Antifungal Activity of Nontoxic Nanocomposite Based on Silver and Reduced Graphene Oxide against Dermatophytes and Candida spp.

Dermatomycoses are typical hair, skin, or nail infections caused mainly by dermatophytes and nondermatophytes: Trichophyton, Microsporum, Epidermophyton, and Candida. In addition to the esthetical impact, pain, and nail deformity, these mycoses can be a source of severe disease. The high cost of treatment, toxicity, and the emergence of resistant infectious agents justifies research into new drugs. This work evaluates the fungicidal activity of nanocomposites (NCs) based on reduced graphene oxide (rGO) loaded with silver (Ag) nanoparticles (rGO/Ag) against clinical isolates of dermatophytes and Candida species. This is an unprecedented study in which, for the first time, hybrid nanocompounds based on Ag/rGO were tested against Epidermophytom, Microsporum, and Trichophyton species (dermatophytes agents). In this paper, we synthesize rGO using different concentrations of Ag by hydrolysis of metal salt AgNO3 and follow the growth of nanocrystals on sheets of rGO provided by the NaBH4. The NCs were analyzed by X-ray diffraction analysis, and the NC morphology, silver distribution on the rGO surface, and crystalline information were investigated by transmission electron microscopy. Antifungal susceptibility assay was performed by the microdilution method based on modified Clinical and Laboratory Standards Institute (CLSI) protocol. Time-kill kinetics was conducted to monitor the effect of the composite to inhibit fungal cells or promote structural changes, avoiding germination. The toxicological evaluation of the NCs was born in an in vivo model based on Galleria mellonella (G. mellonella). Minimum inhibitory concentration (MIC) values of the rGO/Ag NCs ranged from 1.9 to 125 µg/mL. The best inhibitory activity was obtained for rGO/Ag12%, mainly against Candida spp. and Epidermophyton floccosum. In the presence of sorbitol, MIC values of rGO/Ag NCs were higher (ranging from 15.6 to 250 µg/mL), indicating the action mechanism on the cell wall. Both yeast and dermatophytes clinical isolates were inhibited at a minimum of 6 and 24 h, respectively, but after 2 and 12 h, they had initial antifungal interference. All hybrid formulations of rGO/Ag NCs were not toxic for G. mellonella. This study provides insights into an alternative therapeutic strategy for controlling dermatomycoses.

Ninhydrin as a novel DNA hybridization indicator applied to a highly reusable electrochemical genosensor for Candida auris.

This work reports a simple strategy for Candida auris genomic DNA (gDNA) detection, a multi-resistant fungus associated with nosocomial outbreaks in healthcare settings, presenting high mortality and morbidity rates. The platform was developed using gold electrode sensitized with specific DNA capture probe and ninhydrin as a novel DNA hybridization indicator. The genosensor was able to detect C. auris in urine sample by differential pulse voltammetry and electrochemical impedance spectroscopy. The biosensor's analytical performance was evaluated by differential pulse voltammetry, detecting up to 4.5 pg µL-1 of C. auris gDNA in urine (1:10, V/V). Moreover, the genosensor was reused eight times with no loss in the current signal response. The genosensor showed selectivity and stability, maintaining 100% of its response up to 80 days of storage. In order to analyze interactions of single and double-stranded DNA with ninhydrin, SEM, AFM and molecular dynamics studies followed by docking simulations were performed. Theoretical calculations showed ninhydrin interactions more favorably with dsDNA in an A-T rich binding pocket rather than with the ssDNA. Therefore, the proposed system is a promising electrochemical detection device towards a more accurate detection of C. auris gDNA in biological samples.

Agapornis sp. pet birds: Source of dissemination of azole-resistant yeasts.

The objectives of this study were to identify yeasts in 40 feces samples of Agapornis birds and determine their antifungal susceptibility profile by two methodologies. Results indicated 92.5% of positivity (46 isolates). Six genera were identified (14 species): Cryptococcus (39.13%), four species; Candida (36.96%), four species; Rhodotorula (13.05%), two species; Trichosporon (6.52%), two species; Kodamaea ohmeri (2.17%); and Sporobolomyces salmonicolor (2.17%). Regarding the susceptibility profiles, 41.3% of the isolates showed low sensibility to fluconazole and 47.8% to itraconazole. Thus, Agapornis sp. disseminate potentially pathogenic and resistant yeasts. Precautionary measures should be taken regarding breeding and keeping them as pets.

Cryptic Aspergillus from clinical samples in the USA and description of a new species in section Flavipedes.

BACKGROUND: In the last few decades there has been an emergence of cryptic Aspergillus as agents of human infections due to the increase in immunocompromised population and to the improvement of identification tools. METHODS: Continuing our study on Aspergillus isolates from clinical origin deposited in a mycological reference centre in the United States, we selected 37 isolates belonging to less common sections of the genus, to study their species diversity and detect cryptic species by using a polyphasic approach. RESULTS: From this set of isolates, a total of 16 species were identified; the most frequent being A. calidoustus (48.6%, section Usti), A. terreus (13.5%, section Terrei), and A. nidulans (5.7%, section Nidulantes). The remaining isolates corresponded to 13 species of rare or cryptic Aspergillus, i.e. A. europaeus (section Cremei); A. iizukae, A. micronesiensis, A. spelaeus (section Flavipedes); A. pachycristatus, A. quadrilineatus, A. spinulosporus, A. unguis (section Nidulantes); A. alabamensis, A. carneus, A. hortai (section Terrei), A. granulosus (section Usti); and the new species A. suttoniae (section Flavipedes), which is described here. CONCLUSIONS: Correct identification of cryptic species is crucial to reveal new potential pathogens, to gather accurate epidemiological data and to choose an appropriate treatment.

Fast identification of dermatophytes by MALDI-TOF/MS using direct transfer of fungal cells on ground steel target plates.

Dermatophytes cause human infections limited to keratinised tissues. We showed that the direct transfer method allows reliable identification of non-dermatophytes mould and yeast by MALDI-TOF/MS. We aimed at assessing whether the direct transfer method can be used for dermatophytes and whether an own mass spectra library would be superior to the Bruker library. We used the Bruker Biotyper to build a dermatophyte mass spectra library and assessed its performance by 1/testing a panel of mass spectrum produced with strains genotypically identified and, 2/comparing MALDI-TOF/MS identification to morphology-based methods. Identification of dermatophytes using the Bruker library is poor. Our library provided 97% concordance between ITS sequencing and MALDI-TOF/MS analysis with a panel of 1104 spectra corresponding to 276 strains. Direct transfer method using unpolished target plates allowed proper identification of 85% of dermatophytes clinical isolates most of which were common dermatophytes. A homemade dermatophyte MSP library is a prerequisite for accurate identification of species absent in the Bruker library but it also improves identification of species already listed in the database. The direct deposit method can be used to identify the most commonly found dermatophytes such as T. rubrum and T. interdigitale/mentagrophytes by MALDI-TOF/MS.

MLST-Based Population Genetic Analysis in a Global Context Reveals Clonality amongst Cryptococcus neoformans var. grubii VNI Isolates from HIV Patients in Southeastern Brazil.

Cryptococcosis is an important fungal infection in immunocompromised individuals, especially those infected with HIV. In Brazil, despite the free availability of antiretroviral therapy (ART) in the public health system, the mortality rate due to Cryptococcus neoformans meningitis is still high. To obtain a more detailed picture of the population genetic structure of this species in southeast Brazil, we studied 108 clinical isolates from 101 patients and 35 environmental isolates. Among the patients, 59% had a fatal outcome mainly in HIV-positive male patients. All the isolates were found to be C. neoformans var. grubii major molecular type VNI and mating type locus alpha. Twelve were identified as diploid by flow cytometry, being homozygous (AαAα) for the mating type and by PCR screening of the STE20, GPA1, and PAK1 genes. Using the ISHAM consensus multilocus sequence typing (MLST) scheme, 13 sequence types (ST) were identified, with one being newly described. ST93 was identified from 81 (75%) of the clinical isolates, while ST77 and ST93 were identified from 19 (54%) and 10 (29%) environmental isolates, respectively. The southeastern Brazilian isolates had an overwhelming clonal population structure. When compared with populations from different continents based on data extracted from the ISHAM-MLST database (mlst.mycologylab.org) they showed less genetic variability. Two main clusters within C. neoformans var. grubii VNI were identified that diverged from VNB around 0.58 to 4.8 million years ago.

Surface cleaning effectiveness in a walk-in emergency care unit: Influence of a multifaceted intervention.

BACKGROUND: Cleaning of surfaces is essential in reducing environmental bioburdens and health care-associated infection in emergency units. However, there are few or no studies investigating cleaning surfaces in these scenarios. Our goal was to determine the influence of a multifaceted intervention on the effectiveness of routine cleaning of surfaces in a walk-in emergency care unit. METHODS: This prospective, before-and-after interventional study was conducted in 4 phases: phase I (situational diagnosis), phase II (implementation of interventions-feedback on results, standardization of cleaning procedures, and training of nursing staff), phase III (determination of the immediate influence of interventions), and phase IV (determination of the late influence of interventions). The surfaces were sampled before and after cleaning by visual inspection, adenosine triphosphate bioluminescence assay, and microbiologic culture. RESULTS: We sampled 240 surfaces from 4 rooms. When evaluated by visual inspection and adenosine triphosphate bioluminescence, there was a progressive reduction of surfaces found to be inadequate in phases I-IV (P < .001), as well as in culture phases I-III. However, phase IV showed higher percentages of failure by culture than phase I (P = .004). CONCLUSIONS: The interventions improved the effectiveness of cleaning. However, this effect was not maintained after 2 months.

High prevalence of bla(CTX-M) extended spectrum beta-lactamase genes in Klebsiella pneumoniae isolates from a tertiary care hospital: first report of bla(SHV-12), bla(SHV-31), bla(SHV-38), and bla(CTX-M-15) in Brazil.

The aim of this study was to investigate the presence and prevalence of bla(TEM), bla(SHV), and bla(CTX-M) and bla(GES)-like genes, responsible for extended spectrum beta-lactamases (ESBLs) production in clinical isolates of Klebsiella pneumoniae collected from a Brazilian tertiary care hospital. Sixty-five ESBL producing K. pneumoniae isolates, collected between 2005 and 2007, were screened by polymerase chain reaction (PCR). Identification of bla genes was achieved by sequencing. Genotyping of ESBL producing K. pneumoniae was performed by the enterobacterial repetitive intergenic consensus-PCR with cluster analysis by the Dice coefficient. The presence of genes encoding ESBLs was confirmed in 59/65 (90.8%) isolates, comprising 20 bla(CTX-M-2), 14 bla(CTX-M-59), 12 bla(CTX-M-15), 9 bla(SHV-12), 1 bla(SHV-2), 1 bla(SHV-2a), 1 bla(SHV-5), and 1 bla(SHV-31) genes. The ESBL genes bla(SHV-12), bla(SHV-31), and bla(CTX-M-15), and the chromosome-encoded SHV-type beta-lactamase capable of hydrolyzing imipenem were detected in Brazil for the first time. The analysis of the enterobacterial repetitive intergenic consensus-PCR band patterns revealed a high rate of multiclonal bla(CTX-M) carrying K. pneumoniae isolates (70.8%), suggesting that dissemination of encoding plasmids is likely to be the major cause of the high prevalence of these genes among the K. pneumoniae isolates considered in this study.

Câncer cervical: etiologia, diagnóstico e prevenção / Cervical cancer: etiology, diagnosis and prevention

Mundialmente, o câncer cervical constitui o segundo tipo de câncer mais comum entre as mulheres. Sua incidência torna-se evidente na faixa etária entre 20 a 29 anos e o risco aumenta gradualmente com a idade. A etiologia deste tipo de câncer está associada à infecção pelo Papilomavirus Humano (HPV), transmitido sexualmente, e à interação entre diversos fatores de risco. A infecção pelo vírus HPV precede o desenvolvimento de lesões malignas e tem sido associada a lesões precursoras de câncer cervical. O método convencional para rastreamento do câncer cervical é a colpocitologia oncológica ou teste Papanicolaou, considerado um método de baixo custo, simples e de fácil execução. Além da detecção e diagnóstico precocee investigação periódica por meio do exame Papanicolaou, ações preventivas contra o câncer cervical por meio de orientações à população e do tratamento das lesões de colo em suas fases iniciais antes de se tornarem invasivas também são importantes. Vacinas profiláticas contra HPV estão sendo estudadas para impedir a infecção por esses vírus, enquanto que a utilização das vacinas terapêuticas visa tratar o indivíduo já infectado ou até o portador de uma lesão causada por HPV. A expectativa é que em 10 ou 20 anos ocorra redução das taxas de incidência das lesões precursoras desse tipo de neoplasia e, conseqüentemente, a redução da incidência do câncer cervical. Enquanto isso, a expectativa para as próximas décadas é de que a prevenção do câncer cervical continue sendo baseada no rastreamento periódico da população por meio do teste de Papanicolaou, isoladamente ou em conjunto com testes de detecção molecular de HPV, o agenteetiológico desses tumores.

Worldwidely, cervical cancer is the second most common type of cancer among women. Its incidence is higher in the age group of 20 to 29 years; the risk increases gradually with age. The etiology of cervical canceris associated to the Human Papillomavirus infection (HPV), sexually transmitted as well as the interaction among several risk factors. HPV infection precedes the development of malignant lesions and has been associated with precursor lesions of cervical cancer. Oncology colpocitology or Papanicolaou test, a lowcost method, simple and easily performed, is the conventional method for screening cervical cancer. Besides the detection and early diagnosis by Papanicolaou test, preventive actions against cervical cancer through education and treatment of colon lesions in their early phases before they become invasive are also important. Prophylactic vaccines against HPV have been investigated to prevent the virus infection, while the utilization of therapeutical vaccines aims to treat either the individual with the infection or the HPV-lesion carrier. The expectation for the reduction of incidence rates of these precursor lesions and, consequently, the reductionof cervical cancer can occur around the next 10 to 20 years. Meanwhile, the expectation for the next decadesis that the cervical cancer prevention remains based on the population’s periodic screenings by means of Papanicolaou test, alone or in combination with molecular tests for HPV detection, the etiologic agent of these tumors.

Paracoccidioidomicose: correlação entre achados clínicos e laboratoriais na região de São José do Rio Preto / Paracoccidioidomycosis: correlation between clinical and laboratorial findings in São José do Rio Preto region

A paracoccidioidomicose (Pbmicose) é uma doença granulomatosa de comprometimento sistêmico causada pelo fungo dimórfico Paracoccidioides brasiliensis. As manifestações clínicas são diversas e têm relação com a resposta imune do hospedeiro. O diagnóstico de Pbmicose tem evoluído nos últimos anos devido o advento das provas sorológicas. Estas são usadas não só para diagnósticos como também para determinar a eficácia terapêutica antifúngica durante e após o tratamento. O objetivo do estudo foi correlacionar os resultados dos testes sorológicos por imunodifusão dos pacientes com pbmicose com as formas clínicas da doença na população de São José do Rio Preto. Para tanto foram utilizadas informações de 79 prontuários de pacientes atendidos no Hospital de Base da Faculdade de Medicina de São José do Rio Preto, no período de2000 a 2003. O resultado obtido mostra prevalência para população masculina, com idade entre 35 e 55 anos, sendo a maioria trabalhadora ou moradora da zona rural. Os pulmões, a mucosa oral e gânglios foram as áreas anatômicas mais comprometidas. O método diagnóstico mais utilizado foi a biópsia de lesão (70,4%), seguido de imagens radiológicas, compatíveis em 49% dos casos, e prova sorológica por imunodifusão radial positiva em 29 pacientes (40,8%). O tratamento de escolha foi a sulfadiazina, seguida pelo uso de sulfametoxazol +trimetoprim. Foi possível correlacionar os títulos de anticorpos com a forma clínica apresentada, tendo em vista a diminuição destes frente ao sucesso terapêutico.

Paracoccidioidomycosis (Pbmicosis) is a granulomatous disease with systemic impairment caused by dimorphic fungi Paracoccidioides brasiliensis. The clinical manifestations are diverse and have relation with the immune answer of the host. Paracoccidiodomycosis diagnosis has evolved in these last years due to the advent of the serologic tests. These are used not only for diagnosis, but also to determine the antifungical therapeutic effectiveness during and after the treatment. The objective of this study was to correlate the results of immunodiffusion serologic tests of patients with Paracoccidioidomycosis with the clinical forms of the disease in the population of São José do Rio Preto. Seventy nine medical records of patients assisted in the Hospital de Base of Medical School , São José do Rio Preto from 2000 to 2003 were used. The results showed that male population aged between 35 to 55 was the most significant ; the majority was workers or inhabitants from rural area. Lungs, oral mucosa and lymph nodes were the structures more frequently impaired.The biopsy of injury was the diagnostic method most used (70.4%), the radiological images were compatiblein 49% of the cases and serologic tests had been positive in 29 patients (40.8%). The chosen treatment was the sulfadiazine, followed by the administration of sulfametaxazol + trimetoprim. It was possible to correlate antibodies titers with the clinic tests; consequently the reduction of these regarding the therapeutical success.

Infecções Hospitalares por Stenotrophomonas maltophilia: aspectos clínico-epidemiológicos, microbiológicos e de resistência antimicrobiana / Hospital Infection by Stenotrophomonas maltophilia: clinical-epidemiological, microbiology aspects and antimicrobial resistance

A bactéria Stenotrophomonas maltophilia está ocupando papel importante no cenário das infecções hospitalares.É considerada patógeno emergente, sendo responsável por elevada morbi-letalidade, principalmente em pacientes sob terapia imunossupressora ou antibioticoterapia prolongada e de amplo espectro. Outros fatores de risco significativos incluem: longo tempo de internação, procedimentos invasivos, idade avançada e procedimento cirúrgico prévio. O tratamento dessas infecções tem sido objeto de preocupação, uma vez que a bactéria exibe resistência intrínseca à maioria dos antimicrobianos disponíveis, caracterizando-se assim como microrganismo multirresistente. Metodologias de tipagem fenotípica e genotípica têm sido utilizadas na tentativa de definir padrões de transmissão e disseminação intra- e inter-hospitalar, e na comunidade. Nesse contexto, a metodologia molecular é considerada o recurso técnico mais promissor no estabelecimento da epidemiologia desse importante patógeno

Entoropatógenos associados com diarréia aguda em crianças / Enteropathogens associated with acute diarrhead disease in children

Objetivo: A fim de contribuir para um melhor conhecimento da distribuiçäo de agentes etiológicos de diarréia em nosso país, determinou-se a prevalência de enteropatógenos em diarréia aguda de crianças de baixo nível sócio-econômico, moradoras de Säo José do Rio Preto e regiäo. Métodos: Foi realizado um estudo prospectivo em 196 crianças com menos de 5 anos de idade com diarréia aguda e em 33 sem diarréia, durante o ano de 1995, antendidas no Ambulatório Pediátrico do Hospital de Base de Säo José do Rio Preto - Hospital de atendimento secundário, ligado à Faculdade de Medicina, que dá assistência gratuita a pacientes da regiäo, cuja maioria é de nível sócio-econômico baixo (renda máxima familiar de 2 salários mínimos). Resultados: Enteropatógenos foram identificados em 48 por cento dos casos e 27 por cento dos controles. Foram isolados dos grupos diarréico, 41 amostras (21 por cento) de shigella sp, com maior prevalência em criançaas maiores de 1 ano (p<0,01). A freqüência de amostras de EPEC foi de 10,7 por cento nos casos. Näo foram obseervadas diferenças significativas quanto à sua distribuiçäo entre as diferentes faixas etárias. A freqüência de Salmonella foi de 5,6 por cento no grupo diarréico, sendo significativamente maior em crianças menores de 2 anos (p<0,05). Os outros patógenos estudados reopresentaram 12 por cento dos patógenos isolados dos casos diarréicos. No grupo controle, amostras de EPEC atípicas representaram 18,2 por cento dos isolados. A análise de distribuiçäo dos enteropatógenos durante as estaçöes do ano mostra que é altamente significativa a distribuiçäo da freqüência dos mesmos nas diferentes estaçöes do ano. Conclusäo: Uma maior prevalência de Shigella e Salmonella na primavera e veräo foi observada, podendo-se concluir que, provavelmente, há uma relaçäo desses patógenos com a temperatura e umidade do meio ambiente. Entre as crianças estudadas, amostras de Shigella e amostras diarreiogênicas de E. coli (EPEC, ETEC, EAggEC, EIEC) foram as bactérias que mais contribuíram como causa de diarréia, durante todo o ano.