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.

Feeding preference of the shredder Phylloicus sp. for plant leaves of Chrysophyllum oliviforme or Miconia chartacea after conditioning in streams from different biomes / Preferência alimentar do fragmentador Phylloicus sp. por folhas de Chrysophyllum oliviforme e Miconia chartacea após condicionadas em riachos de dois biomas

Abstract Macroinvertebrate shredders consume preferably leaves conditioned by fungi and bacteria which offer greater palatability to them. Plant species in Cerrado present high concentration of chemical elements such as lignin and cellulose, phenols and tanins thus making them less attractive for shredders consumption and limiting the palatability. This study aimed to evaluate the feeding preference of a macroinvertebrate shredder of the genus Phylloicus for plant material from two different biomes (Cerrado and Mata Atlântica), after conditioning in a stream of Mata Atlântica and observing their physical and chemical characteristics. Senescent leaves were collected, monthly from the litterfall of riparian vegetation in a 500 m stretch of a stream in each biome from August 2014 to January 201. The most abundant species in each stream was selected for the experiment. The experimental design consisted in with two treatments. The first (T1) comprised leaf discs from Chrysophyllum oliviforme (Cerrado species) together with leaf discs of Miconia chartacea (Atlantic Forest species) which were conditioned in the Atlantic Forest stream. The second treatment (T2) involved leaf discs of Miconia chartacea conditioned in Mata Altlântica and Cerrado streams. Both tests had showed significant differences between the two treatments (T1 and T2). For T1, there was consumption of M. chartacea leaf discs by Phylloicus sp., but there was no consumption of C. oliviforme discs. For T2, there was preference for M. chartaceae leaves conditioned in a stream of Mata Atlântica than in Cerrado stream. The results showed that Phylloicus sp., had presented preference for food detritus of the Mata Antlântica biome and rejection to the one from Cerrado biome.

Resumo Macroinvertebrados fragmentadores consomem folhas preferencialmente condicionadas por fungos e bactérias que lhes oferece uma maior palatabilidade. Nas espécies do cerrado esse condicionamento está também associado às altas concentrações de elementos químicos limitantes à palatabilidade como alto teor de lignina e celulose, que tornam as folhas menos atrativas para os fragmentadores. O trabalho teve como objetivo avaliar a preferência alimentar de macroinvertebrados fragmentador ( Phylloicus sp.) por material vegetal de dois diferentes biomas (Cerrado e Mata Atlântica), após condicionamento em riacho de Mata Atlântica, observando suas características físicas e químicas. Foram coletadas folhas senescentes do aporte vegetal (AV) de espécies nativas da vegetação ripária nesses dois biomas, com periodicidade mensal em um trecho de 500 m de um córrego em cada bioma. O experimento foi delineado com dois tratamentos. O primeiro (T1) compreendeu discos de folhas do Cerrado (Chrysophyllum oliviforme) mais discos de folhas da Mata Atlântica (Miconia chartacea) que foram condicionadas em córrego de Mata Atlântica. O segundo tratamento (T2) envolveu discos de folhas da Mata Atlântica condicionadas em córrego da Mata Atlântica mais discos de folhas da Mata Atlântica condicionadas em córrego do Cerrado. Os dois testes apontaram diferenças significativas entre os dois tratamentos (T1 e T2). Para T1 houve consumo de discos de folha de M. chartacea por Phylloicus sp, mas não houve consumo dos discos de C. oliviforme , de Cerrado. Para T2, houve o consumo, porém a preferência pelas folhas de M. chartaceae condicionadas no córrego da Mata Atlantica foi consideravelmete maior. Os resultados apontam que Phylloicus sp, apresentou preferência alimentar pelo detrito de bioma de Mata Atlântica e rejeição pelo detrito do bioma Cerrado.

Feeding preference of the shredder Phylloicus sp. for plant leaves of Chrysophyllum oliviforme or Miconia chartacea after conditioning in streams from different biomes

Abstract Macroinvertebrate shredders consume preferably leaves conditioned by fungi and bacteria which offer greater palatability to them. Plant species in Cerrado present high concentration of chemical elements such as lignin and cellulose, phenols and tanins thus making them less attractive for shredders consumption and limiting the palatability. This study aimed to evaluate the feeding preference of a macroinvertebrate shredder of the genus Phylloicus for plant material from two different biomes (Cerrado and Mata Atlântica), after conditioning in a stream of Mata Atlântica and observing their physical and chemical characteristics. Senescent leaves were collected, monthly from the litterfall of riparian vegetation in a 500 m stretch of a stream in each biome from August 2014 to January 201. The most abundant species in each stream was selected for the experiment. The experimental design consisted in with two treatments. The first (T1) comprised leaf discs from Chrysophyllum oliviforme (Cerrado species) together with leaf discs of Miconia chartacea (Atlantic Forest species) which were conditioned in the Atlantic Forest stream. The second treatment (T2) involved leaf discs of Miconia chartacea conditioned in Mata Altlântica and Cerrado streams. Both tests had showed significant differences between the two treatments (T1 and T2). For T1, there was consumption of M. chartacea leaf discs by Phylloicus sp., but there was no consumption of C. oliviforme discs. For T2, there was preference for M. chartaceae leaves conditioned in a stream of Mata Atlântica than in Cerrado stream. The results showed that Phylloicus sp., had presented preference for food detritus of the Mata Antlântica biome and rejection to the one from Cerrado biome.

Resumo Macroinvertebrados fragmentadores consomem folhas preferencialmente condicionadas por fungos e bactérias que lhes oferece uma maior palatabilidade. Nas espécies do cerrado esse condicionamento está também associado às altas concentrações de elementos químicos limitantes à palatabilidade como alto teor de lignina e celulose, que tornam as folhas menos atrativas para os fragmentadores. O trabalho teve como objetivo avaliar a preferência alimentar de macroinvertebrados fragmentador ( Phylloicus sp.) por material vegetal de dois diferentes biomas (Cerrado e Mata Atlântica), após condicionamento em riacho de Mata Atlântica, observando suas características físicas e químicas. Foram coletadas folhas senescentes do aporte vegetal (AV) de espécies nativas da vegetação ripária nesses dois biomas, com periodicidade mensal em um trecho de 500 m de um córrego em cada bioma. O experimento foi delineado com dois tratamentos. O primeiro (T1) compreendeu discos de folhas do Cerrado (Chrysophyllum oliviforme) mais discos de folhas da Mata Atlântica (Miconia chartacea) que foram condicionadas em córrego de Mata Atlântica. O segundo tratamento (T2) envolveu discos de folhas da Mata Atlântica condicionadas em córrego da Mata Atlântica mais discos de folhas da Mata Atlântica condicionadas em córrego do Cerrado. Os dois testes apontaram diferenças significativas entre os dois tratamentos (T1 e T2). Para T1 houve consumo de discos de folha de M. chartacea por Phylloicus sp, mas não houve consumo dos discos de C. oliviforme , de Cerrado. Para T2, houve o consumo, porém a preferência pelas folhas de M. chartaceae condicionadas no córrego da Mata Atlantica foi consideravelmete maior. Os resultados apontam que Phylloicus sp, apresentou preferência alimentar pelo detrito de bioma de Mata Atlântica e rejeição pelo detrito do bioma Cerrado.

Nutrient addition does not enhance leaf decomposition in a Southeastern Brazilian stream (Espinhaço mountain range)

A decomposition experiment using eucalyptus leaves was carried out in a Southeastern Brazilian mountain stream located at the transition between the Cerrado and the Atlantic Forest to test whether nutrient addition increases microbial and invertebrate colonisation and accelerates breakdown rates. The results show that none of the tested variables was significantly affected by nutrient addition, despite the average increase in ATP concentrations and invertebrate colonisation observed in the fertilised leaf bags. This could mean that breakdown in the stream was already at its maximum due to the relatively high water temperature and nutrient content, or that the breakdown rate of eucalyptus leaves was too fast to allow the detection of any effects of nutrient addition. Breakdown rates of eucalyptus leaves were much faster than the values reported in literature for most species in Brazilian Cerrado streams, suggesting that the replacement of the natural vegetation by eucalyptus may affect nutrient dynamics in the region.

Um experimento de decomposição de folhas de eucalipto foi efetuado em um riacho do sudeste do Brasil, localizado na transição entre o Cerrado e a Mata Atlântica para testar se a adição de nutrientes aumenta a colonização das folhas por microrganismos e por invertebrados e se acelera a sua taxa de decomposição. Nenhuma das variáveis testadas foi significativamente afetada pela adição de nutrientes, apesar do aumento médio verificado nas concentrações de ATP e na colonização por invertebrados dos sacos contento detritos fertilizados. Isto pode significar que a decomposição no riacho já ocorria à taxa máxima devido à temperatura e concentração de nutrientes relativamente elevadas da água ou que a taxa de decomposição das folhas de eucalipto foi demasiado rápida para permitir detectar qualquer efeito da adição de nutrientes. Os coeficientes de decomposição das folhas de eucalipto (0,1127 - 0,1192 dia-1) foram mais elevados que os verificados na literatura para a maioria das espécies em riachos do Cerrado, sugerindo que o reflorestamento com eucalipto pode alterar a dinâmica dos nutrientes na região.

Evaluación prospectiva de pacientes con SDRA con sospecha de infección por influenza a H1N1 en terapia intensiva. Seguimiento a los 30 días del alta / Prospective evaluation of patients with ARDS with suspected H1N1 influenza infection in intensive therapy. Follow-up 30 days after discharge

El objetivo del presente es la caracterización de pacientes en terapia intensiva en el hospital de alta complejidad, y portadores de insuficiencia respiratoria aguda con requerimiento de asistencia ventilatoria mecánica, a su vez, el comportamiento de pacientes con infección por el virus influenza H1N1 que desarrollan síndrome de dificultad respiratorio agudo (SDRA).

Hospital strain colonization by Staphylococcus epidermidis

The skin and mucous membranes of healthy subjects are colonized by strains of Staphylococcus epidermidis showing a high diversity of genomic DNA polymorphisms. Prolonged hospitalization and the use of invasive procedures promote changes in the microbiota with subsequent colonization by hospital strains. We report here a patient with prolonged hospitalization due to chronic pancreatitis who was treated with multiple antibiotics, invasive procedures and abdominal surgery. We studied the dynamics of skin colonization by S. epidermidis leading to the development of catheter-related infections and compared the genotypic profile of clinical and microbiota strains by pulsed field gel electrophoresis. During hospitalization, the normal S. epidermidis skin microbiota exhibiting a polymorphic genomic DNA profile was replaced with a hospital-acquired biofilm-producer S. epidermidis strain that subsequently caused repetitive catheter-related infections.

Clinical, epidemiological, and microbiological characteristics of bacteremia caused by high-level gentamicin-resistant Enterococcus faecalis

Enterococcus spp bacteremia is associated with high mortality and the appearance of high-level gentamicin resistance (HLGR) created additional challenges for the treatment of these infections. We evaluated the epidemiological and clinical characteristics of patients with bacteremias caused by HLGR and non_HLGR Enterococcus faecalis isolates at a teaching hospital in the State of São Paulo, Brazil. Patients with bacteremia due to E. faecalis diagnosed between January 1999 and December 2003 were included in the study. We collected clinical, epidemiological, and microbiological data from medical records. Banked isolates were typed using pulsed-field gel electrophoresis. We identified 145 cases of E. faecalis bacteremia: 66 (45.5 percent) were caused by HLGR isolates and 79 (54.5 percent) by non_HLGR. In the univariate analysis, patients with HLGR infection were older, had higher rates of bladder catheterization, and more often had treatment with cephalosporin, quinolone, and/or carbapenem compared with patients with non_HLGR infection (P < 0.05). Multivariate analysis indicated that older age, hematological malignancy, and previous use of vancomycin were independently associated with HLGR (P < 0.05). Mortality rates were not significantly different among patients with HLGR (50 percent) and non_HLGR (43 percent) infections (P = 0.40). Of the 32 genotyped isolates, 16 were distributed into 6 main electrophoresis patterns and 16 others had distinct patterns. E. faecalis bacteremia is associated with high mortality and is frequently caused by HLGR isolates at this teaching hospital. The variability among genotyped isolates suggests that endogenous infections, rather than patient-to-patient transmission of E. faecalis, are more common at this institution.

Use of molecular epidemiology to monitor the nosocomial dissemination of methicillin-resistant Staphylococcus aureus in a University Hospital from 1991 to 2001

Methicillin-resistant Staphylococcus aureus (MRSA) has been the cause of major outbreaks and epidemics among hospitalized patients, with high mortality and morbidity rates. We studied the genomic diversity of MRSA strains isolated from patients with nosocomial infection in a University Hospital from 1991 to 2001. The study consisted of two periods: period I, from 1991 to 1993 and period II from 1995 to 2001. DNA was typed by pulsed-field gel electrophoresis and the similarity among the MRSA strains was determined by cluster analysis. During period I, 73 strains presented five distinctive DNA profiles: A, B, C, D, and E. Profile A was the most frequent DNA pattern and was identified in 55 (75.3 percent) strains; three closely related and four possibly related profiles were also identified. During period II, 80 (68.8 percent) of 117 strains showed the same endemic profile A identified during period I, 18 (13.7 percent) closely related profiles and 18 (13.7 percent) possibly related profiles and, only one strain presented an unrelated profile. Cluster analysis showed a 96 percent coefficient of similarity between profile A from period I and profile A from period II, which were considered to be from the same clone. The molecular monitoring of MRSA strains permitted the determination of the clonal dissemination and the maintenance of a dominant endemic strain during a 10-year period and the presence of closely and possibly related patterns for endemic profile A. However, further studies are necessary to improve the understanding of the dissemination of the endemic profile in this hospital.

Clonal dissemination of VanA-type glycopeptide-resistant Enterococcus faecalis between hospitals of two cities located 100 km apart

Nosocomial dissemination of glycopeptide-resistant enterococci represents a major problem in hospitals worldwide. In Brazil, the dissemination among hospitals in the city of São Paulo of polyclonal DNA profiles was previously described for vancomycin-resistant Enterococcus faecium. We describe here the dissemination of VanA phenotype E. faecalis between two hospitals located in different cities in the State of São Paulo. The index outbreak occurred in a tertiary care university hospital (HCUSP) in the city of São Paulo and three years later a cluster caused by the same strain was recognized in two patients hospitalized in a private tertiary care hospital (CMC) located 100 km away in the interior of the state. From May to July 1999, 10 strains of vancomycin-resistant E. faecalis were isolated from 10 patients hospitalized in the HCUSP. The DNA genotyping using pulsed-field gel electrophoresis (PFGE) showed that all isolates were originated from the same clone, suggesting nosocomial dissemination. From May to July 2002, three strains of vancomycin-resistant E. faecalis were isolated from two patients hospitalized in CMC and both patients were colonized by the vancomycin-resistant Enterococcus in skin lesions. All isolates from CMC and HCUSP were highly resistant to vancomycin and teicoplanin. The three strains from CMC had minimum inhibitory concentration >256 æg/ml for vancomycin, and 64 (CMC 1 and CMC 2) and 96 æg/ml (CMC 3) for teicoplanin, characterizing a profile of VanA resistance to glycopeptides. All strains had the presence of the transposon Tn1546 detected by PCR and were closely related when typed by PFGE. The dissemination of the E. faecalis VanA phenotype among hospitals located in different cities is of great concern because E. faecalis commonly colonizes the gastrointestinal tract of patients and healthy persons for periods varying from weeks to years, which, together with the persistence of vancomycin-resistant Enterococcus in hospital rooms after standard cleaning procedures, increases the risk of the dissemination and reservoir of the bacteria.