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 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.

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.

First isolation of Cryptococcus gattii molecular type VGII and Cryptococcus neoformans molecular type VNI from environmental sources in the city of Belém, Pará, Brazil

Cryptococcus neoformans and Cryptococcus gattii are important agents of meningoencephalitis in humans in the city of Belém. This clinical data suggests that the region may be a highly endemic area for the pathogenic Cryptococcus species within the state of Pará (PA), Northern Brazil. Preliminary analysis of 11 environmental samples from the city of Belém showed two positive locations, including a hollow of a kassod tree (Senna siamea) colonized simultaneously by C. gattii molecular type VGII and C. neoformans molecular type VNI, and a birdcage in a commercial aviary positive for C. neoformans, molecular type VNI. This is the first evidence of an environmental occurrence of molecular types VNI and VGII in PA.

Suscetibilidade in vitro e resistência a antifúngicosestudo comparativo entre os tipos moleculares VNI de Cryptococcus neoformans e VGI, VGII de Cryptococcus gattii pela citometria de fluxo

A criptococose é uma micose sistêmica adquirida pela inalação de basidiosporos ou leveduras desidratadas de Cryptococus neoformans e Cryptococus gattii, estas duas espécies podem causar criptococose oportunista e primária respectivamente. C. neoformans está constituído de tipos moleculares VNI-VNIV e C. gattii de VGI-VGIV que apresentam distribuição geográfica diferenciada, como por exemplo, o tipo VNI é cosmopolita e está associado a AIDS e VGI predominando na Austrália e EUA, o tipo VGII predominando no Brasil e America Latina. Este trabalho tem por objetivo realizar estudo comparado dos tipos moleculares VNI de C. neoformans, VGI e VGII de C. gattii analisando diferentes aspectos tais como: 1- Determinar o perfil da suscetibilidade in vitro da concentração inibitória mínima (CIM) de fluconazol (FLZ), itraconazol (ITZ), 5-fluorocitosina (5FC) e anfotericina B (AMB), isoladamente e de forma combinada de AMB com 5FC e AMB com Voriconazol (VRZ); 2- Determinar CIM pela citometria de fluxo (CMF) frente a FLZ, ITZ e AMB; 3- Definir a concentração mínima letal (CML) de AMB e 5FC, isoladamente e em combinação; 4- Avaliar a ação da melanina frente a 5FC e AMB na forma combinada e isolada de 5FC; 5- Induzir a resistência in vitro para FLZ e padronizar os fluorocromos: acetoximetil - calceína (calceina-AM), acetoximetil - 2\2019, 7\2019 -bis-(2-carboxietil)-5-(e -6)- carboxifluoresceína (BCECF-AM), rodamina 123 (Rh123) e iodeto de 3, 3\2019 \2013dipentiloxacarbocianina (DiOC5) na CMF para verificar a expressão de bombas de efluxo; 6- Comparar a expressão de bombas de efluxo Os resultados permitiram identificar diferentes fenótipos de suscetibilidade que foram analisados e comparados entre as duas espécies e os tipos moleculares, permitindo a produção de quatro artigos; sendo assim, concluímos que: 1- Na análise das CIMs o tipo molecular VGII apresentou-se menos suscetível em relação a VGI e VNI; já na combinação in vitro de AMB e VRZ foi observado 100% de indiferença, e na combinação de AMB e 5FC observou-se necessidade de padronização da concentração da glicose para obter testes que possam ser futuramente relacionados a casos clínicos; 2- O método de CMF demonstrou ser alternativa de leitura automatizada, reprodutível, para os testes de suscetibilidade antifúngica com uso de Laranja de Acridina e FUN-1; 3- Foi verificada a importância da realização do teste da CML para verificar a ação protetora da melanina frente a combinação de AMB e 5FC. 4- A expressão da melanina, na combinação de AMB e 5FC reduz a detecção do sinergismo e o efeito aditivo in vitro. 5. Os isolados induzidos à resistência ao FLZ permitiram obter resultados estatisticamente significativos na verificação de Bombas de efluxo in vitro na CMF com uso de fluorocromo Dioc5 e bloqueador de protonóforos CCCP; 6- Foi verificado que 65% de isolados não induzidos a resistência e 90% de isolados induzidos a resistência do tipo molecular VGII expulsam o FLZ com certa vantagem em relação aos tipos moleculares VGI e VNI. Os resultados deste trabalho contribuem para compreensão do comportamento in vitro de C. neoformans e C. gattii frente a drogas antifúngicas cujos resultados poderão ser aplicados em estudos clínicos de correlação in vitro e in vivo para melhor compreensão da terapêutica antifúngica da criptococose e validação dos testes de suscetibilidade para estas duas espécies

First isolation of Cryptococcus gattii molecular type VGII and Cryptococcus neoformans molecular type VNI from environmental sources in the city of Belém, Pará, Brazil

Cryptococcus neoformans and Cryptococcus gattii are important agents of meningoencephalitis in humans in the city of Belém. This clinical data suggests that the region may be a highly endemic area for the pathogenic Cryptococcus species within the state of Pará (PA), Northern Brazil. Preliminary analysis of 11 environmental samples from the city of Belém showed two positive locations, including a hollow of a kassod tree (Senna siamea) colonized simultaneously by C. gattii molecular type VGII and C. neoformans molecular type VNI, and a birdcage in a commercial aviary positive for C. neoformans, molecular type VNI. This is the first evidence of an environmental occurrence of molecular types VNI and VGII in PA.

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.

Genotype and mating type distribution within clinical Cryptococcus neoformans and Cryptococcus gattii isolates from patients with cryptococcal meningitis in Uberaba, Minas Gerais, Brazil

We molecularly characterized 81 cryptococcal isolates recovered from cerebrospinal fluid samples of 77 patients diagnosed between 1998 and 2007 as having cryptococcal meningitis in Uberaba Minas Gerais, Brazil. Fifty-seven (74%) were male with a mean age 35.6 years. Seventy-two (88.9%) of the isolates were from 68 AIDS patients and cryptococcosis was the first AIDS-defining condition in 38 (55.9%) patients. Cryptococcosis and AIDS were simultaneously diagnosed in 25 (65.8%) of these 38 patients. Genotypes were characterized through the use of URA5 restriction fragment length polymorphisms analysis, the genetic variability was determined using PCR-fingerprinting with the minisatellite-specific primer M13, and the mating type and serotypes were established by PCR. Seventy-six of the 81 isolates were Cryptococcus neoformans (93.8%), while the remaining five were C. gattii (6.1%), but all were mating type a. C. neoformans isolates were genotype VNI (serotype A), while C. gattii isolates were VGII. Four of the latter isolates were identical, but only two were from AIDS patients. Six of the nine isolates from non-AIDS patients were VNI. PCR fingerprints of the isolates from two of the three AIDS patients with clinical relapse were 100% identical. The predominance of VNI and mating type a is in accordance with data from other parts of the world. The occurrence of VGII in Minas Gerais indicates a geographical expansion within Brazil.

Phylogeny and phenotypic characterization of pathogenic cryptococcus species and closely related saprobic taxa in the tremellales

Eukaryotic Cell, Vol.8, Nº3

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.

Regional pattern of the molecular types of Cryptococcus neoformans and Cryptococcus gattii in Brazil

The molecular types of 443 Brazilian isolates of Cryptococcus neoformans and Cryptococcus gattii were analyzed to determine their geographic distribution within Brazil and their underlying host conditions. The following data, imported from previous epidemiological studies as well as two culture collections, were analyzed for: place of isolation, source (clinical or environmental), host risk factors, species, serotype, mating type, and molecular type. Molecular typing by PCR-fingerprinting using primers for the minisatellite-specific core sequence of the wild-type phage M13 or microsatellites [(GACA)4, (GTG)5], restriction fragment length polymorphism of URA5 gene analysis, and/or amplified fragment length polymorphism (AFLP) identified eight major genotypes: VNI/AFLP1, VNII/AFLP1A, VNIII/AFLP2, and VNIV/AFLP3 for C. neoformans, and VGI/AFLP4, VGII/AFLP6, VGIII/AFLP5, and VGIV/AFLP7 for C. gattii. The most common molecular type found in Brazil was VNI (64%), followed by VGII (21%), VNII (5%), VGIII (4%), VGI and VNIV (3% each), and VNIII (< 1%). Primary cryptococcosis caused by the molecular type VGII (serotype B, MAT) prevails in immunocompetent hosts in the North and Northeast regions, disclosing an endemic regional pattern for this specific molecular type in the Northern 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%) were human immunodeficiency virus (HIV)-positive and 26 (41.3%) were HIV-negative. URA5-restriction fragment length polymorphism analysis identified 37/63 (58.7%) isolates as the C. neoformans VNI genotype, predominantly in HIV-positive patients (32/37, 86.5%), and 24/63 (38.1%) as the C. gattii VGII genotype, mostly in HIV-negative patients (21/26, 80.8%). 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%) of the HIV-positive subjects and in 13/26 (50%) of the HIV-negative patients. Our results provide new information on the molecular epidemiology of C. neoformans and C. gattii in Brazilian endemic areas.

Cryptococcosis in Colombian children and literature review

Cryptococcosis is reported in adults and is often acquired immune deficiency syndrome (AIDS)-associated; however, its frequency in children is low. Based on the National Survey on Cryptococcosis conducted in Colombia, an epidemiological and clinical analysis was performed on cases of the disease observed in children less than 16 years old between 1993-2010. We found 41 affected children (2.6% prevalence) from the 1,578 surveys received. The country mean annual incidence rate was 0.017 cases/100,000 children under 16 years, while in Norte de Santander the incidence rate was 0.122 cases/100,000 (p < 0.0001). The average age of infected children was 8.4 and 58.5% were male. In 46.3% of cases, a risk factor was not identified, while 24.4% had AIDS. The most frequent clinical manifestations were headache (78.1%), fever (68.8%), nausea and vomiting (65.6%), confusion (50%) and meningeal signs (37.5%). Meningitis was the most frequent clinical presentation (87.8%). Amphotericin B was given to 93.5% of patients as an initial treatment. Positive microbiological identification was accomplished by India ink (94.7%), latex in cerebrospinal fluid (100%) and culture (89.5%). Out of 34 isolates studied, Cryptococcus neoformans var. grubii (VNI 85.3%, VNII 8.8%) was isolated in 94.1% of cases and Cryptococcus gattii (VGII) was isolated in 5.9% of cases. These data are complemented by a literature review, which overall suggests that cryptococcosis in children is an unusual event worldwide.

Ancient dispersal of the human fungal pathogen Cryptococcus gattii from the Amazon rainforest

Over the past two decades, several fungal outbreaks have occurred, including the high-profile 'Vancouver Island' and 'Pacific Northwest' outbreaks, caused by Cryptococcus gattii, which has affected hundreds of otherwise healthy humans and animals. Over the same time period, C. gattii was the cause of several additional case clusters at localities outside of the tropical and subtropical climate zones where the species normally occurs. In every case, the causative agent belongs to a previously rare genotype of C. gattii called AFLP6/VGII, but the origin of the outbreak clades remains enigmatic. Here we used phylogenetic and recombination analyses, based on AFLP and multiple MLST datasets, and coalescence gene genealogy to demonstrate that these outbreaks have arisen from a highly-recombining C. gattii population in the native rainforest of Northern Brazil. Thus the modern virulent C. gattii AFLP6/VGII outbreak lineages derived from mating events in South America and then dispersed to temperate regions where they cause serious infections in humans and animals.

Molecular typing of IberoAmerican Cryptococcus neoformans isolates

A network was established to acquire basic knowledge of Cryptococcus neoformans in IberoAmerican countries. To this effect, 340 clinical, veterinary, and environmental isolates from Argentina, Brazil, Chile, Colombia, Mexico, Peru, Venezuela, Guatemala, and Spain were typed by using M13 polymerase chain reaction-fingerprinting and orotidine monophosphate pyrophosphorylase (URA5) gene restriction fragment length polymorphsm analysis with Hhal and Sau961 in a double digest. Both techniques grouped all isolates into eight previously established molecular types. The majority of the isolates, 68.2% (n=232), were VNI (var. grubii, serotype A), which accords with the fact that this variety causes most human cryptococcal infections worldwide. A smaller proportion, 5.6% (n=19), were VNII (var. grubii, serotype A); 4.1% (n=14), VNIII (AD hybrid), with 9 isolates having a polymorphism in the URA5 gene; 1.8% (n=6), VNIV (var. neoformans, serotype D); 3.5% (n=12), VGI; 6.2% (n=21), VGII; 9.1% (n=31), VGIII, and 1.5% (n=5) VGIV, with all four VG types containing var. gattii serotypes B and C isolates.

Tipagem molecular, perfis de sensibilidade e caracterização de transcritos diferencialmente expressos durante a infecção de Cryptococcus neoformans

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)