Criptococose: estudo clínico-epidemiológico, laboratorial e das variedades do fungo em 96 pacientes

Estudo prospectivo foi realizado no Hospital de Clínicas da Universidade Federal de Uberlândia, entre março de 1998 e novembro de 2003, em 96 pacientes com diagnóstico clínico e laboratorial de criptococose, sendo 81,3% portadores de Aids. Cepas de Cryptococcus neoformans foram obtidas de diferentes amostras, sendo 77% em líquido cefalorraquidiano. A var neoformans foi isolada em 89 casos e a var gattii em 7. A meningoencefalite criptocócica (56,3% dos casos), foi a manifestação clínica mais descrita, seguida da fungemia (13,5%). Entre os fatores de risco, a AIDS (81,3%) foi o mais comumente associado à micose. A pesquisa direta do fungo realizada em 121 amostras demonstrou o microrganismo em 98,3%, com cultura (+) em todas. Dos pacientes, 59,4% foram tratados com anfotericina B ou derivados triazólicos, sendo que 72,9% evoluíram para óbito, em particular os portadores de AIDS (62,5%). Atualmente, a criptococose tem sido diagnosticada com muita freqüência em nosso meio e constitui uma das doenças oportunísticas de maior morbidade e mortalidade nos pacientes com AIDS.

Chemical composition, toxicity and larvicidal and antifungal activities of Persea americana (avocado) seed extracts

The present study had the aim of testing the hexane and methanol extracts of avocado seeds, in order to determine their toxicity towards Artemia salina, evaluate their larvicidal activity towards Aedes aegypti and investigate their in vitro antifungal potential against strains of Candida spp, Cryptococcus neoformans and Malassezia pachydermatis through the microdilution technique. In toxicity tests on Artemia salina, the hexane and methanol extracts from avocado seeds showed LC50 values of 2.37 and 24.13mg mL-1 respectively. Against Aedes aegypti larvae, the LC50 results obtained were 16.7mg mL-1 for hexane extract and 8.87mg mL-1 for methanol extract from avocado seeds. The extracts tested were also active against all the yeast strains tested in vitro, with differing results such that the minimum inhibitory concentration of the hexane extract ranged from 0.625 to 1.25mg L-¹, from 0.312 to 0.625mg mL-1 and from 0.031 to 0.625mg mL-1, for the strains of Candida spp, Cryptococcus neoformans and Malassezia pachydermatis, respectively. The minimal inhibitory concentration for the methanol extract ranged from 0.125 to 0.625mg mL-1, from 0.08 to 0.156mg mL-1 and from 0.312 to 0.625mg mL-1, for the strains of Candida spp., Cryptococcus neoformans and Malassezia pachydermatis, respectively.

Micoses sistêmicas: fatores associados ao óbito em pacientes com infecção pelo vírus da imunodeficiência humana, Cuiabá, Estado de Mato Grosso, 2005-2008

A prevalência de micose sistêmica entre 1.300 pacientes portadores de HIV/Aids de Cuiabá, Mato Grosso foi de 4,6%, no período de 2005-2008. As espécies de fungos isoladas foram o Cryptococcus neoformans (50%), Cryptococcus gattii (1,6%), Cryptococcus spp (6,6%), Histoplasma capsulatum (38,3%) e Paracoccidioides brasiliensis (3,3%). Óbito foi registrado em 32 (53,3%) pacientes, sendo a criptococose a principal causa. A contagem de linfócitos T CD4+ foi baixa e semelhante entre os pacientes que sobreviveram ou faleceram por micose sistêmica. O etilismo (OR:8,2; IC95%: 1,4-62,1; p=0,005) e o nível médio de desidrogenase lática [758 (182) U/L vs 416 (268) U/L; p<0,001] foram as características independentemente associadas ao óbito dos pacientes do estudo. Os resultados mostram alta letalidade por micoses sistêmicas em pacientes portadores de HIV/Aids de Cuiabá e sugerem que características clínico-laboratoriais tais como o etilismo e a elevação precoce da desidrogenase lática podem ser fatores relacionados ao pior prognóstico nessas condições.

Evaluation of the histopathological hepatic lesions and opportunistic agents in Brazilian HIV patients

INTRODUCTION: to evaluated the type histopathological hepatic lesions and opportunistic agents in Brazilian HIV-infected patients. METHODS: we examined 52 percutaneous liver biopsies of 50 HIV-infected patients who had at least two of the following conditions: fever of unknown origin, unexplained severe emaciation, hepatomegaly or abnormal liver chemistry. The specimens were cultured for mycobacteria and fungi and stained by standard procedures. RESULTS: reactive patterns, granulomatous hepatitis and chronic active hepatitis were verified in 28 (54%), 11 (21%) and 8 (15%) of the patients respectively. Opportunistic infections were diagnosed in 18 (36%) patients: mycobacteria in 12 (24%), Cryptococcus neoformans in 5 (10%) patients and mycobacteria and yeast was isolated from the same liver fragment in one patient. CONCLUSIONS: mycobacteriosis was the most common opportunistic infection and liver tissue culture is an important method to detect opportunistic agents, even in the absence of histological lesions.

Fungemia in a university hospital: an epidemiological approach

INTRODUCTION: Fungemia corresponds to the isolation of fungi in the bloodstream and occurs mostly in immunosuppressed patients. The early diagnosis and treatment of these infections are relevant given the serious threat to the affected patients and possible spread to other organs, often becoming fatal. The growing number of fungemia associated with poor prognosis resulted in this research aiming to diagnose and assess the epidemiological aspects of hematogenous infections by fungi. METHODS: The study included 58 blood samples collected within a 1-year period, from patients at the Hospital das Clinicas, Federal University of Pernambuco, by venipuncture in vacuum tubes. Blood samples were processed for direct examination and culture and identification, conducted by observing the macroscopic and microscopic characteristics, as well as physiological characteristics when necessary. RESULTS: Eight (13.8%) episodes of fungemia were identified, accounting for the total sample, and these pathogens were Candida, Histoplasma, Trichosporon, Cryptococcus, and a dematiaceous fungus. C. albicans was the prevalent species, accounting for 37.5% of the cases. Most affected patients were adult males. There was no predominance for any activity, and the risk of acquired immunodeficiency syndrome was the underlying pathology most often cited. CONCLUSIONS: The isolation of fungi considered as emergent species, such as C. membranifaciens and dematiaceous species, highlights the importance of epidemiological monitoring of cases of fungemia in immunocompromised patients, as the therapy of choice depends on the knowledge of the aethiological agent.

Clinical and microbiological features of cryptococcal meningitis

Introduction In this study, the clinical features, underlying diseases and clinical outcomes of patients with cryptococcosis were investigated. In addition, a molecular analysis of the Cryptococcus neoformans species complex isolated from these patients was performed. Methods A prospective study of 62 cases of patients with cryptococcal infection was conducted at the Hospital de Doenças Tropicais de Goiás Dr. Anuar Auad from 2009-2010. Cryptococcal meningitis cases were diagnosed by direct examination and cerebrospinal fluid (CSF) sample culture. The profiling of these patients was assessed. The CSF samples were submitted to India ink preparation and cultured on Sabouraud dextrose agar, and C. neoformans was identified by the production of urease, a positive phenoloxidase test and assimilation of carbohydrates. C. neoformans and C. gattii isolates were distinguished by growth on L-canavanine-glycine-bromothymol blue medium, and molecular analysis was conducted via PCR fingerprinting reactions using M13 and (GACA)4 primers. Results From the 62 patients with cryptococcosis, 71 isolates of CSF were obtained; 67 (94.4%) isolates were identified as C. neoformans var. grubii/VNI, and 4 (5.6%) were identified as C. gattii/VGII. Of these patients, 53 had an HIV diagnosis. The incidence of cryptococcosis was higher among patients 20-40 years of age, with 74.2% of the cases reported in males. Cryptococcus-related mortality was noted in 48.4% of the patients, and the symptoms were altered sensorium, headache, fever and stiff neck. Conclusions The high morbidity and mortality observed among patients with cryptococcosis demonstrate the importance of obtaining information regarding the epidemiological profile and clinical course of the disease in the State of Goiás, Brazil.

In vitro antifungal activities of leaf extracts of Lippia alba (Verbenaceae) against clinically important yeast species

Introduction There are few studies reporting the antifungal activities of Lippia alba extracts. Methods A broth microdilution assay was used to evaluate the antifungal effects of Lippia alba extracts against seven yeast species of Candida and Cryptococcus. The butanol fraction was investigated by gas chromatography-mass spectrometry. Results The butanol fraction showed the highest activity against Candida glabrata. The fraction also acted synergistically with itraconazole and fluconazole against C. glabrata. The dominant compounds in the butanol fraction were 2,2,5-trimethyl-3,4-hexanedione, 3,5-dimethyl-4-octanone and hexadecane. Conclusions The butanol fraction may be a good candidate in the search for new drugs from natural products with antifungal activity.

Puerperal brain cryptococcoma in an HIV-negative woman successfully treated with fluconazole: a case report

Cryptococcus spp. cerebral abscesses are uncommon in immunocompetent subjects. The recommended induction treatment is the administration of amphotericin B plus flucytosine combined with resection for lesions ≥3cm. In this paper, we describe an HIV-negative woman diagnosed with a large cryptococcoma in the immediate postpartum period. The lesion was not resected, and due to amphotericin B intolerance, she received an extended course of fluconazole monotherapy. There was no disease recurrence during the 4 years of follow-up. The abrupt onset of her symptoms following delivery suggests that she developed a postpartum immune reconstitution syndrome. This case also demonstrates that in specific situations fluconazole monotherapy can be attempted in immunocompetent patients with cryptococcoma.

Ventriculoperitoneal shunts for treating increased intracranial pressure in cryptococcal meningitis with or without ventriculomegaly

Introduction Cryptococcosis is an opportunistic mycosis, especially in patients that are human immunodeficiency virus (HIV)-positive, and frequently involves the central nervous system. Methods We assessed the potential of ventriculoperitoneal shunting (VPS) in preventing mortality due to uncontrollable intracranial hypertension (ICH) in 15 patients with acquired immunodeficiency syndrome (AIDS)-related cryptococcal meningitis. Results After 2 weeks of antifungal therapy consisting of amphotericin B deoxycholate with or without fluconazole, patients with persistent ICH underwent VPS, despite having persistent Cryptococcus neoformans infection. In 12 patients, the uncontrollable ICH was resolved by VPS. Conclusions Patients with cryptococcal meningoencephalitis who have ICH must be considered for VPS even with positive cerebrospinal fluid cultures.

Cryptococcosis in Atlántico, Colombia: an approximation of the prevalence of this mycosis and the distribution of the etiological agent in the environment

ABSTRACTINTRODUCTION:Cryptococcosis is an invasive disease acquired by inhalation of infectious propagules from the environment. Currently, compulsory notification of the spread of this disease is not required in Colombia. However, reporting of human immunodeficiency virus (HIV)/acquired immune deficiency syndrome cases to the National Surveillance System has suggested that there is a growing population at risk of contracting cryptococcosis. Few studies have described the occurrence of cryptococcosis in Colombia. Therefore, in this study, we examined the pathology of this disease in Atlántico, Colombia and determined the distributions of Cryptococcus neoformans and Cryptococcus gattii in the environment.METHODS:Clinical samples/isolates were gathered from cases of cryptococcosis previously diagnosed at health institutions in Atlántico, and surveys were completed by clinicians. The environmental study considered 32 sampling points and three tree species, i.e., Quickstick ( Gliricidia sepium ), Almond ( Terminalia catappa ), and Pink trumpet ( Tabebuia rosea ). Environmental and clinical samples/isolates were analyzed for phenotypic and genotypic confirmation.RESULTS:From 1997-2014, 41 cases of cryptococcosis were reported. The mean patient age was 40.5 years (range: 18-63 years); 76% were men, and 78% were HIV positive. Isolation was possible in 38 cases ( C. neoformans , molecular type VNI in 37 cases and C. gattii , molecular type VGI in one case). In 2012-2014, 2,068 environmental samples were analyzed with a positivity of 0.4% ( C. neoformans , molecular type VNI) in Almond and Pink trumpet trees.CONCLUSIONS:Cryptococcus neoformans , molecular type VNI had a higher prevalence than C. gattii and was associated with human exposure and the pathogenesis of cryptococcosis in this geographical region.

Modificación de la actividad de células inmunocompetentes en criptococosis y distomatosis experimental

El objetivo de este proyecto es profundizar los conocimientos en las células que regulan las interacciones huésped-parásito en dos modelos experimentales diferentes donde se involucra la participación de Cryptococcus neoformans y Fasciola hepatica. En el primer modelo se continuará analizando el modelo de infección experimental con Cryptococcus neoformans. Para ello se estudiarán las modificaciones que ocurran en los mecanismos de activación del macrófago respecto a la generación de derivados tóxicos del oxígeno y derivados tóxicos del nitrógeno. Por otra parte, considerando que en células tímicas y células esplénicas de animales infectados con Cryptococcus neoformans se ha evideniciado la presencia de células con funciones supresoras; se determinará si el metabolismo de la L-arginina participa en esta actividad supresora. Cultivando células de timo y bazo se determinará la sensibilidad de estas células al óxido nítrico por medio de dadores químicos de NO y por el NO generado por macrófagos activados. Por otra parte se desarrollará un modelo de distomatosis experimental en ratas, en el cual se estudiarán las funciones accesorias de CP de ratas infectadas con Fasciola hepatica y la participación de los productos de excreción-secreción de este parásito en la modificación de estas funciones. Se analizarán las capacidades fagocíticas y presentadoras de antígeno en CP de ratas infectadas como así también en CP de animales inoculados con los productos excretores-secretores del parásito. Además se estudiará la acción de la CP de ratas infectadas con Fasciola hepatica sobre la respuesta proliferativa de células mononucleares de bazo de ratas normales estimuladas con mitógenos.

Primary endemic Cryptococcosis gattii by molecular type VGII in the state of Pará, Brazil

In order to study the infectious agents causing human disseminated cryptococcosis in the state of Pará, North Brazil, 56 isolates of Cryptococcusspp. (54 isolated from cerebral spinal fluid and two from blood cultures) from 43 cases diagnosed between 2003-2007 were analysed. The species were determined through morphological and physiological tests and genotypes were determined by URA5-RFLP and PCR-fingerprinting (wild-type phage M13). The following species and genotypes were identified: Cryptococcus neoformans VNI (28/56, 50%), Cryptococcus gattii VGII (25/56, 44.64%) and C. gattii VGI (3/56, 5.26%). The genotype VNI occurred in 12 out of 14 HIV-positive adults, whereas the genotype VGII occurred in 11 out of 21 HIV-negative adults (p < 0.02, OR = 6.6 IC95% 0.98-56.0). All patients less than 12 years old were HIV negative and six cases were caused by the VGII genotype, one by the VGI and one by VNI. Therefore, endemic primary mycosis in HIV-negative individuals, including an unexpectedly high number of children, caused by the VGII genotype deserves further study and suggests the need for surveillance on cryptococcal infection in the state of Pará, Eastern Amazon.

Inteins in pathogenic fungi: a phylogenetic tool and perspectives for therapeutic applications

Inteins or "internal proteins" are coding sequences that are transcribed and translated with flanking sequences (exteins). After translation, the inteins are excised by an autocatalytic process and the host protein assumes its normal conformation and develops its expected function. These parasitic genetic elements have been found in important, conserved proteins in all three domains of life. Most of the eukaryotic inteins are present in the fungi kingdom and the PRP8 intein is one of the most widespread inteins, occurring in important pathogens such as Cryptococcus neoformans (varieties grubii and neoformans), Cryptococcus gattii, Histoplasma capsulatum and Paracoccidioides brasiliensis. The knowledge of conserved and non-conserved domains in inteins have opened up new opportunities for the study of population variability in pathogenic fungi, including their phylogenetic relationships and recognition or diagnoses of species. Furthermore, inteins in pathogenic fungi should also be considered a promising therapeutic drug target, since once the autocatalytic splicing is inhibited, the host protein, which is typically vital, will not be able to perform its normal function and the fungal cell will not survive or reproduce.

The antimicrobial activity of lapachol and its thiosemicarbazone and semicarbazone derivatives

Lapachol was chemically modified to obtain its thiosemicarbazone and semicarbazone derivatives. These compounds were tested for antimicrobial activity against several bacteria and fungi by the broth microdilution method. The thiosemicarbazone and semicarbazone derivatives of lapachol exhibited antimicrobial activity against the bacteria Enterococcus faecalis and Staphylococcus aureus with minimal inhibitory concentrations (MICs) of 0.05 and 0.10 µmol/mL, respectively. The thiosemicarbazone and semicarbazone derivatives were also active against the pathogenic yeast Cryptococcus gattii (MICs of 0.10 and 0.20 µmol/mL, respectively). In addition, the lapachol thiosemicarbazone derivative was active against 11 clinical isolates of Paracoccidioides brasiliensis, with MICs ranging from 0.01-0.10 µmol/mL. The lapachol-derived thiosemicarbazone was not cytotoxic to normal cells at the concentrations that were active against fungi and bacteria. We synthesised, for the first time, thiosemicarbazone and semicarbazone derivatives of lapachol. The MICs for the lapachol-derived thiosemicarbazone against S. aureus, E. faecalis, C. gattii and several isolates of P. brasiliensis indicated that this compound has the potential to be developed into novel drugs to treat infections caused these microbes.

Silver nanoparticle production by the fungus Fusarium oxysporum: nanoparticle characterisation and analysis of antifungal activity against pathogenic yeasts

The microbial synthesis of nanoparticles is a green chemistry approach that combines nanotechnology and microbial biotechnology. The aim of this study was to obtain silver nanoparticles (SNPs) using aqueous extract from the filamentous fungus Fusarium oxysporum as an alternative to chemical procedures and to evaluate its antifungal activity. SNPs production increased in a concentration-dependent way up to 1 mM silver nitrate until 30 days of reaction. Monodispersed and spherical SNPs were predominantly produced. After 60 days, it was possible to observe degenerated SNPs with in additional needle morphology. The SNPs showed a high antifungal activity against Candida and Cryptococcus , with minimum inhibitory concentration values ≤ 1.68 µg/mL for both genera. Morphological alterations of Cryptococcus neoformans treated with SNPs were observed such as disruption of the cell wall and cytoplasmic membrane and lost of the cytoplasm content. This work revealed that SNPs can be easily produced by F. oxysporum aqueous extracts and may be a feasible, low-cost, environmentally friendly method for generating stable and uniformly sized SNPs. Finally, we have demonstrated that these SNPs are active against pathogenic fungi, such as Candida and Cryptococcus .