that is prevalent in Southeast Asia and Northern Australia, has been sporadically reported in Brazil since 2003. We report a case of aortic aneurysm with blood culture positive for B. pseudomallei. The phylogenetic analysis of 16S ribosomal DNA showed this isolate to be evolutionarily grouped with the MSHR346 strains from Thailand.

Characterization of the gastrointestinal yeast microbiota of cockatiels (Nymphicus hollandicus): a potential hazard to human health.

Cockatiels are the world's second most popular psittacine pet bird, but no data characterizing their gastrointestinal microbiota have been found. Thus, the aim of this work was to characterize the yeast gastrointestinal microbiota of cockatiels and to evaluate the relevance of cockatiels as carriers of potentially pathogenic yeasts. A total of 60 cockatiels, from 15 different premises, were assessed. A thorough clinical examination was performed with each bird, and samples were collected from oral cavity, crop and cloaca. The stools were collected from cages where the birds were kept. The isolates were identified according to morphological and biochemical characteristics. Yeasts were isolated from at least one anatomical site of 65% of the birds and 64.3% of the stool samples. The oral cavity (53.3%) and the crop (58.3%) were the anatomical sites with the highest prevalence and the highest number of yeast isolates. Overall, 120 yeast isolates, belonging to 13 species, were obtained. The most frequently isolated species were Candida albicans, with 39 (32.5%) isolates, followed by Candida tropicalis (20%), Trichosporon asteroides (12.5%), Candida famata (10%) and others. Mixed yeast colonies were isolated from 23.3% of the birds and C. albicans was seldom found in association with other species (P<0.05). The results of this work demonstrated that cockatiels harbour potentially pathogenic yeasts throughout their gastrointestinal tract and in stools, and are prone to disseminating them in the environment.

Fungal microbiota dynamics as a postmortem investigation tool: focus on Aspergillus, Penicillium and Candida species.

AIMS: To investigate the presence of fungi during three human decomposition stages: bloated, putrefaction and skeletonization. METHODS AND RESULTS: The samples were gathered in the city of Fortaleza, Ceará, Brazil, from the public morgue and cemeteries. The material was submitted to conventional mycological procedures by direct examination and macro/micro morphological and biochemical analyses. The main fungi isolated were Aspergillus spp., Penicillium spp. and Candida spp. in the bloated stage (n = 34 cadavers) and in the putrefaction stage (n = 6 cadavers), while in the skeletonization stage (n = 20 cadavers), the main fungi were Aspergillus spp., Penicillium spp. and Mucor sp. CONCLUSIONS: Aspergillus, Penicillium and Candida species were associated with decomposed human cadavers. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings enable tracing out the profile of fungal communities of human cadavers for the first time. However, much more research will be necessary to develop this new segment of mycology and to enable its routine use in forensic science.

Ants (Hymenoptera: Formicidae) as carriers of fungi in hospital environments: an emphasis on the genera Tapinoma and Pheidole.

The aim of this study was to evaluate the presence of filamentous fungi and yeasts on the external surface of ants at hospitals. From March 2007 to February 2008, 2,899 ants were evaluated in two public hospitals in the city of Fortaleza, Ceará, in northeastern Brazil. The ants were attracted by nontoxic baits, distributed within critical and semicritical hospital areas. The fungi were identified through macro- and micromorphological analysis, biochemical profile, and growth in chromogenic medium. From this study, 5 genera and 13 species of ants were identified, from critical (8% of the collected ants) and semicritical (92%) areas, during the daytime (48%) and nighttime (52%) periods. In the mycological analysis, 75% of the ants were fungi carriers, with the species Tapinoma melanocephalum and species from the genus Pheidole having the most potential as carriers of airborne fungi (75 and 18%, respectively) and yeasts (6 and 1%, respectively). In summary, ants act as carriers of airborne fungi and yeasts, including some pathogenic species.