Molecular types of Cryptococcus species isolated from patients with cryptococcal meningitis in a Brazilian tertiary care hospital

ABSTRACT There are limited data on the molecular epidemiology of cryptococcosis in Brazil. Here, we report on the identification of the molecular pattern of the Cryptococcus species that caused meningitis in patients admitted in a Brazilian reference tertiary care hospital, and review the published studies addressing the molecular epidemiology of Cryptococcus in Brazil. Our study has shown the predominance of molecular type VNII in HIV-infected patients with cryptococcal meningoencephalitis. Molecular types VNII and VGII were occasionally detected in HIV-infected and non-infected patients with meningoencephalitis. In contrast, previous studies have shown that several regions exhibited a high prevalence of the VNI molecular type and sporadic cases of the VNII and VGII molecular types in patients with cryptococcosis in Brazil. Additional studies including VNII isolates will contribute to understanding the epidemiology and phylogenetic relationship of these genotype compared to the other ones. So far, no clear correlation has been established between genotypes, antifungal susceptibility for Cryptococcus and clinical outcome in cryptococcosis.

Cryptococcal meningitis epidemiology: 17 years of experience in a State of the Brazilian Pantanal

Abstract INTRODUCTION: This study aimed to describe cryptococcal meningitis (CM) cases and the associated demographic, clinical, and microbiological data obtained from cities in the State of Mato Grosso do Sul in the Midwestern region of Brazil. METHODS: The data from 129 patients with laboratory-confirmed CM admitted from 1997 to 2014 were retrospectively reviewed. The molecular types of Cryptococcus neoformans and Cryptococcus gattii isolated from cerebrospinal fluid were analyzed to determine their geographic distribution. RESULTS: The patients had a mean age of 37 years and consisted mostly of men (76.7%). Most of the Cryptococcus isolates were obtained from patients infected with human immunodeficiency virus (HIV) and included 105 (87.5%) and 5 (55.6%) isolates of C. neoformans and C. gattii complexes, respectively. A restriction fragment length polymorphism (RFLP) analysis of URA5 revealed that most of the isolates were C. neoformans molecular type VNI (89.1%), whereas the molecular types VGII (7%) and VNII (3.9%) were observed less frequently. Notably, 65% of the cases with a time from symptom onset to laboratory diagnosis of more than 60 days resulted in fatalities, and sequelae were observed among the patients who survived. CONCLUSIONS: The present study documents the occurrence of neurocryptococcosis, which is mainly caused by C. neoformans VNI, in Mato Grosso do Sul, Brazil, with probable autochthonous cases in the Brazilian Pantanal, the world's largest tropical wetland, and a biome where cryptococcosis has not yet been explored.

Caracterización molecular de los aislamientos de Cryptococcus neoformans de pacientes con HIV, Guayaquil, Ecuador / Molecular characterization of Cryptococcus neoformans isolates from HIV patients, Guayaquil, Ecuador

Resumen Introducción. La neurocriptococosis es una infección fúngica oportunista que representa un alto costo en vidas humanas y para la economía de los países. Sus agentes causales, las especies del complejo Cryptococcus neoformans/Cryptococcus gattii, tienen una fase sexuada y otra asexuada, cuatro serotipos principales y siete variedades moleculares con diferencias clínico-epidemiológicas, fenotípicas y de sensibilidad a los antifúngicos. Objetivo. Caracterizar molecularmente los aislamientos clínicos de C. neoformans de Guayaquil, Ecuador. Materiales y métodos. Se determinó el tipo de apareamiento, el serotipo y la variedad molecular mediante reacción en cadena de la polimerasa y análisis del polimorfismo de los fragmentos de restricción de 27 aislamientos levaduriformes previamente identificados como C. neoformans mediante métodos convencionales. Los aislamientos fueron recuperados del líquido cefalorraquídeo de pacientes con síndrome neurológico seropositivos para HIV, internados en el Hospital de Infectología "Dr. José Daniel Rodríguez Maridueña", entre diciembre de 2013 y enero de 2015. Resultados. Se demostró el amplio predominio de C. neoformans del serotipo A, MATα y el genotipo VNI entre los aislamientos estudiados. Conclusiones. Estos datos son similares a los obtenidos en otros países y son los primeros de su tipo en Guayaquil, Ecuador, por lo cual constituyen un aporte importante al conocimiento de la criptococosisen esta ciudad.

Abstract Introduction: Neurocryptococcosis is an opportunistic fungal infection that represents a high cost in human lives and for the economy of countries. Its causative agent, the Cryptococcus neoformans/Cryptococcus gattiispecies complex, has a sexual and an asexual phase, four major serotypes and seven molecular varieties with phenotypic, clinical-epidemiological and antifungal susceptibility differences. Objective: To characterize by molecular methods clinicalisolates of C. neoformans from Guayaquil, Ecuador. Materials and methods: We determined mating types, serotypes and molecular varieties by PCR and RFLP in 27 yeast isolates previously identified as C. neoformans by conventional methods. The isolates were recovered from cerebrospinal fluid of HIV seropositive patients with neurological syndrome admitted at "Dr. José Daniel Rodríguez Maridueña" Hospital from December, 2013, to January, 2015. Results: We established a wide prevalence of C. neoformans serotype A, MATαand genotype VNI among the studied isolates. Conclusions: These data are similar to those obtained in other countries and the first identified by molecular characterization in Guayaquil, Ecuador. Therefore, they constitute an important contribution to the knowledge on cryptococcosis in this country.

Molecular diagnosis of cryptococcal meningitis in cerebrospinal fluid: comparison of primer sets for Cryptococcus neoformans and Cryptococcus gattii species complex

Aim: This study evaluated the use of polymerase chain reaction for cryptococcal meningitis diagnosis in clinical samples. Materials and methods: The sensitivity and specificity of the methodology were evaluated using eight Cryptococcus neoformans/C. gattii species complex reference strains and 165 cere- brospinal fluid samples from patients with neurological diseases divided into two groups: 96 patients with cryptococcal meningitis and AIDS; and 69 patients with other neurological opportunistic diseases (CRL/AIDS). Two primer sets were tested (CN4-CN5 and the multiplex CNa70S-CNa70A/CNb49S-CNb-49A that amplify a specific product for C. neoformans and another for C. gattii). Results: CN4-CN5 primer set was positive in all Cryptococcus standard strains and in 94.8% in DNA samples from cryptococcal meningitis and AIDS group. With the multiplex, no 448-bp product of C. gattii was observed in the clinical samples of either group. The 695 bp products of C. neoformans were observed only in 64.6% of the cryptococcal meningitis and AIDS group. This primer set was negative for two standard strains. The specificity based on the negative samples from the CTL/AIDS group was 98.5% in both primer sets. Conclusions: These data suggest that the CN4/CN5 primer set was highly sensitive for the identification of C. neoformans/C. gattii species complex in cerebrospinal fluid samples from patients with clinical suspicion of cryptococcal meningitis. .

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. .

Genotypes of Cryptococcus neoformans and Cryptococcus gattii as agents of endemic cryptococcosis in Teresina, Piauí (northeastern Brazil)

Throughout Brazil, Cryptococcus neoformans is the cause of cryptococcosis, whereas Cryptococcus gattii is endemic to the northern and northeastern states. In this study, the molecular types of 63 cryptococcal isolates recovered from the cerebrospinal fluid of meningitis patients diagnosed between 2008-2010 in Teresina, Piauí, Brazil, were analysed. Out of the 63 patients, 37 (58.7 percent) were human immunodeficiency virus (HIV)-positive and 26 (41.3 percent) were HIV-negative. URA5-restriction fragment length polymorphism analysis identified 37/63 (58.7 percent) isolates as the C. neoformans VNI genotype, predominantly in HIV-positive patients (32/37, 86.5 percent), and 24/63 (38.1 percent) as the C. gattii VGII genotype, mostly in HIV-negative patients (21/26, 80.8 percent). The occurrence of C. gattii VGII in six apparently healthy children and in seven adolescents/young adults in this region reaffirms the endemic occurrence of C. gattii VGII-induced primary cryptococcosis and early cryptococcal infection. Lethality occurred in 18/37 (48.6 percent) of the HIV-positive subjects and in 13/26 (50 percent) of the HIV-negative patients. Our results provide new information on the molecular epidemiology of C. neoformans and C. gattii in Brazilian endemic areas.

Cryptococcus gattii molecular type VGII as agent of meningitis in a healthy child in Rio de Janeiro, Brazil: report of an autochthonous case / Cryptococcus gattii tipo molecular VGII como agente causador de meningoencefalite em criança saudável no Rio de Janeiro, Brasil: relato de um caso autóctone

Cryptococcus gattii causes meningoencephalitis in immunocompetent hosts, occurring endemically in some tropical and subtropical regions. Recently, this fungus was involved in an outbreak in Vancouver Island and British Columbia (Canada). In this temperate region, the VGII type is predominant. The paper describes an autochthonous case of meningoencephalitis by C. gattii VGII in a previously health child in Rio de Janeiro, considered nonendemic region of Brazil. The fungus was identified by biochemical tests and the molecular type was determined by URA5-RFLP. The present report highlights the need for clinical vigilance for primary cryptococcal meningitis in nonendemic areas.

Cryptococcus gattii é causa de meningoencefalite em hospedeiros imunocompetentes, ocorrendo endemicamente em regiões tropicais e subtropicais. Recentemente foi causador de surtos na Ilha de Vancouver e na Columbia Britânica (Canadá). Nesta região de clima temperado, o tipo VGII é predominante. Relatamos um caso de meningoencefalite pelo C.gattii tipo VGII acometendo criança previamente saudável autóctone do Rio de Janeiro, região não endêmica do Brasil. O agente foi identificado por testes bioquímicos e o tipo molecular determinado através de URA5-RFLP. O presente relato enfatiza a necessidade de vigilância clínica para a meningite criptocóccica primária em áreas não endêmicas.

Clinical and mycological profile of cryptococcosis in a tertiary care hospital.

This study examined the extent of cryptococcosis in clinically diagnosed cases of meningitis in HIV-1 seropositive and apparently immunocompetent patients. One hundred and forty-six samples, obtained from 126 chronic meningitis patients comprised of cerebrospinal fluid (CSF), blood, sputum and urine. The samples were processed by standard microbiological procedures. Cryptococcal isolates were identified by microscopy, cultural characteristics, melanin production on niger seed agar and hydrolysis of urea. The isolates were further speciated on cannavanine glycine bromothymol blue (CGB) media. Cryptococcal antigen detection of CSF samples was performed by latex agglutination test (LAT). Minimum inhibitory concentration (MIC) of amphotericin B for the isolates was also tested. Cryptococcosis was diagnosed in 13 patients (eight HIV-1 seropositive and five apparently immunocompetent). Cryptococcus neoformans var. neoformans was the predominant isolate. Cryptococcal antigen was detected in all, whereas microscopy could detect yeast cells in nine patients. The isolates were sensitive to amphotericin B. CD4 cell counts ranged from 8 to 96/cu mm. The study concludes that all CSF samples with clinical diagnosis of subacute and chronic meningitis should be subjected to tests for detection of Cryptococcus in clinical laboratory irrespective of the immune status.

Criptococosis diseminada en pacientes con SIDA. Análisis clÝnico, microbiológico e inmunológico de 51 pacientes / Disseminated cryptococcosis in patients with AIDS. Clinical, microbiological, and immunological analysis of 51 patients

In the context of HIV infection, cryptococcal meningitis is the most common mycosis threatening the patient's life. We conducted a retrospective evaluation to determine the epidemiological, microbiological, immunological and clinical characteristics of disseminated cryptococcosis in 51 hospitalised HIV seropositive patients. All the individuals (n = 51) presented reactive serology for HIV (ELISA and/or Western blot) and none fulfilled strict HAART treatment, previous to the opportunistic infection. CD4+ lymphocyte T counts showed levels between 361 and 0 cells/microliter (mean = 45). All patients but one had counts lower than 100 cells/microliter. Cryptococcosis presented as unique episode in 35 patients (68.6) and in 16 as relapse (31.3). In all of them we detected central nervous system involvement. The induction treatment was carried out with amphotericin B (AMB), continued with maintenance therapy with fluconazole. Lethality rate was 36.7, slightly superior among patients in relapse (40) compared to those who presented a first episode of the mycosis (35.2). In those individuals for whom data were available, 65.2 of blood cultures, 94.1 of CSF cultures and 79.06 of microscopic CSF examination with India ink were positive. Titers of Cryptococcus neoformans capsular antigen in CSF > or = 1/1000 were found in 36.1 and > or = 1/1000 in 73.6 of serum samples. In conclusion, manifestations and severity of disseminated cryptococcosis continue maintaining the characteristics of half a decade behind, in those patients who are not treated with HAART. Neurological involvement existed in all patients of this cohort. Treatment is not able to modify the parameters of mortality seen in previous communications. Diagnostic methods applied in this study are in accordance with those in the bibliography.

Meningitis por Criptococcus neoformans en pacientes con SIDA en el Hospital Santo Tomas / Cryptococcus neoformans meningitis in patients with AIDS at the Saint Thomas Hospital

The authors studied the clinical histories of 17 patients with AIDS who were hospitalized with the diagnosis of Meningoencephalitis. Laboratory studies showed the causative agent to be Cryptococcus neoformans. All patients had fever and most had localized headache. Some patients had nausea and vomiting, nuchal rigidity and convulsions. One each had blurred vision, photophobia, periods of disorientation, ataxia, lumbar or cervical pain. Cell count, chemical analysis, India ink preparation and culture of the cerebrospinal fluid confirmed the diagnosis and the etiologic agent. Blood cultures were negative in the few patients on whom it was performed. The best results of therapy were obtained in the patients who received Amphotericin B and Fluocytosine (80%) in dosages of 0.3 to 1 mg/k/day and 150 mg/day respectively, for 21 days

Cryptococcal intracerebral mass lesions associated with cryptococcal meningitis.

12 patients with cryptococcal intracerebral mass lesions associated with cryptococcal meningitis are reported. There were 6 males and 6 females with a mean age of 41 years. Most of them presented with symptoms and signs of meningitis initially without focal neurological signs. Computed tomography revealed single or multiple, well or poorly marginated hypodense lesions without perilesional edema, except 1 case which had marked edema, with or without ring enhancement. One case had small hyperdense, diffuse, enhanced nodules. New lesions or progression of the existing lesions occurred during medical therapy in some patients. The clinical manifestations of patients with intracranial mass lesions did not significantly differ from patients without mass lesions, but the mortality was higher (50% vs 29%).