Molecular Verification of Trichophyton in the Brazilian URM Culture Collection.

Trichophyton species cause dermatophytosis in humans, with a high, worldwide frequency of reports and important public health relevance. We evaluated 61 Trichophyton strains from different sources deposited in the University Recife Mycology (URM) culture collection of the Universidade Federal de Pernambuco, Brazil. Strains were phenotypically identified and confirmed by sequencing Internal Transcribed Spacers rDNA and partial beta-tubulin 2-exon. Additionally, we evaluated their susceptibility to terbinafine and itraconazole. Physiological analyses included urease activity and growth in casein medium. Phenotypic methods allowed the reliable identification of T. rubrum only, whereas, for other species, molecular methods were mandatory. All Trichophyton species exhibited susceptibility profiles to itraconazole (0.04-5.33 µg/mL) and terbinafine (0.17-3.33 µg/mL). Our analyses revealed a heterogeneous distribution of T. mentagrophytes, which does not support the current distribution within the species complex of T. mentagrophytes and its genotypes.

Constant testing for Pestivirus in cell lines reveals different routes of contamination.

Pestivirus can contaminate cell cultures and sera and cause serious problems that evolve the integrity of studies, confidence in diagnostic results, and safety of human and animal vaccines. Contaminations by Pestivirus and other viruses may occur at any time and regular assays of monitoring in cell cultures and your supplies are necessary. This study aimed to analyze the phylogeny of Pestivirus detected from cell cultures, calf serum, and standard strains of three laboratories in Brazil that carry out frequent tests for the monitoring of cellular contaminations. These samples were submitted to phylogenetic analysis to understand the genetic relationship between contaminants occurring in these facilities. As result, the Pestivirus found in samples were Bovine viral diarrhea virus (BVDV-1 and BVDV-2), Hobi-like viruses (often named BVDV-3), and Classical swine fever virus (CSFV), and the phylogenetic analysis help us to infer at three possible routes of contamination in this work.

Validation of real-time PCR technique for detection of Mycobacterium bovis and Brucella abortus in bovine raw milk.

Brucellosis and tuberculosis are diseases of great economic impact in cattle herds and are controlled by governmental programs in many countries. The validation of a diagnostic technique is fundamental for its application in official control programs of these diseases. The aim of the present study was to validate a polymerase chain reaction in real time (qPCR) for detection of Mycobacterium bovis and Brucella abortus in samples of artificially contaminated raw milk. The technique was evaluated using tests of analytical sensitivity and specificity, repeatability, internal reproducibility, and robustness. Initially, five DNA extraction methodologies were tested, and the DNeasy Mericon Food Kit-Qiagen and the Maxwell® 16 Tissue DNA Purification Kit-Promega presented the best analytical specificity of all the commercial kits tested and were used exclusively in subsequent tests. The lowest limits of detection obtained in the qPCR were 2.3 pg for M. bovis DNA and 20.7 fg for B. abortus DNA. The repeatability and reproducibility associated with the robustness indicate that the evaluated methods are applicable as rapid tools for the official in vivo diagnosis of bovine tuberculosis and brucellosis in raw milk from dairy herds in Brazil.

Virome analyses of Hevea brasiliensis using small RNA deep sequencing and PCR techniques reveal the presence of a potential new virus.

BACKGROUND: Hevea brasiliensis is an important commercial crop due to the high quality of the latex it produces; however, little is known about viral infections in this plant. The only virus described to infect H. brasiliensis until now is a Carlavirus, which was described more than 30 years ago. Virus-derived small interfering RNA (vsiRNAs) are the product of the plant's antiviral defense triggered by dsRNA viral intermediates generated, during the replication cycle. These vsiRNAs are complementar to viral genomes and have been widely used to identify and characterize viruses in plants. METHODS: In the present study, we investigated the virome of leaf and sapwood samples from native H. brasiliensis trees collected in two geographic areas in the Brazilian Amazon. Small RNA (sRNA) deep sequencing and bioinformatic tools were used to assembly, identify and characterize viral contigs. Subsequently, PCR amplification techniques were performed to experimentally verify the presence of the viral sequences. Finally, the phylogenetic relationship of the putative new virus with related viral genomes was analyzed. RESULTS: Our strategy allowed the identification of 32 contigs with high similarity to viral reference genomes, from which 23 exhibited homology to viruses of the Tymoviridae family. The reads showed a predominant size distribution at 21 nt derived from both strands, which was consistent with the vsiRNAs profile. The presence and genome position of the viral contigs were experimentally confirmed using droplet digital PCR amplifications. A 1913 aa long fragment was obtained and used to infer the phylogenetic relationship of the putative new virus, which indicated that it is taxonomically related to the Grapevine fleck virus, genus Maculavirus. The putative new virus was named Hevea brasiliensis virus (HBrV) in reference to its host. CONCLUSION: The methodological strategy applied here proved to be efficient in detecting and confirming the presence of new viral sequences on a 'very difficult to manage' sample. This is the second time that viral sequences, that could be ascribed as a putative novel virus, associated to the rubber tree has been identified.

Development of a droplet digital RT-PCR for the quantification of foot-and-mouth virus RNA.

Foot-and-mouth-disease (FMD) is a highly contagious disease of domestic animals which can result in substantial economic losses, caused by the FMD virus (FMDV). The aim of this study was to develop and standardize a novel reverse transcriptase droplet digital PCR (RT-ddPCR) assay for the quantification of FMDV RNA. This assay was based upon an OIE-recognized real-time RT-PCR that detects the 3D-encoding region of FMDV. The limit of detection at 101.4 TCID50/mL and 26.5 copies was determined using FMDV-A24-Cruzeiro-virus and a plasmid containing the 3D-FMDV sequences, respectively. FMDV O, A and C serotypes and 11 species of non-FMDV were used to confirm the sensitivity and specificity of the assay. The RT-ddPCR was standardized using 60 bovine samples (representing negative and positive samples of epithelium and/or oesophageal-pharyngeal [OP] fluid) from animals suspected of vesicular diseases and previously tested by RT-qPCR. The RT-ddPCR showed robustness, sensitivity, specificity and accuracy, with similar results to the RT-qPCR. Moreover, the new RT-ddPCR diagnostic tool allowed the absolute quantification of FMDV RNA from epithelium and OP-fluid samples, as well as having the advantages of direct quantification by endpoint, eliminating the need for a calibration standard curve required in quantitative real-time RT-PCR.

Rescue therapy with stent retrievers for thromboembolism during endovascular treatment of intracranial aneurysms.

Thromboembolism is the most frequent complication in endovascular treatment of intracranial aneurysms, causing disability and death. As stent retrievers have achieved high rates of arterial recanalization in the management of ischemic stroke, these devices were tested as rescue therapy of thromboembolism during aneurysm embolization. We retrospectively analyzed 10 consecutive patients with transprocedural arterial occlusion, treated with mechanical thrombectomy at a single center. Good angiographic recanalization was achieved in eight cases, mTICI 3, 2b and 2a in five, three and two patients, respectively, without additional complications or any deaths. Five patients showed complete recovery (mRS 0) and all patients showed improvement of disability (average mRS 1.1) over a mean follow-up period of 31 months. Eight patients had good clinical recovery, while two remained with deficits (mRS 3 and 4). The study found that the stent retriever is a valuable, rapid and effective tool for restoring blood flow, improving the safety of endovascular treatment.

Rescue therapy with stent retrievers for thromboembolism during endovascular treatment of intracranial aneurysms / Terapia de resgate com stent para tromboembolismo durante tratamento endovascular de aneurismas intracranianos

ABSTRACT Thromboembolism is the most frequent complication in endovascular treatment of intracranial aneurysms, causing disability and death. As stent retrievers have achieved high rates of arterial recanalization in the management of ischemic stroke, these devices were tested as rescue therapy of thromboembolism during aneurysm embolization. We retrospectively analyzed 10 consecutive patients with transprocedural arterial occlusion, treated with mechanical thrombectomy at a single center. Good angiographic recanalization was achieved in eight cases, mTICI 3, 2b and 2a in five, three and two patients, respectively, without additional complications or any deaths. Five patients showed complete recovery (mRS 0) and all patients showed improvement of disability (average mRS 1.1) over a mean follow-up period of 31 months. Eight patients had good clinical recovery, while two remained with deficits (mRS 3 and 4). The study found that the stent retriever is a valuable, rapid and effective tool for restoring blood flow, improving the safety of endovascular treatment.

RESUMO Tromboembolismo é a complicação mais frequente no tratamento endovascular de aneurismas cerebrais, podendo causar morte ou sequelas. Como os stent retrievers alcançaram altas taxas de recanalização arterial no tratamento do acidente vascular encefálico isquêmico, testamos esses dispositivos para tratar eventos tromboembólicos ocorridos durante a embolização de aneurismas. Foram analisados retrospectivamente 10 pacientes apresentando oclusão arterial transoperatória, tratados com trombectomia mecânica em um único centro. Obtivemos recanalização angiográfica em oito casos, mTICI 3, 2b e 2a em cinco, três e dois pacientes, respectivamente, sem complicações adicionais ou óbito. Cinco casos apresentaram recuperação completa (mRS 0) e todos os pacientes apresentaram melhora dos déficits (mRS médio 1.1) durante acompanhamento médio de 31 meses. Oito pacientes apresentaram boa recuperação clínica, enquanto dois permaneceram com déficits (mRS 3 e 4). O estudo concluiu que stents são uma ferramenta valiosa, rápida e eficaz para restaurar o fluxo sanguíneo, aumentando a segurança do tratamento endovascular.

Molecular epidemiology of senecavirus A associated with vesicular disease in pigs in Brazil.

Senecavirus A (SV-A) may cause vesicular disease and neonatal mortality in pigs, and was first detected in Brazil in 2015. Samples including tissues and serum from pigs with suspected vesicular diseases were collected from January to August in 2015 from farms in the states of Minas Gerais, Santa Catarina, Goiás and Rio Grande do Sul, Brazil, and tested for the presence of SV-A by reverse transcriptase PCR. All samples were negative for foot and mouth disease virus, as well as 13 other infectious agents associated with vesicular diseases in pigs. SV-A was detected by PCR in 65/265 (24.5%) specimens. A 530 base pair fragment sequenced from the VP1 protein coding region indicated a high genetic distance from SV-A in other countries, but a common origin among the Brazilian isolates.