Cryptococcosis by Cryptococcus neoformans/Cryptococcus gattii Species Complexes in non-HIV-Infected Patients in Southeastern Brazil

Abstract INTRODUCTION: The clinical manifestations of cryptococcosis are usually associated with the infecting agents Cryptococcus neoformans (CN) and C. gattii (CG) species complexes and the host. In this study, non-HIV-infected patients, at a university hospital in southeastern Brazil, had epidemiological and clinical data associated with cryptococcal disease and isolated Cryptococcus species: CN - 24 patients and CG - 12 patients. METHODS: The comparison was comprised of demographic data, predisposing factors, clinical and laboratory manifestations, and outcomes of cryptococcosis patients treated between 2000 and 2016. Immunocompetent and immunosuppressed patients were also compared, irrespective of the infecting species. Cryptococcus spp. were genotyped by PCR-RFLP analysis of the URA5 gene. RESULTS: Infections by the CN species complex (100% VNI genotype) were associated with drug immunosuppression and fungemia, and patients infected with the CG species complex (83% VG II and 17% VGI genotypes) had more evident environmental exposure and higher humoral response. CN and CG affected patients with or without comorbidities. CONCLUSIONS: Diabetes mellitus, other chronic non-infectious diseases, and alcoholism were likely predisposing factors for infection by both CN and CG species. Immunocompetent patients, independent of the infecting Cryptococcus species complexes, showed a higher occurrence of meningitis and a trend toward less fungal dissemination and longer survival than immunosuppressed hosts.

Frequency and geographical distribution of genotypes and mating types of Cryptococcus neoformans and Cryptococcus gattii species complexes in Argentina / Frecuencia y distribución geográfica de genotipos y tipos sexuales de los complejos de especies Cryptococcus neoformans y Cryptococcus gattii en Argentina

Abstract The aim of this work was to know the frequency and geographical distribution of genotypes and mating types of Cryptococcus neoformans and Cryptococcus gattii species complexes isolated from human infections in Argentina during the period from April 2009 to April 2011. A multicenter study was conducted, in which 372 isolates were obtained from 61 laboratories throughout the country. Of those, 98.8% of the isolates belonged to the C. neoformans species complex and 1.1% to the C. gattii species complex. Genotype VNI (MATa) was the most frequently isolated (n = 326, 87.6%), followed by VNII (MATa) (n = 22, 5.9%), the recently described VNII-VNIV (aADa) hybrid (n = 14, 3.8%), VNIV (MATa) (n=4, 1.1%), VNIII (aADa) hybrid (n = 1, 0.3%), and VNIII (aADa) hybrid (n = 1, 0.3%). The Argentine Central region showed the greatest number of cases and genotype diversity. Interestingly, a relative high frequency was observed in genotype VNII (MATa) in the Cuyo, Northeast and Northwest regions and, also in VNII-VNIV (aADa) hybrids in the Northwest region. C. gattii species complex was isolated at a low rate; 3 VGI (MATa) and 1 VGII (MATa) isolates were obtained from the Northwest and Central regions. In conclusion, this study shows that genotype frequencies seem to vary among regions in Argentina and reveals a relatively high frequency of rare hybrids in the Northwest region. Further regional clinical and environmental studies may help to elucidate if those varia-tions in frequencies are associated with the existence of regional ecological niches or any other regional factors.

Resumen El objetivo de! trabajo fue conocer la frecuencia y la distribución geográfica de genotipos y tipos sexuales de aislados pertenecientes a los complejos de especies Cryptococcus neoformansy Cryptococcus gattii obtenidos de infecciones humanas en Argentina. Entre abril de 2009 y abril de 2011 se realizó un estudio multicéntrico del que se obtuvieron 372 aislados de 61 laboratorios de diferentes zonas del país. El 98,8% de los aislados pertenecieron al complejo C. neoformansy el 1,1% al complejo C. gattii. El genotipo VNI (MATa) fue el más frecuente (n = 326; 87,6%), le siguieron VNII (MATa) (n = 22; 5,9%), el híbrido VNII-VNIV (aADa) (n = 14; 3,8%), VNIV (MATa) (n =4; 1,1%) y los híbridos VNIII (aADa) (n = 1; 0,3%) y VNIII (aADa) (n = 1; 0,3%). La región Centro mostró el mayor número de casos y la mayor diversidad de genotipos. Cabe destacar que el genotipo VNII (MATa) tuvo una frecuencia relativamente alta en las regiones de Cuyo, Noreste y Noroeste. En esta última región, también fue alta la frecuencia del híbrido VNII-VNIV (aADa). La frecuencia de aislamiento de miembros del complejo C. gattii fue baja: se obtuvieron 3 aislados VGI (MATa) y 1 VGII (MATa) de las regiones Centro y Noroeste. En conclusión, este estudio muestra que las frecuencias de genotipos varían entre las distintas regiones de Argentina y señala la presencia de híbridos poco comunes en una frecuencia relativamente alta dentro de la región Noroeste. Contar con mayor número de estudios clínicos y ambientales regionales podría ayudar a elucidar si tales variaciones están asociadas a la existencia de nichos ecológicos particulares o a algún otro factor regional.

Clinical and microbiological characteristics of cryptococcosis at an university hospital in China from 2013 to 2017

ABSTRACT Background: This study aims to explore the epidemiology, clinical profile and strain characteristics of cryptococcosis from 2013 to 2017 in a major teaching hospital in China. Methods: Trends in antifungal drug susceptibility of 217 consecutive non-repetitive cryptococcal isolates collected from patients of an university hospital in China were analyzed between 2013 and 2017. Of those, 98 isolates were conserved for identification by internal transcribed spacer (ITS) sequencing and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) system. Multilocus sequence typing (MLST) was used to designate molecular types. Clinical characteristics of the 98 patients with cryptococcosis during the period of 2013-2017 were retrospectively evaluated. Results: There was a trend for gradual increase in the MIC range of fluconazole was from 2013 to 2017. The conserved 98 clinical cryptococcal isolates included 97 C. neoformans and one C. gattii, and 90 (91.8%) isolates belonged to ST5 genotype VNI. Out of the 98 patients with cryptococcosis, 28 (28.6%) were HIV-infected and 32 (32.7%) had no underlying diseases. HIV-infected patients had higher mortality than HIV-uninfected patients (28.6% vs 14.3%, p = 0.147). Conclusions: Most of the patients with cryptococcosis were not HIV-infected in this study, while patients with HIV had a higher mortality. Reduced susceptibility to fluconazole was observed among C. neoformans isolates, most of them belonged to ST5 genotype VNI having an impact on the effective dose of fluconazole.

Visible DNA microarray and loop-mediated isothermal amplification (LAMP) for the identification of Cryptococcus species recovered from culture medium and cerebrospinal fluid of patients with meningitis

Cryptococcal meningitis affects normal hosts and immunocompromised patients exhibiting high mortality rates. The objective of this study was to design two molecular assays, visible microarray platforms and loop-mediated isothermal amplification (LAMP), to identify Cryptococcus spp. and the species neoformans and gattii from the cerebral spinal fluid (CSF). To identify Cryptococcus and the two species, we designed two microarrays DNA platforms based on the internal transcribed spacer (ITS) region and CAP59 gene and LAMP assays specific for Cryptococcus species. The assays were tested using CSF from patients with cryptococcal meningitis. CSF from patients with cryptococcal meningitis was cultured in Sabouraud culture medium, and the Cryptococcus spp. grown in the culture medium were also tested for LAMP and microarray platforms. The results were compared to DNA sequencing of the same genetic regions. A total of 133 CSF samples were studied. Eleven CSFs were positive for Cryptococcus (9 C. neoformans and 2 C. gattii), 15 were positive for bacteria, and 107 were negative. The CAP59 platform correctly identified 73% of the CSF samples, while the ITS platform identified 45.5%. CAP59 platform correctly identified 100% of the Cryptococcus isolates, and ITS platform identified 70%. The two sets of LAMP primers correctly identified 100% of the Cryptococcus isolates. However, for CSF samples, the amplification occurred only in 55.5% of C. neoformans. The methodologies were reliable in the identification of Cryptococcus species, mainly for isolates from culture medium, and they might be applied as adjunctive tests to identify Cryptococcus species.

Epidemiological and TNFα polymorphism evaluation in patients with cryptococcal meningitis treated at a referral hospital in North Brazil

Abstract INTRODUCTION: The present study evaluated the epidemiology of cryptococcal meningitis and TNFα gene polymorphisms in patients at a reference hospital in northern Brazil. METHODS: Samples from 25 patients infected with Cryptococcus spp. were collected to confirm the infection and to analyze the TNFα gene polymorphisms. RESULTS: Cryptococcus neoformans was detected as the predominant etiological agent (100%) in HIV-positive patients. No genetic polymorphic changes were found. CONCLUSIONS: No correlation was observed between the analyzed TNFα polymorphisms and cryptococcal meningitis.

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.

Meningitis criptocóccica en pacientes sin infección por VIH: presentación de dos casos y revisión de la literatura / Meningeal cryptococcosis in patients non infected with HIV: presentation of two cases and review of the literature

Resumen La criptococosis es una enfermedad fúngica de distribución global con una mayor incidencia en países en vía de desarrollo, donde constituye una de las principales causas de meningitis, asociado a una alta morbi-mortalidad. Afecta principalmente a pacientes con inmunosupresión (por infección por VIH, tratamiento con glucocorticoides, sometidos a trasplantes, cáncer, etc). Sin embargo, en los últimos años se ha presentado un incremento de casos en inmunocompetentes, siendo tanto o más grave que en inmunocomprometidos. Presentamos dos casos de meningitis criptocóccica. Un varón de 48 años, en quien se descartó una infección por VIH u otra causa de inmunosupresión y una mujer de 67 años con antecedentes de cirrosis hepática. Debe considerarse Cryptococcus spp. como posible agente causal de meningitis, tanto en pacientes inmunocomprometidos como inmunocompetentes.

Cryptococcosis is a fungal disease of global distribution with a higher incidence in developing countries, where it represents one of the main causes of meningitis, associated with high morbidity and mortality. It mainly occurs in patients with immunosuppression (due to HIV infection, glucocorticoid treatment, transplants, cancer, etc.). However, in recent years there has been an increase in cases in immunocompetent, which is as serious or more severe than in immunocompromised. We report two cases of cryptococcal meningitis. A 48-year-old male with no pathological history, in whom HIV infection or another cause of immunosuppression was ruled out, and a 67-year-old woman with a previous diagnosis of liver cirrhosis. The above mentioned highlights the importance of always considering Cryptococcus spp. as possible causative agent of meningitis, in both immunocompromised and immunocompetent patients.

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.

The epidemiology of cryptococcosis and the characterization of Cryptococcus neoformans isolated in a Brazilian University Hospital

ABSTRACT Cryptococcosis, a systemic disease caused by the fungus Cryptococcus neoformans/ Cryptococcus gattii is more severe in immunocompromised individuals. This study aimed to analyze the epidemiology of the disease, the molecular characteristics and the antifungal susceptibility of C. neoformans isolated from patients treated in a Brazilian university hospital. This retrospective study was conducted in the Clinical Hospital, Federal University of Uberlândia, and evaluated cases of cryptococcosis and strains of C. neoformans isolated from 2004 to 2013. We evaluated 41 patients, 85% of whom were diagnosed with AIDS. The fungus was isolated from the cerebrospinal fluid (CSF) of 21 patients (51%); 19.5% had fungemia and in 24% the agent was isolated from the CSF and blood, concurrently. Meningoencephalitis was the most frequent (75%) manifestation of infection. Despite adequate treatment, the mortality of the disease was 58.5%. Most isolates (97.5%) presented the VNI genotype (serotype A, var. grubii) and one isolate was genotyped as C. gattii (VGI); all the isolates were determined as mating type MATa and showed susceptibility to the tested antifungals (fluconazole, voriconazole, amphotericin B and 5-flucytosine). Although AIDS detection rates remain stable, opportunistic infections such as cryptococcosis remain as major causes of morbidity and mortality in these patients.

Distribution and association between environmental and clinical isolates of Cryptococcus neoformans in Bogotá-Colombia, 2012-2015

The propagules of the fungal species Cryptococcus neoformans and C. gattii, whose varieties are distributed world wide, are the primary cause of cryptococcosis, a life threatening disease. The study of environmental and clinical isolates of Cryptococcosis is an important contribution to the epidemiology and ecology of the fungus. The aim of this work was to determine the presence of C. neoformans and C. gattii in the environment in Bogotá, Colombia’s capital city and to establish the relation between clinical and environmental isolates in the period 2012-2015. From a total of 4.116 environmental samples collected between October 2012 - March 2014, 35 were positive for C. neoformans var. grubii. From 55 cryptococcosis cases reported in Bogotá during 2012-2015, 49 isolates were recovered. From those, 94% were identified as C. neoformans var. grubii molecular type VNI; 4% as VNII and 1,2% as C. neoformans var neoformans VNIV. The 84 detected clinical and environmental isolates studied had a similarity between 49-100% according with molecular typing. The correlation between environmental and clinical samples confirms the hypothesis that patients acquire the disease from environmental exposure to the fungal propagules.

Validation and clinical application of a molecular method for the identification of Cryptococcus neoformans/Cryptococcus gattii complex DNA in human clinical specimens

ABSTRACT The diagnosis of cryptococcosis is usually performed based on cultures of tissue or body fluids and isolation of the fungus, but this method may require several days. Direct microscopic examination, although rapid, is relatively insensitive. Biochemical and immunodiagnostic rapid tests are also used. However, all of these methods have limitations that may hinder final diagnosis. The increasing incidence of fungal infections has focused attention on tools for rapid and accurate diagnosis using molecular biological techniques. Currently, PCR-based methods, particularly nested, multiplex and real-time PCR, provide both high sensitivity and specificity. In the present study, we evaluated a nested PCR targeting the gene encoding the ITS-1 and ITS-2 regions of rDNA in samples from a cohort of patients diagnosed with cryptococcosis. The results showed that in our hands, this Cryptococcus nested PCR assay has 100% specificity and 100% sensitivity and was able to detect until 2 femtograms of Cryptococcus DNA.

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. .

FIRST REPORT ON Cryptococcus neoformans IN PIGEON EXCRETA FROM PUBLIC AND RESIDENTIAL LOCATIONS IN THE METROPOLITAN AREA OF CUIABÁ, STATE OF MATO GROSSO, BRAZIL / Primeiro registro de Cryptococcus neoformans em excretas de pombos provenientes de locais públicos e residenciais de área metropolitana de Cuiabá, Estado do Mato Grosso, Brasil

SUMMARY Cryptococcosis is a severe systemic mycosis caused by two species of Cryptococcus that affect humans and animals: C. neoformans and C. gattii. Cosmopolitan and emergent, the mycosis results from the interaction between a susceptible host and the environment. The occurrence of C. neoformans was evaluated in 122 samples of dried pigeon excreta collected in 49 locations in the City of Cuiabá, State of Mato Grosso, Brazil, including public squares (n = 5), churches (n = 4), educational institutions (n = 3), health units (n = 8), open areas covered with asbestos (n = 4), residences (n = 23), factory (n = 1) and a prison (n = 1). Samples collected from July to December of 2010 were seeded on Niger seed agar (NSA). Dark brown colonies were identified by urease test, carbon source assimilation tests and canavanine-glycine-bromothymol blue medium. Polymerase chain reaction primer pairs specific for C. neoformans were also used for identification. Cryptococcus neoformans associated to pigeon excreta was isolated from eight (6.6%) samples corresponding to six (12.2%) locations. Cryptococcus neoformans was isolated from urban areas, predominantly in residences, constituting a risk of acquiring the disease by immunocompromised and immunocompetent individuals. .

RESUMO A criptococose é micose sistêmica potencialmente grave causada por duas espécies do gênero Cryptococcus que acometem tanto homens como animais: Cryptococcus neoformans e C. gattii. São infecções cosmopolitas e emergentes, resultantes da interação do hospedeiro - humano e animal versus meio ambiente. A proposta deste trabalho foi avaliar a ocorrência de C. neoformans em 122 amostras de excretas secas de pombos coletadas em 49 locais na cidade de Cuiabá, Estado do Mato Grosso, Brasil, incluindo: praças públicas (n = 5), igrejas (n = 4), instituições de ensino (n = 3), unidades de saúde (n = 8), áreas abertas exibindo cobertura de amianto (n = 4), conjuntos residenciais domiciliares (n = 23), uma fábrica (n = 1) e um presídio (n = 1). Semeadura de suspensão de amostras em meio ágar niger (NSA), identificação fenotípica por provas bioquímicas e teste em meio de canavanina-glicina-azul de bromotimol, das colônias isoladas com pigmentação marrom escura. Foi também utilizada a técnica da reação em cadeia da polimerase com pares de iniciadores específicos para identificação de C. neoformans. As amostras foram coletadas de julho a dezembro de 2010. Cryptococcus neoformans foi isolado em oito (6,6%) de 122 amostras correspondendo a seis (12,2%) dos 49 sítios analisados. Cryptococcus neoformans associado a excretas de pombos ocorre em áreas de Cuiabá, predominando em residências nas amostras analisadas, constituindo fator de risco potencial para aquisição da doença tanto para indivíduos imunocomprometidos como imunocompetentes. .

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. .

Genotipificación de aislamientos clínicos del complejo Cryptococcus neoformans/Cryptococcus gattii obtenidos en el Hospital ½Dr. Julio C. Perrando», de la ciudad de Resistencia (Chaco, Argentina) / [Genotyping of Cryptococcus neoformans/Cryptococcus gattii complex clinical isolates from Hospital \"Dr. Julio C. Perrando\", Resistencia city (Chaco, Argentina)].

Cryptococcosis is a fungal infection caused by yeast species of Cryptococcus genus, particularly Cryptococcus neoformans/Cryptococcus gattii species complex. The knowledge of the cryptococcosis casuistic in northeastern Argentina is scarce and there is no information about the molecular types circulating in this area. The aim of this study was to genotyping C. neoformans/C. gattii complex clinical isolates obtained at Hospital "Dr. Julio C. Perrando", Resistencia city (Chaco, Argentina), in order to determine species, variety and molecular type. During two years and one month 26 clinical isolates were studied. Using conventional and molecular methods one isolate was identified as C. gattii VGI type, and 25 isolates as C. neoformans var. grubii; 23 of these belonged to VNI type and two belonged to VNII type. This data is a contribution to the knowledge of cryptococcosis epidemiology in Argentina and the first report about C. neoformans/ C. gattii complex molecular types from clinical isolates in northeastern Argentina.

Cryptococcus neoformans carried by Odontomachus bauri ants

Cryptococcus neoformans is the most common causative agent of cryptococcosis worldwide. Although this fungus has been isolated from a variety of organic substrates, several studies suggest that hollow trees constitute an important natural niche for C. neoformans. A previously surveyed hollow of a living pink shower tree (Cassia grandis) positive for C. neoformans in the city of Rio de Janeiro, Brazil, was chosen for further investigation. Odontomachus bauri ants (trap-jaw ants) found inside the hollow were collected for evaluation as possible carriers of Cryptococcus spp. Two out of 10 ants were found to carry phenoloxidase-positive colonies identified as C. neoformans molecular types VNI and VNII. The ants may have acted as a mechanical vector of C. neoformans and possibly contributed to the dispersal of the fungi from one substrate to another. To the best of our knowledge, this is the first report on the association of C. neoformans with ants of the genus Odontomachus.

Antifungal susceptibilities of Cryptococcus species complex isolates from AIDS and non-AIDS patients in Southeast China

Cryptococcus spp. are common causes of mycoses in immunocompromised patients. To determine the drug susceptibilities of clinical Cryptococcus spp. isolates, the characteristics of 61 clinical Cryptococcus spp. complex isolates and their antifungal susceptibilities were investigated, including 52 C. neoformans and 9 C. gattii isolates collected at Shanghai between 1993 and 2009. Antifungal susceptibility of clinical isolates to amphotericin B, fluconazole, itraconazole, and flucytosine were determined by the microdilution method M27-A2 and the ATB FUNGUS 3 kit. The 90% minimum inhibitory concentration (MIC90) and susceptibility ranges were as follows: 1 (0.0625-1) µg/mL for amphotericin B, 4 (0.125-16) µg/ mL for fluconazole, 0.25 (0.0313-4) µg/mL for itraconazole, and 4 (0.125-8) µg/mL for flucytosine. Fluconazole, itraconazole, and flucytosine have excellent in vitro activity against all tested clinical Cryptococcus spp., and we also found a high rate of tolerance to amphotericin B (MICs ranging from 0.55-1 µg/mL). Furthermore, C. neoformans isolates from acquired immune deficiency syndrome (AIDS) patients were less susceptible to fluconazole and flucytosine than those from non-AIDS patients. These data suggest that use of amphotericin B may lead to tolerance or resistance of the pathogen over time. There were also no significant associations between species, genotypes, and in vitro susceptibilities of these clinical isolates.