Multi-locus sequence typing and phylogenetics of Cryptococcus neoformans AD hybrids.

Hybrid AD strains of the human pathogenic Cryptococcus neoformans species complex have been reported from many parts of the world. However, their origin, diversity, and evolution are incompletely understood. In this study, we analyzed 102 AD hybrid strains representing 21 countries on five continents. For each strain, we obtained its mating type and its allelic sequences at each of the seven loci that have been used for genotyping haploid serotypes A and D strains of the species complex by the Cryptococcus research community. Our results showed that most AD hybrids exhibited loss of heterozygosity at one or more of the seven analyzed loci. Phylogenetic and population genetic analyses of the allelic sequences revealed multiple origins of the hybrids within each continent, dating back to one million years ago in Africa and up to the present in other continents. We found evidence for clonal reproduction and long-distance dispersal of these hybrids in nature. Comparisons with the global haploid serotypes A and D strains identified new alleles and new haploid multi-locus genotypes in AD hybrids, consistent with the presence of yet-to-be discovered genetic diversity in haploid populations of this species complex in nature. Together, our results indicate that AD hybrids can be effectively genotyped using the same multi-locus sequencing type approach as that established for serotypes A and D strains. Our comparisons of the AD hybrids among each other as well as with the global haploid serotypes A and D strains revealed novel genetic diversity as well as evidence for multiple origins and dynamic evolution of these hybrids in nature.

Ultrasound-guided percutaneous biopsy of thoracic lesions: high diagnostic yield and low complication rate.

AIM: To investigate the use of ultrasound (US)-guided biopsy of thoracic lesions aiming to determine diagnostic success and complication rates and to identify factors that may affect sample adequacy and safety. MATERIAL AND METHODS: This was a retrospective study of consecutive percutaneous US-guided biopsy over 10-year period, including 147 procedures in 146 patients (66 ± 7 years, 83 men, 63 women) with lesions located in the lung (67/147), chest wall (54/147), mediastinum (14/147) and pleura (12/147). Overall diagnostic success, yield for benign and malignant diagnoses and diagnostic success according to lesion location, biopsy type (fine-need aspiration [FNA] or core-needle biopsy [CNB]) and number of specimens were calculated. Presence of complications and effect of age, lesion location, biopsy type, and number of specimens were measured. RESULTS: The overall diagnostic success rate was 90.5% and was similar for malignant (90.6%) and benign (87.5%) diagnoses. Specimen adequacy was similar for FNA and CNB (91.2% and 88.9%, p=0.66); number of specimens did not affect yield. Diagnostic success was highest for mediastinal and chest wall lesions (92.9% and 94.4%) and lowest for pleural lesions (75%), albeit not statistically significant (p=0.45). Complications occurred in 4/147(2.7%) cases, exclusively in lung lesions, and were not associated with any covariates analysed. CONCLUSIONS: US-guided biopsy is an effective and safe technique for diagnosis of thoracic lesions, with high diagnostic yield and low complication rate. In the presence of an adequate acoustic window, US guidance can be valuable for diagnosis of peripheral lung and mediastinal lesions. Radiologists performing thoracic biopsy should be encouraged to implement or expand the use of US guidance in their practice.

Diversity and phylogeny of basidiomycetous yeasts from plant leaves and soil: Proposal of two new orders, three new families, eight new genera and one hundred and seven new species.

Nearly 500 basidiomycetous yeast species were accepted in the latest edition of The Yeasts: A Taxonomic Study published in 2011. However, this number presents only the tip of the iceberg of yeast species diversity in nature. Possibly more than 99 % of yeast species, as is true for many groups of fungi, are yet unknown and await discovery. Over the past two decades nearly 200 unidentified isolates were obtained during a series of environmental surveys of yeasts in phyllosphere and soils, mainly from China. Among these isolates, 107 new species were identified based on the phylogenetic analyses of nuclear ribosomal DNA (rDNA) [D1/D2 domains of the large subunit (LSU), the small subunit (SSU), and the internal transcribed spacer region including the 5.8S rDNA (ITS)] and protein-coding genes [both subunits of DNA polymerase II (RPB1 and RPB2), the translation elongation factor 1-α (TEF1) and the mitochondrial gene cytochrome b (CYTB)], and physiological comparisons. Forty-six of these belong to 16 genera in the Tremellomycetes (Agaricomycotina). The other 61 are distributed in 26 genera in the Pucciniomycotina. Here we circumscribe eight new genera, three new families and two new orders based on the multi-locus phylogenetic analyses combined with the clustering optimisation analysis and the predicted similarity thresholds for yeasts and filamentous fungal delimitation at genus and higher ranks. Additionally, as a result of these analyses, three new combinations are proposed and 66 taxa are validated.

HIV/AIDS-Associated Cryptococcosis in Portugal Spanning the Pre- to Post-HAART Era: A Retrospective Assessment at the Genotypic Level Based on URA5-RFLP.

Cryptococcosis caused by the fungus Cryptococcus neoformans is an opportunistic mycosis, infecting mainly immunodepressed individuals. Molecular epidemiology studies of cryptococcosis in Europe are limited. This paper presents a retrospective study of cryptococcosis in 105 cryptococcal isolates from two hospitals in Lisbon, Portugal, among HIV/AIDS patients, from 1991 to 2007. Among these patients, the number of cases of cryptococcosis increased from 5.1 to 6.9 cases per year from the pre- to post-highly active antiretroviral therapy (HAART) era. As expected, the median age of the patients increased, from 32 (mean: 33 ± 8) to 39 (mean: 41 ± 10) years, and the ratio of male to female patients remained high (7.7 and 7.6, respectively). Strain genotyping based on restriction fragment length polymorphism of the orotidine monophosphate pyrophosphorylase (URA5-RFLP) gene showed that, in general, the relative frequencies of the genotypes VNI-IV are similar to those from other European countries. These frequencies were, respectively, for the pre- and post-HAART periods: 41.7 and 43.5 % for VNI; 2.8 and 17.4 % for VNII; 38.9 and 30.4 % for VNIII; 16.7 and 7.2 % for VNIV and 0 and 1.4 % for VGII. Some apparent although statistically insignificant differences among these values were observed between both periods. The genotypic frequencies were not also statistically different according to the patients' gender or age range. Of note are the high proportion of VNIII isolates (common in Europe) and the high increase in the frequency of the VNII genotype in the post-HAART. Ultimately, these results may have implications in disease therapy, management and control.

A low cost, safe, disposable, rapid and self-sustainable paper-based platform for diagnostic testing: lab-on-paper.

There is a strong interest in the use of biopolymers in the electronic and biomedical industries, mainly towards low-cost applications. The possibility of developing entirely new kinds of products based on cellulose is of current interest, in order to enhance and to add new functionalities to conventional paper-based products. We present our results towards the development of paper-based microfluidics for molecular diagnostic testing. Paper properties were evaluated and compared to nitrocellulose, the most commonly used material in lateral flow and other rapid tests. Focusing on the use of paper as a substrate for microfluidic applications, through an eco-friendly wax-printing technology, we present three main and distinct colorimetric approaches: (i) enzymatic reactions (glucose detection); (ii) immunoassays (antibodies anti-Leishmania detection); (iii) nucleic acid sequence identification (Mycobacterium tuberculosis complex detection). Colorimetric glucose quantification was achieved through enzymatic reactions performed within specific zones of the paper-based device. The colouration achieved increased with growing glucose concentration and was highly homogeneous, covering all the surface of the paper reaction zones in a 3D sensor format. These devices showed a major advantage when compared to the 2D lateral flow glucose sensors, where some carryover of the coloured products usually occurs. The detection of anti-Leishmania antibodies in canine sera was conceptually achieved using a paper-based 96-well enzyme-linked immunosorbent assay format. However, optimization is still needed for this test, regarding the efficiency of the immobilization of antigens on the cellulose fibres. The detection of Mycobacterium tuberculosis nucleic acids integrated with a non-cross-linking gold nanoprobe detection scheme was also achieved in a wax-printed 384-well paper-based microplate, by the hybridization with a species-specific probe. The obtained results with the above-mentioned proof-of-concept sensors are thus promising towards the future development of simple and cost-effective paper-based diagnostic devices.

Insights on the genotype distribution among Cryptococcus neoformans and C. gattii Portuguese clinical isolates.

This study provides a comprehensive picture of the C. neoformans/C. gattii molecular types most often associated with human cryptococcosis in Portugal and assesses the impact of C. gattii in these infections. One hundred and twenty-two clinical isolates, from distinct patients, were identified as C. neoformans and genotyped by URA5-RFLP, with the molecular types VNI (45.5 %) and VNIII (30.9 %) being the most commonly found ones. The molecular types VNII (11.4 %) and VNIV (11.4 %) were less abundant. One patient was found to be infected with a VGII isolate. This patient exhibited unusual clinical symptoms of cryptococcosis, reinforcing the suspicion for the presence of a different genotypic pattern, as determined afterwards. This case was detected in 2007 and is the first report of a potential autochthonous C. gattii infection case in Portugal, as the patient revealed no historical record of travelling outside the country.

Ant identity determines the fungi richness and composition of a myrmecochorous seed.

Myrmecochory-seed dispersal by ants-is a mutualistic interaction in which ants attracted by seed appendices take them away from the parental plant location, where seeds usually have better development odds. Not all ant species benefit plants, and the mechanisms of those divergent outcomes are still unclear, especially from the perspective of microbial third parties. Here, we explore the effects of seed manipulation on fungi communities promoted by two ant species with contrasting effects on seed germination and antimicrobial cleaning strategies. We hypothesize that: i) fungi richness is higher in seeds manipulated by Acromyrmex subterraneus (species that negatively affect seed germination), followed by unmanipulated seeds and seeds manipulated by Atta sexdens (ant species that increase seed germination) and ii) seeds manipulated by A. sexdens, Ac. subterraneus and unmanipulated seeds present dissimilar fungi compositions. We identified fungal morphotypes in three groups of seeds: i) manipulated by A. sexdens; ii) manipulated by Ac. subterraneus; iii) unmanipulated. Seeds manipulated by Ac. subterraneus exhibited higher fungal richness than those manipulated by A. sexdens and unmanipulated seeds, indicating that the ant species known to impair germination increases the fungal load on seeds. Additionally, we found that A. sexdens ants were unable to reduce fungal richness compared to unmanipulated seeds. Furthermore, fungal composition differed among all three treatments. Our results underscore the significance of ant species identity in shaping the fungal communities associated with myrmecochorous seeds. Given the potential influence of microbial infection on seed fate, we suggest considering manipulation strategies when evaluating the overall quality of an ant as a seed disperser.

Direct and specific identification of Cryptococcus neoformans in biological samples using fluorescently labelled DNA probes.

Fluorescence in situ hybridisation (FISH) is a suitable technique for the rapid, reliable and cultivation-independent identification of microbial pathogens. This study describes the development of fluorescently labelled rRNA-targeted oligonucleotides and a FISH assay to detect and identify Cryptococcus neoformans in culture and biological samples. All C. neoformans reference and clinical isolates gave positive signals with the specific oligonucleotide probes, whereas all non-target yeast species gave negative reactions with the same probes. The assay was also successfully applied to the detection of C. neoformans cells in cerebrospinal samples from patients with clinical diagnosis of cryptococcosis. The described FISH-based assay revealed to be practical, sensitive and specific for the detection and identification of C. neoformans yeasts.

Is end-organ surveillance necessary in patients with well-functioning metal-on-metal hip resurfacings? A cardiac MRI survey.

AIMS: Cardiac magnetic resonance (CMR) was used to assess whether cardiac function or tissue composition was affected in patients with well-functioning metal-on-metal hip resurfacing arthroplasties (MoMHRA) when compared with a group of controls, and to assess if metal ion levels correlated with any of the functional or structural parameters studied. PATIENTS AND METHODS: In all, 30 participants with no significant cardiac history were enrolled: 20 patients with well-functioning MoMHRA at mean follow-up of 8.3 years post-procedure (ten unilateral, ten bilateral; 17 men, three women) and a case-matched control group of ten non-MoM total hip arthroplasty patients (six men, four women). The mean age of the whole cohort (study group and controls) at the time of surgery was 50.6 years (41.0 to 64.0). Serum levels of cobalt and chromium were measured, and all patients underwent CMR imaging, including cine, T2* measurements, T1 and T2 mapping, late gadolinium enhancement, and strain measurements. RESULTS: None of the MoMHRA patients showed clinically significant cardiac functional abnormality. The MoMHRA patients had larger indexed right and left end diastolic volumes (left ventricular (LV): 74 ml/m2vs 67 ml/m2, p = 0.045; right ventricular: 80 ml/m2vs 71 ml/m2, p = 0.02). There was a small decrease in T2 time in the MoMHRA patients (median 49 ms vs 54 ms; p = 0.0003). Higher metal ion levels were associated with larger LV volumes and with shorter T2 time. CONCLUSION: Although cardiac function is not clinically adversely affected in patients with well-functioning MoMHRA, modern imaging is able to demonstrate subtle changes in structure and function of the heart. As these changes correlate with systemic ion measurements, they may be consequences of wear debris deposition. Longer, longitudinal studies are necessary to determine whether cardiac function will become affected. Cite this article: Bone Joint J 2019;101-B:540-546.

Epidemiological situation of vesicular stomatitis virus infection in cattle in the state of Paraíba, semiarid region of Brazil.

The aim of this survey was to estimate the apparent herd-level and animal-level prevalences, as well as to identify risk factors and spatial clustering of vesicular stomatitis virus (VSV) positive herds in the state of Paraíba, semiarid of Brazil. The state was divided into three sampling strata: Sertão, Borborema and Zona da Mata/Agreste. For each sampling stratum, herd-level and animal-level prevalences were estimated by a two-stage sampling survey. First, a pre-established number of herds (primary sampling units) were randomly selected; second, within each herd, a pre-established number of cows aged ≥ 24 months were systematically selected (secondary sampling units). In total, 2279 animals were sampled from 468 herds. Serum samples were submitted to virus neutralization (VN) test for detection of antibodies to VSV using three viral strains: VSIV-3 2013SaoBento/Paraiba E, strain Indiana (VSIV-1) and VSNJV. A herd was considered positive for VSV if it included at least one positive animal in herds of up to 10 females, two positive animals in herds of 11-99 females, and three positive in herds with more than 99 females. The spatial clustering was assessed using the Cuzick-Edwards' k-nearest neighbor method and spatial scan statistics. The apparent herd-level prevalence in the state of Paraíba was 38.5% (95% CI = 35.5-41.6%), 80.6% (95% CI = 73.6-86.2%) in the region of Sertão, 7.0% (95% CI = 3.9-12.2%) in Borborema, and 2.6% (95% CI = 1.0-6.7%) in Agreste/Zona da Mata. The apparent animal-level prevalence was 26.2% (95% CI = 20.6-32.8%) in the state of Paraíba, 48.2% (95% CI = 41.5-54.9%) in Sertão, 6.3% (95% CI = 2.7-14%) in Borborema, and 3.2% 1.9% (95% CI = 0.4-8.4%) in Agreste/Zona da Mata. The risk factors identified were as follows: mixed production (milk/beef) (OR = 4.54), herd size > 23 animals (OR = 3.57), presence of cervids (OR = 15.24), rental of pastures (OR = 3.02), sharing of water sources (OR = 2.57) and presence of horses (OR = 1.69). Two significant clusters of positive herds were detected: the primary cluster covered the Sertão region and the secondary cluster covered part of the Sertão and Borborema regions. Our results suggest high VSV circulation in the bovine population of the state of Paraíba, semiarid region of Brazil, mainly in the Sertão mesoregion, and based on risk factor analysis it was possible to identify important associations that deserve more investigation on causal factors.

Herd-level seroprevalence and associated risk factors for bovine cysticercosis in the State of Paraíba, Northeastern Brazil.

This study focused on estimating the herd-level and animal-level prevalences, and identifying risk factors associated with bovine cysticercosis in the State of Paraíba, Northeastern Brazil. The state was divided into three sampling strata: Sertao, Borborema and Zona da Mata/Agreste. For each sampling stratum, herd-level and animal-level prevalences were estimated by a two-stage sampling survey. First, a pre-established number of herds (primary sampling units) were randomly selected; second, within each herd a pre-established number of cows aged ≥24months were systematically selected (secondary sampling units). Ten animals were sampled in herds with up to 99 cows aged over 24 months; 15 animals were sampled in herds with 100 or more cows aged over 24 months; and all animals were sampled in those with up to 10 cows aged over 24 months. In total, 2382 animals were sampled from 474 herds. Serological diagnosis was initially performed by the indirect ELISA, and positive sera were confirmed by immunoblot. A herd was deemed positive if it included at least one positive animal in herds of up to 29 females, and two positive animals in herds with more than 29 females. The herd-level prevalence in the State of Paraíba was 10.8% (95% CI=8.1%-14.1%), 10.3% (95% CI=6.4%-16.1%) in the region of Sertão, 6.9% (95% CI=3.9%-12.1%) in Borborema, and 13.8% (95% CI=9.3%-20.2%) in Agreste/Zona da Mata. The animal-level prevalence was 2.3% (95% CI=1.6%-3.3%) in the State of Paraíba, 1.4% (95% CI=0.8%-2.5%) in Sertão, 3.6% (95% CI=1.7%-7.4%) in Borborema, and 3.2% (95% CI=1.9%-5.4%) in Agreste/Zona da Mata. The risk factors identified were as follows: animal purchasing (OR=2.19) and presence of flooded pastures (OR=1.99). Our findings suggest that bovine cysticercosis herd-level seroprevalence in the State of Paraíba, Northeastern Brazil, is high, and support the idea that prevention measures should be applied at herd level and farmers should restrict the access of their cattle to flooded pastures.

Application of fluorescence in situ hybridisation (FISH) to the analysis of yeast population dynamics in winery and laboratory grape must fermentations.

To analyse the yeast population diversity during wine fermentations, specific fluorescein-labelled oligonucleotide probes targeted to the D1/D2 region of the 26S rRNA of different yeast species known to occur frequently in this environment were designed and tested with reference strains. The probes were then used to identify wine must isolates and to follow, in combination with plate counts, the evolution of yeast populations in two winery fermentations of white and red grape musts. In both cases, a high diversity of non-Saccharomyces yeast species was detected, including Candida stellata, Hanseniaspora uvarum, H. guilliermondii, Kluyveromyces marxianus, K. thermotolerans and Torulaspora delbrueckii. Some of these species (e.g., K. marxianus, K. thermotolerans and T. delbrueckii) were present in significant amounts during the tumultuous fermentation stage, despite the predominance of Saccharomyces cerevisiae cells following the inoculation of the wine musts with a starter strain. To further clarify the yeast population dynamics at the late phase of the fermentations, and because winery conditions do not allow a reliable control of experimental variables, strains isolated from the industrial musts were used to conduct two laboratory microvinifications in synthetic grape juice, using different ratios of S. cerevisiae/non-Saccharomyces in the inocula. Under these conditions, the results were similar to those obtained in the winery, showing a yeast profile with mixed species throughout the first fermentation stage, i.e. until about 40-50% of the total sugar was consumed. Non-Saccharomyces yeasts were outgrown by S. cerevisiae only after ethanol reached concentrations around 4-5% (v/v), which argues in favour of a potential important role of non-Saccharomyces in the final organoleptic characteristics of the wine.

Herd-level prevalence and associated risk factors for Mycobacterium avium subsp. paratuberculosis in cattle in the State of Paraíba, Northeastern Brazil.

A cross-sectional study based on a planned sampling was carried out to determine herd-level and animal-level prevalences, and to identify risk factors associated with herd-level prevalence for bovine paratuberculosis in the State of Paraíba, Northeastern Brazil. The state was divided into three sampling groups: sampling stratum 1 (mesoregion of Sertão), sampling stratum 2 (mesoregion of Borborema), and sampling stratum 3 (mesoregions of Zona da Mata and Agreste). For each sampling stratum, herd-level and animal-level prevalences were estimated by a two-stage sampling survey. In the first stage, a pre-established number of herds (primary sampling units) were randomly selected; in the second stage, a pre-established number of cows aged ≥24 months were randomly selected (secondary sampling units). Ten animals were sampled in herds with up to 99 cows aged over 24 months; 15 animals were sampled in herds with 100 or more cows aged over 24 months; and all animals were sampled in those with up to 10 cows aged over 24 months. In total, 2504 animals were sampled from 480 herds. Enzyme-linked immunosorbant assay (ELISA) test kits were used for Mycobacterium avium subsp. paratuberculosis (MAP) antibody detection. A herd was deemed positive for the presence of MAP if it included at least one positive animal in herds of up to 24 females, and two positive animals in herds with more than 24 females. The herd-level prevalence in the State of Paraíba was 34.5% (95% CI=30.2-39.1%), 26.6% (95% CI=20.2-34.2%) in the region of Borborema, 30.5% (95% CI=23.9-38.0%) in Agreste/Mata, and 41.4% (95% CI=34.0-49.1%) in Sertão. The animal-level prevalence was 10.7% (95% CI=7.3-15.4%) in the State of Paraíba, 7.9% (95% CI=5.2-11.7%) in the region of Borborema, 9.4% (95% CI=7.3-12.1%) in Sertão, and 13.9% (95% CI=6.2--28.3%) in Agreste/Mata. The frequency of seropositive animals per herd ranged from 6.7% to 100% (median of 20%). The risk factors identified were as follows: Sertão region (OR=1.9), more than 12 adult animals in the herd (OR=1.9), and not using maternity pens (OR=1.7). Our findings suggest that MAP herd-level seroprevalence in the State of Paraíba, Northeastern Brazil, is high, and support the idea that the use of maternity pens will be important for preventing transmission of MAP in the herds.

Differentiation of mobile and immobile pesticides on anionic clays by 1H HR MAS NMR spectroscopy.

Adsorbed vs. intercalated MCPA (4-chloro-2-methylphenoxyacetic acid) in highly hydrated clays taken as a soil model were clearly distinguished by 1H HR MAS NMR; adsorbed herbicide gave sharp signals indicating high mobility while intercalated herbicide gave very wide unresolved spectra due to its strong interaction with the solid matrix.

Intravenous hydroxyethylrutosides combined with long-term oral anticoagulation in atherosclerotic nonreconstructable critical leg ischemia: a retrospective study.

OBJECTIVE: To evaluate in a group of seriously diseased patients with nonreconstructable chronic critical leg ischemia (CLI), treated by a combination of i.v. hydroxyethylrutosides (HR)* and oral anticoagulation (AC) by warfarin, the short-term effects on the cutaneous microvascular blood perfusion of the soles of feet and especially the long-term clinical outcome in terms of amputation and death. DESIGN: A retrospective comparison between two groups of patients, HR + AC and a comparable reference group, fulfilling the same inclusion and exclusion criteria corresponding to the definition of CLI according to the Second European Consensus Document (1991). Clinical follow-up in both groups was made after 1, 3, 6, 12, and 24 months. SETTING: Patients were examined at university departments of clinical physiology with special interest in peripheral vascular disease, in cooperation with colleagues at university departments of surgery, internal medicine and dermatology of Karolinska Hospital, Södersjukhuset and Huddinge Hospital. PATIENTS: A total of seventy patients with CLI according to the definition of the Second European Consensus Document, 1991, ie, besides severe rest pain or ischemic lesions also a toe blood pressure < 30 mg Hg. Group with HR + anticoagulation (AC): 42 patients (19 diabetics, 23 nondiabetics). Reference group: 28 patients (18 diabetics, 10 nondiabetics). For distribution of age and toe blood pressure at baseline, see Table I. INTERVENTIONS: Therapy group: besides ordinary standard therapy, daily HR infusions for a mean period of 3.6 weeks + oral anticoagulation continued to the end of the study at 24 months. A comparable reference group on the same basic therapy but without the combination HR + AC. PARAMETERS IN EVALUATION: Short-term parameters: clinical data, skin temperature, and fluorescein imaging. Long-term outcome: amputation or death. RESULTS: Short-term and long-term results with HR + AC indicated that patients with severe CLI and very poor prognosis benefited in terms of survival and limb salvage from initial therapy with HR infusion combined with long-term oral anticoagulation. Results of this combined treatment seem at least comparable with those with i.v. prostacyclin analogies.

Spatial cluster analysis for bovine paratuberculosis in Paraiba State, Northeastern Brazil / Análise de aglomerados espaciais para paratuberculose bovina no Estado da Paraíba, Nordeste do Brasil

The aim of this survey was to identify spatial clustering of bovine paratuberculosis positive herds in the State of Paraíba, Northeastern Brazil. The state was divided into three sampling groups: sampling stratum 1 (mesoregion of Sertão), sampling stratum 2 (mesoregion of Borborema), and sampling stratum 3 (mesoregions of Zona da Mata and Agreste). Ten animals were sampled in herds with up to 99 cows aged over 24 months; 15 animals were sampled in herds with 100 or more cows aged over 24 months; and all animals were sampled in those with up to 10 cows aged over 24 months. In total, 2504 cows aged ≥ 24 months were sampled from 480 herds. Indirect enzyme-linked immunosorbant assay (ELISA) test kits were used for Mycobacterium avium subsp. paratuberculosis (MAP) antibody detection. A herd was deemed positive for paratuberculosis if it included at least one positive animal in herds of up to 24 females, and two positive animals in herds with more than 24 females. Spatial clustering was assessed using the Cuzick-Edwards' k-nearest neighbor method and spatial scan statistics. Two significant clustering of positive herds were detected in Northern part of Borborema mesoregion, a border region with the State of Rio Grande do Norte, in which there is a large animal movement from different locations without knowing the sanitary condition of animals. As serological tests for MAP diagnosis are not widely available and are very expensive, as well as replacement or maintenance of livestock by animal purchasing is common in the region, it is concluded that prevention measures should be applied at herd level.(AU)

O objetivo deste estudo foi identificar agrupamentos espaciais de rebanhos positivos para paratuberculose bovina no Estado da Paraíba, Nordeste do Brasil. O estado foi dividido em três grupos amostrais: estrato amostral 1 (mesorregião do Sertão), estrato amostral 2 (mesorregião da Borborema), e estrato amostral 3 (mesorregiões da Zona da Mata e Agreste). Dez animais foram amostrados em rebanhos com até 99 vacas com idade maior ou igual a 24 meses; 15 animais foram amostrados em rebanhos com 100 ou mais vacas com idade maior ou igual a 24 meses; e todos os animais foram amostrados naqueles rebanhos com até 10 vacas. No total, foram amostradas 2504 vacas com idade ≥24 meses de 480 rebanhos. Para a detecção de anticorpos anti-Mycobacterium avium subsp. paratuberculosis (MAP) foram utilizados kits do teste imunoenzimático indireto (ELISA). Um rebanho foi considerado positivo para paratuberculose se apresentasse pelo menos um animal positivo em rebanhos de até 24 fêmeas, e dois animais positivos em rebanhos com mais de 24 fêmeas. Os agrupamentos espaciais foram avaliados com o uso da metodologia k-vizinhos mais próximos de Cuzick-Edwards e estatística espacial de varredura. Dois agrupamentos significativos de rebanhos positivos foram detectados na parte norte da mesorregião da Borborema, uma região de fronteira com o Estado do Rio Grande do Norte onde há intenso movimento de animais de diferentes locais sem o conhecimento do estado sanitário desses animais. Tendo em vista que os testes sorológicos para diagnóstico de MAP não são amplamente disponíveis e muito caros, bem como é comum na região a reposição e manutenção dos rebanhos por compra de animais, conclui-se que medidas de prevenção devem ser aplicadas em nível de rebanho.(AU)

Gold nanoprobe assay for the identification of mycobacteria of the Mycobacterium tuberculosis complex.

Members of the Mycobacterium tuberculosis complex (MTC) are causative agents of human and animal tuberculosis. This complex encompasses several phylogenetically related species, including M. tuberculosis, the main aetiological agent of human tuberculosis, and Mycobacterium bovis, the causative agent of bovine tuberculosis, a relevant worldwide zoonosis. Clear epidemiological evaluation of appropriate and effective treatment requires unambiguous differentiation between MTC members. Routine diagnosis has been increasingly relying on the molecular identification of MTC members. In the present study, we report the use of a gold nanoparticle-based approach for the sensitive, specific and fast identification of MTC and for the differentiation of M. bovis and M. tuberculosis using the gyrB locus as target. This gold nanoprobe strategy relies on the colorimetric differentiation of specific DNA sequences based on differential aggregation profiles in the presence or absence of specific target hybridization. Three nanoprobes were designed and successfully used for the specific identification of members of MTC, M. bovis and M. tuberculosis.

Differentiation of Cryptococcus neoformans varieties and Cryptococcus gattii using CAP59-based loop-mediated isothermal DNA amplification.

Members of the Cryptococcus species complex (C. neoformans and C. gattii) are opportunistic pathogens responsible for frequently fatal cases of meningoencephalitis. These yeasts have been classified into five serotypes. Serotypes A and D are assigned to C. neoformans var. grubii and C. neoformans var. neoformans, respectively, Serotype AD strains are hybrids and serotype B and C strains are considered to belong to the related but distinct species C. gattii. Previous studies have identified 'serotype-associated' alleles of several genes in the Cryptococcus species complex. We developed a loop-mediated isothermal DNA amplification method using CAP59 allele-specific primers to identify the serotypes A, D and B/C of the Cryptococcus species complex.

Leptospirose bovina no Estado da Paraíba: prevalência e fatores de risco associados à ocorrência de propriedades positivas / Bovine leptospirosis in Paraíba State: prevalence and risk factors associated with the occurrence of positive herds

Foram determinadas as prevalências de propriedades positivas e de animais positivos e identificados fatores de risco associados à leptospirose em vacas no Estado da Paraíba, Nordeste do Brasil. Foram aleatoriamente selecionadas 2.317vacas com idade ≥ 24 meses, procedentes de 450propriedades. Para o diagnóstico sorológico da infecção por Leptospira spp. foi empregado o teste de soroaglutinação microscópica (SAM), utilizando-se 24 sorovares como antígenos. Uma propriedade foi considerada positiva quando apresentou pelo menos um animal soropositivo. Das 450 propriedades investigadas 398 (89,7 por cento; IC 95 por cento = 86,6-92,2 por cento) apresentaram pelo menos um animal reagente na SAM para qualquer sorovar, e 1.349 (61,1 por cento; IC por cento = 56,6-65,4 por cento) animais foram soropositivos. O sorovar Hardjo foi o mais prevalente nas propriedades e nos animais, com frequências de 58,17 por cento e 54,69 por cento, respectivamente. Propriedade ser localizada no Sertão (odds ratio = 3,20; p = 0,003), presença de animais silvestres (odds ratio =2,89; p=0,005), não resfriar o leite (odds ratio =3,83; p=0,034) e presença de pastos alagados (odds ratio =2,36; p<0,001) foram identificados como fatores de riscos associados à prevalência de propriedades positivas. Conclui-se que a leptospirose encontra-se amplamente difundida em bovinos do Estado da Paraíba, o que reforça a necessidade de intensificação de medidas de prevenção e controle, como a vacinação dos rebanhos. De acordo com os resultados da análise de fatores de risco, sugere-se que o controle sanitário antes da introdução de animais, drenagem de áreas alagadas e melhora nas condições de manejo são importantes medidas para a prevenção da infecção.

Herd-level and animal-level prevalences were determined and risk factors associated with leptospirosis were identified in cows in the State of Paraíba, Northeastern Brazil. A total of 2,317 cows with ≥ 24 months of age from 450 herds were randomly sampled. For the serological diagnosis of Leptospira spp. infection, the microscopic agglutination test (MAT) was carried out using 24 serovars as antigens. A herd was considered positive when presented at least one seropositive animal. Of the 450 investigated herds, 398 (89.7 percent; 95 percent CI = 86.6-92.2 percent) presented at least one reactant animal at MAT to any serovar, and 1,349 (61.1 percent; 95 percent CI = 56.6-65.4 percent) animals were seropositive. Serovar Hardjo was the most prevalent in herds and animals, with frequencies of 58.17 percent and 54.69 percent respectively. Location of the herd in the Sertão (odds ratio = 3.20; p=0.003), presence of wildlife (odds ratio =2.89; p=0.005), not cooling milk (odds ratio =3.83; p=0.034) and presence of flooded pastures (odds ratio =2.36; p<0.001) were identified as risk factors for herd-level prevalence. It is concluded that leptospirosis is widely spread in cattle in State of Paraíba, which reinforces the need for increased prevention and control measures, such as vaccination of herds. According to the results of risk factors analysis it is suggested that the sanitary control before the introduction of animals, drainage of wetlands and improvement in management conditions are important measures to preventing the infection.

Computerized evaluation of the peripheral blood flow during maximal vasodilatation in humans using venous occlusion plethysmography.

Forearm blood flow was studied with venous occlusion plethysmography after ischaemic exercise in 10 healthy volunteers and 10 hypertensive patients. The peak blood flow was calculated by three methods: the manual conventional graphic technique and by fitting of a tangent to the inflow curve with the help of a computer. In addition a computer-aided derivation of an exponential curve adapted to the initial part of the inflow curve was used for the calculation of the maximal blood flow. With the two tangential techniques the peak mean blood flow values did not differ significantly and were 58 ml min-1 100 ml-1 tissue while the exponential derivation procedure gave in both groups about 40% higher maximal flow values. The effect of venous occlusion on the linear velocity of the brachial arterial blood flow blood was studied with Ultrasound Doppler Flowmetry. The maximal flow velocity after ischaemic exercise decreased by about 18% during 50 mmHg venous occlusion; the influence of the venous stasis was least during the first heart beats after the onset of the venous occlusion. The effect of the venous stasis was similar but less pronounced in the hypertensive patients. In conclusion, forearm blood flow during maximal vasodilatation is reduced by venous occlusion and the best estimate of the flow is obtained from the earliest part of the venous occlusion inflow curve, as indicated by the use of the computer-aided exponential derivation technique.