Prevalence and Recovery of Microorganisms from Containers used for the Collection of Forensic Biological Samples

Background: Microbes play a significant role in the degradation of biological evidence collected for forensic analysis. The present study is aimed to isolate and identify the microbes present inside the empty container used for the biological evidence collection. Methods: Bacterial isolation from the selected containers was done by cotton swab over the inner surface of the containers. Streaking was done on the surface of the three different culture plates as a Blood agar plate, Nutrient plate and MacConkey plate. The plates were placed in an incubator shaker at 37ºC for 48 hours. The colonies grown on the surface of the media were counted on and used for further study. Various biochemical assays were performed to characterize isolated bacteria. Results: Staining results suggested that the presence of Gram-positive stain (Staphylococcus, Bacillus, Corynebacterium, Clostridium) and Gram negative stain (E. coli, Enterobacteriaceae, Pseudomonas, Salmonella, Shigella, Stenotrophomonas, Bdellovibrio, Acetic acid bacteria). The Catalase and Coagulase test suggested the presence of Staphylococcus aureus, S. epidermis and S. sapropyticus. Moreover, the indole test suggested the presence of Citrobacter koseri, Kebsiella oxytoca, Proteus vulgaris etc. Some of the bacteria were urea metabolizing, including Proteus spp, Helicobacter pylori, Cryptococcus spp, Corynebacterium spp. Conclusion: This study recommends that there should be proper maintenance of the chain of custody from the collection to analysis so that evidence properly prevents degradation or contamination in the biological evidence. Extra care is needed for the collection and packing of biological evidence from the crime scene. Moreover, the collection containers, if left wide open, lead to contamination and degradation of biological evidence.

PERFIL EPIDEMIOLÓGICO DE FUNGEMIA EM HEMOCULTURA AUTOMATIZADA E EM CULTURA MICOLÓGICA

INTRODUÇÃO: As micoses sistêmicas representam um grande desafio mundial e ainda são um tema negligenciado pelas autoridades de saúde pública. Estima-se que mais de foram 300 milhões de pessoas em todo o mundo sofrem de infecções fúngicas a cada ano com cerca de 1,0 a 2,0 milhões de mortes. Sabe-se que o atraso do diagnóstico das micoses sistêmicas impossibilita o início da terapia antifúngica imediata podendo ocasionar mortes ou o agravamento destas. O diagnóstico micológico, usualmente, baseia-se na identificação dos fungos por meio da observação de suas características morfológicas, isolados do material clínico cultivado. No entanto, em se tratando de amostras sanguíneas, sistemas de hemocultura automatizados são utilizados. OBJETIVO: Este estudo teve como objetivo avaliar o perfil epidemiológico dos fungos isolados de amostras sanguíneas cultivados no sistema de hemocultura automatizado e nos meios usuais da micologia clássica. METODOLOGIA: Amostras sanguíneas de 596 pacientes internados no Hospital Eduardo de Menezes, Minas Gerais, no período de março de 2017 a dezembro de 2019, foram inoculadas pareadas nos meios ágar Sabouraud e ágar Mycosel e no frasco aeróbio do sistema BacT/ALERT®. RESULTADOS: Candida spp. 0,3% (2/596, IC 95% 0,09 - 1,22) Cryptococcus spp. 0,8% (5/596, IC 95% 0,36 - 1,95) e Sporothrix spp. 0,2 % (1/596, IC 95% 0,03 - 0,94) foram os fungos isolados na hemocultura automatizada. Por outro lado, recuperamos Cryptococcus spp. 1,0% (6/596, IC 95% 0,46 - 2,18), Histoplasma spp. 3,0% (18/596, IC 95% 1,92 - 4,72), Paracoccidioides spp. 0,5% (3/596, IC 95% 0,17 - 1,47) e Sporothrix spp. 0,2 % (1/596, IC 95% 0,03 - 0,94) nas culturas micológicas clássicas. CONCLUSÃO: Os fungos mostraram notável variabilidade entre os gêneros na preferência das metodologias utilizadas. Na prática, podemos concluir que as metodologias se complementam e dessa forma, a aplicação simultânea da hemocultura automatizada e das culturas micológicas em Sabouraud e Mycosel oferecem a possibilidade de detectar uma diversidade maior de agentes fúngicos e melhora a sensibilidade do diagnóstico. Palavras-chave: micoses sistêmicas, diagnóstico micológico, hemocultura automatizada.

Cryptococcus neoformans VNII as the Main Cause of Cryptococcosis in Domestic Cats from Rio de Janeiro, Brazil

Cryptococcosis is a systemic fungal disease acquired from contaminated environments with propagules of the basidiomycetous yeasts of the Cryptococcus neoformans and C. gattii species complexes. The C. neoformans species complex classically comprises four major molecular types (VNI, VNII, VNIII, and VNIV), and the C. gattii species complex comprises another four (VGI, VGII, VGIII, and VGIV) and the newly identified molecular type VGV. These major molecular types differ in their epidemiological and ecological features, clinical presentations, and therapeutic outcomes. Generally, the most common isolated types are VNI, VGI, and VGII. The epidemiological profile of cryptococcosis in domestic cats is poorly studied and cats can be the sentinels for human infections. Therefore, the present study aimed to determine the molecular characterization of Cryptococcus spp. isolated from domestic cats and their dwellings in the metropolitan area of Rio de Janeiro, Brazil. A total of 36 Cryptococcus spp. strains, both clinical and environmental, from 19 cats were subtyped using multilocus sequence typing (MLST). The ploidy was identified using flow cytometry and the mating type was determined through amplification with specific pheromone primers. All strains were mating type alpha and 6/36 were diploid (all VNII). Most isolates (63.88%) were identified as VNII, a rare molecular type, leading to the consideration that this genotype is more likely related to skin lesions, since there was a high percentage (68.75%) of cats with skin lesions, which is also considered rare. Further studies regarding the molecular epidemiology of cryptococcosis in felines are still needed to clarify the reason for the large proportion of the rare molecular type VNII causing infections in cats.

Titan Cells and Yeast Forms of Cryptococcus neoformans and Cryptococcus gattii Are Recognized by GXMR-CAR

Cryptococcosis, a systemic mycosis that affects both the immunocompromised and immunocompetent, is caused by the inhalation of dehydrated yeasts or fungal spores of Cryptococcus gattii or Cryptococcus neoformans. The Cryptococcus spp. polysaccharide capsule is composed mainly of glucuronoxylomannan—GXM, its major virulence factor. The capsule thickness increases to more than 15 μm during titanization, favoring the pathogenesis of cryptococcosis. Previous studies demonstrated that cytotoxic T cells that had been bioengineered with GXM-targeting chimeric antigen receptor (GXMR-CAR) were able to recognize C. neoformans by promoting the control of titanization. GXMR-CAR, a second-generation CAR, contains a single-chain variable fragment that originates from a 18B7 clone: a human IgG4 hinge, followed by a human CD28 (transmembrane/cytoplasmic domains) and a CD3ς chain. In the current study, we redirected T cells to target distinct C. neoformans and C. gattii cell types by GXMR-CAR. Lentiviral particles carrying the GXMR-CAR sequence were used to transduce Jurkat cells, and these modified cells interacted with the GXM of the C. gattii R265 strain. Moreover, GXMR-CAR mediated the recognition of C. gattii and C. neoformans yeasts with both thin and thick polysaccharide capsules, and GXMR-CAR Jurkat cells interacted with titan cells sourced from both Cryptococcus spp. Thus, bioengineered cells using CAR can improve the treatment of cryptococcosis.

Overview on the Prevalence of Fungal Infections, Immune Response, and Microbiome Role in COVID-19 Patients

Patients with severe COVID-19, such as individuals in intensive care units (ICU), are exceptionally susceptible to bacterial and fungal infections. The most prevalent fungal infections are aspergillosis and candidemia. Nonetheless, other fungal species (for instance, Histoplasma spp., Rhizopus spp., Mucor spp., Cryptococcus spp.) have recently been increasingly linked to opportunistic fungal diseases in COVID-19 patients. These fungal co-infections are described with rising incidence, severe illness, and death that is associated with host immune response. Awareness of the high risks of the occurrence of fungal co-infections is crucial to downgrade any arrear in diagnosis and treatment to support the prevention of severe illness and death directly related to these infections. This review analyses the fungal infections, treatments, outcome, and immune response, considering the possible role of the microbiome in these patients. The search was performed in Medline (PubMed), using the words “fungal infections COVID-19”, between 2020–2021.

Estudio epidemiológico de histoplasmosis, paracoccidioidomicosis y criptococosis en una zona urbana de Ciudad Guayana, estado Bolívar, Venezuela

Se realizó un estudio prospectivo con el objetivo de determinar la prevalencia de infecciones por Histoplasma capsulatum, Complejo Paracoccidioides spp. y Complejo Cryptoccocus en individuos que viven o trabajan en la Plaza de las Ciencias del Sur, Puerto Ordaz, en el estado Bolívar, Venezuela, donde se refugian aves migratorias. Se aplicó una encuesta clínico-epidemiológica y se administraron pruebas intradérmicas de histoplasmina y paracoccidiodina, a todos los sujetos participantes en el estudio. A aquellos individuos sintomáticos respiratorios crónicos, se les realizó una evaluación clínica, radiológica, cultivo micológico y estudio serológico (anticuerpos específicos para Histoplasma capsulatum, Complejo Paracoccidioides spp. y del antígeno capsular del Complejo Cryptococcus spp.). Se aplicaron 632 pruebas intradérmicas a 316 individuos. De estos, sólo 296 (93,7%) acudieron a la lectura de las pruebas. Los sujetos tenían una media de edad de 40,3 años (rango: 7-76 años). Las manifestaciones clínicas más frecuentes fueron: tos (n=49; 16,5%), expectoración (n=17; 5,7%) y fiebre (n=8; 2,7%). La prueba para la histoplasmina fue positiva en el 47,3% (n=140) y a la paracoccidioidina, en el 32,4% (n=96). Los factores de riesgos fueron: contacto con palomas 30,4% (n=90) y visitas a fincas con gallineros 29,1% (n=86). Se identificaron dos casos de paracoccidioidomicosis pulmonar (0,7%) y un caso (0,3%) de criptococosis pulmonar. La elevada prevalencia de infección por Histoplasma capsulatum (47,3%) y Paracoccidioides spp. (32,4%) en ambientes urbanos del estado Bolívar, demuestra la necesidad de promover la búsqueda activa de estos hongos patógenos en quienes tienen síntomas respiratorios crónicos, que no mejoran con terapia antibacteriana y que habitan en zonas con características ambientales y geográficas, propicias para el desarrollo de estos hongos.

Fungal Biofilms as a Valuable Target for the Discovery of Natural Products That Cope with the Resistance of Medically Important Fungi—Latest Findings

The development of new antifungal agents that target biofilms is an urgent need. Natural products, mainly from the plant kingdom, represent an invaluable source of these entities. The present review provides an update (2017–May 2021) on the available information on essential oils, propolis, extracts from plants, algae, lichens and microorganisms, compounds from different natural sources and nanosystems containing natural products with the capacity to in vitro or in vivo modulate fungal biofilms. The search yielded 42 articles; seven involved essential oils, two Brazilian propolis, six plant extracts and one of each, extracts from lichens and algae/cyanobacteria. Twenty articles deal with the antibiofilm effect of pure natural compounds, with 10 of them including studies of the mechanism of action and five dealing with natural compounds included in nanosystems. Thirty-seven manuscripts evaluated Candida spp. biofilms and two tested Fusarium and Cryptococcus spp. Only one manuscript involved Aspergillus fumigatus. From the data presented here, it is clear that the search of natural products with activity against fungal biofilms has been a highly active area of research in recent years. However, it also reveals the necessity of deepening the studies by (i) evaluating the effect of natural products on biofilms formed by the newly emerged and worrisome health-care associated fungi, C. auris, as well as on other non-albicans Candida spp., Cryptococcus sp. and filamentous fungi; (ii) elucidating the mechanisms of action of the most active natural products; (iii) increasing the in vivo testing.

Optimizing Transformation Frequency of Cryptococcus neoformans and Cryptococcus gattii Using Agrobacterium tumefaciens

The transformation of Cryptococcus spp. by Agrobacterium tumefaciens has proven to be a useful genetic tool. A number of factors affect transformation frequency. These factors include acetosyringone concentration, bacterial cell to yeast cell ratio, cell wall damage, and agar concentration. Agar concentration was found to have a significant effect on the transformant number as transformants increased with agar concentration across all four serotypes. When infection time points were tested, higher agar concentrations were found to result in an earlier transfer of the Ti-plasmid to the yeast cell, with the earliest transformant appearing two h after A. tumefaciens contact with yeast cells. These results demonstrate that A. tumefaciens transformation efficiency can be affected by a variety of factors and continued investigation of these factors can lead to improvements in specific A. tumefaciens/fungus transformation systems.

Infections due to Rare Cryptococcus Species. A Literature Review

Infections due to rare Cryptococcus species (other than C. neoformans species complex, C. gattii species complex, C. albidus or C. laurentii) are barely reported. The aim of this work is to present a comprehensive literature review of all the papers describing infections due to these species referenced in the main databases (PubMed/MEDLINE, ScienceDirect, Scopus, and Google Scholar). Clinical and epidemiological data together with laboratory findings (identification and antifungal susceptibility) of each isolate were analyzed. Fifty-eight cryptococosis due to rare species were described in 46 papers between 1934–2018. These reports included 16 rare Cryptococcus spp. that were generally associated with nervous system infections and fungemias. Some species are non-capsulated and are not able to grow at 37 °C. Few species were identified by commercially available methods, making internal transcriber spacer (ITS) and D1/D2 regions sequencing mandatory. The most potent antifungal was amphotericin B (although some species showed high MIC values). The studied strains showed high MICs values to 5-fluorocytosine (all >64 µg/mL), echinocandins (all >8 µg/mL), and fluconazole (>80% of the MICs >4 µg/mL). Due to the scarcity of the data and the absence of guidelines for the treatment of these infections, this review could be informative and could help in the diagnosis and treatment of these infections.

Resistance mechanisms of Cryptococcus spp. and plant compounds as tools to combat them

Cryptococcus is a genus of dimorphic basidiomycete fungi found in the form of yeasts and filaments. Cryptococcosis has as main etiological agents the species Cryptococcus neoformans and Cryptococcus gattii. This disease is considered a public health problem and has becoming more alarming because of the limitations of antimicrobials available to its treatment, in addition to an increase in reports of fungal resistance. In this sense, the present review sought to survey information on the resistance mechanisms of Cryptococcus spp. against the main drugs used in cryptococcosis therapy as well as on the antimicrobial activities of plants against these fungi. Studies have reported that several mechanisms may be involved in fungal resistance to drugs including drug inactivation by enzymes, expression of efflux pumps and others drug transporters, as well as changes in the drug target and/or implementation of alternative metabolic pathways. As an alternative to conventional antimicrobials, substances and molecules extracted from plants have demonstrated potential for controlling these pathogens. These phytochemicals can trigger the inhibition and/or death of Cryptococcus through morphological changes on fungi cells, inhibition of ergosterol synthesis, cell leakage, capsular decrease, interference in cell division, reduction of activity of several enzymes such as laccase and urease, inhibition of biofilm formation, among others. In this sense, plants are an important source of bioactive compounds with antimicrobial activity that can be studied in the search for new drugs that are increasingly effective, specific and less toxic in the control of cryptococcosis.