Single-cell sequencing unveils the lifestyle and CRISPR-based population history of Hydrotalea sp. in acid mine drainage.

Acid mine drainage (AMD) is characterized by an acid and metal-rich run-off that originates from mining systems. Despite having been studied for many decades, much remains unknown about the microbial community dynamics in AMD sites, especially during their early development, when the acidity is moderate. Here, we describe draft genome assemblies from single cells retrieved from an early-stage AMD sample. These cells belong to the genus Hydrotalea and are closely related to Hydrotalea flava. The phylogeny and average nucleotide identity analysis suggest that all single amplified genomes (SAGs) form two clades that may represent different strains. These cells have the genomic potential for denitrification, copper and other metal resistance. Two coexisting CRISPR-Cas loci were recovered across SAGs, and we observed heterogeneity in the population with regard to the spacer sequences, together with the loss of trailer-end spacers. Our results suggest that the genomes of Hydrotalea sp. strains studied here are adjusting to a quickly changing selective pressure at the microhabitat scale, and an important form of this selective pressure is infection by foreign DNA.

Identification and characterization of genes related to cellulolytic activity in basidiomycetes.

Enzymes produced by basidiomycetes that are involved in the cellulose degradation process, and their respective codifying genes, must be identified to facilitate the development of novel biotechnological strategies and applications in the agro-industry. The objective of this study was to identify prospective cellulase-producing genes and characterize their cellulolytic activity, in order to elucidate the potential biotechnological applications (with respect to vegetal residues) of basidiomycetes. The basidiomycete strains Lentinula edodes U8-1, Lentinus crinitus U9-1, and Schizophyllum commune U6-7 were analyzed in this study. The cellulolytic activities of these fungi were evaluated based on the halo formation in carboxymethyl cellulose culture medium after dyeing with Congo red. The presence of cellulase-codifying genes (cel7A, cel6B, cel3A, and egl) in these fungal strains was also evaluated. L. edodes and S. commune presented the highest cellulolytic halo to mycelial growth radius ratio, followed by L. crinitus. Four genes were amplified in the L. edodes strain, whereas three and one genes were isolated from L. crinitus and S. commune, respectively. The cel6B gene (L. edodes) presented the conserved domain glyco_hydro_6 and characterized as cellobiohydrolase gene. The results of this study contribute to the existing knowledge on cellulases in basidiomycetes, and serve as a basis for future studies on the expression of these genes and the characterization of the catalytic activity of these enzymes. This allows for better utilization of these fungi in degrading vegetal fibers from agro-industrial residues and in other biotechnological applications.

Avaliação da dosagem de própolis pulverizada nas folhas de amoreira (morus sp.) sobre o desenvolvimento biológico e produtivo do bicho-da-seda (bombyx mori L. ) / Evaluación de la dosis de propóleos esparcidas en las hojas de morera (morus sp.) en el desarrollo biológico y productivo del gusano de seda (bombyx mori L.) / Evaluation of the doses of propolis pulverization in mulberry leaves (morus sp.) on the biological and productive development of silkworm (bombyx mori L.)

Este experimento foi realizado no Laboratório de Sericicultura, no Campus Sede da Universidade Paranaense (UNIPAR) de Umuarama, no período de 17/09/2009 a 17/10/2009, com o objetivo de verificar o efeito da própolis em diferentes dosagens na alimentação durante o desenvolvimento biológico do bicho-da-seda (Bombxy mori L). O método empregado na parte experimental foi a pulverização do extrato alcoólico de própolis, diluído em 500 mL de água destilada nas folhas de amoreira, nas seguintes dosagens, água-controle, 5mL, 10mL, 15mL e 20mL compondo os tratamentos T1, T2, T3, T4, T5, respectivamente. As folhas de amoreira foram fornecidas cinco vezes ao dia, durante o manejo alimentar. Verificou-se pelos resultados obtidos, que as diferentes dosagens de própolis utilizadas interferem no ganho de peso das lagartas, no peso dos casulos verdes e crisálidas quando comparado ao tratamento controle, influenciando também no número de casulos formados e, para os teores de seda bruto e líquido não apresentaram resultados significativos, quando comparados com o tratamento controle. Portanto, verificou-se que a própolis, nas dosagens utilizadas, não trouxe efeitos depressivos à biologia e produção do bicho-da-seda.

This experiment was conducted at the Sericulture Laboratory, in the Campus Sede of the Universidade Paranaense (UNIPAR) in Umuarama, from 17/09/2009 to 17/10/2009, in order to verify the effects of propolis in different dosages during the development of silkworm (Bombyx mori L). The method used during the experiment was the dillution of an alcohoolic solution of propolis, in 500 mL of destilled water with its further pulverization on mullberry leaves, with the following doses: water control; 5 mL; 10 mL; 15 mL; 20 mL, composing the following treatments: T1, T2, T3, T4, T5, respectively. The mullberry leaves were sprinkled five times a day, during the feed management. It was verified by the results that the different doses of propolis used interfere with gain-weight of the larvae, weight of cocoons and chrysalis, when compared to control. The treatment also influenced the number of cocoons formed. The contento fraw and liquid silk were not significant when compared with the control. Therefore, it was concluded that the propolis, used in those dosages, brought no depressive effects to the development and the production of silkworm.

Este experimento se realizó en el Laboratorio de Sericicultura del Campus Sede de la Universidad Paranaense (UNIPAR) de Umuarama, en el período de 17/09/2009 a 17/10/2009, con el objetivo de verificar el efecto de propóleos en diferentes dosis en la alimentación durante el desarrollo biológico del gusano de seda (Bombyx mori L.). El método usado en la parte experimental fue la pulverización del extracto alcohólico de propóleos, diluido en 500 ml de agua destilada en las hojas de morera, en las siguientes dosis: agua control, 5 ml, 10 ml, 15 ml y 20 ml componiendo los tratamientos T1, T2, T3, T4, T5, respectivamente. Las hojas de morera fueron suministradas cinco veces al día, durante el manejo alimentar. Por los resultados obtenidos se verificó que las dosis diferentes de propóleos utilizadas interfieren en el gano de peso de los gusanos, en el peso de los capullos verdes y crisálidas cuando comparado al tratamiento control, influenciando también en el número de capullos formados y, para contenidos de seda bruta y líquida no presentan resultados significativos, mientras comparado con el tratamiento control. Sin embargo, se puede verificar que propóleos, en dosis utilizadas, no trajo efectos depresivos a la biología y producción del gusano.

Leishmania identification by PCR of Giemsa-stained lesion imprint slides stored for up to 36 years.

This study examined the ability of PCR to amplify Leishmania DNA, stored on Giemsa-stained slides, from American cutaneous leishmaniasis (ACL) patients. In total, 475 slides stored for up to 36 years were obtained from an outpatient clinic in a Brazilian ACL-endemic region, and Leishmania DNA was amplified from 395 (83.2%) of the DNA samples using primers specific for the minicircle kinetoplast DNA. Restriction fragment length polymorphism analysis of these amplicons demonstrated that Leishmania (Viannia) braziliensis was the only species present in these samples. The results demonstrated that archived Giemsa-stained slides can provide a Leishmania DNA source for performing clinical and epidemiological studies of leishmaniasis.

Leishmania infantum-induced primary and challenge infections in rhesus monkeys (Macaca mulatta): a primate model for visceral leishmaniasis.

Visceral leishmaniasis (VL) was experimentally induced in rhesus macaques (Macaca mulatta) by intravenously inoculating 2 x 10(7)amastigotes/kg of body weight of Leishmania infantum. The macaques developed a systemic disease showing characteristic features of human VL such as fever, diarrhoea, body weight loss, anaemia, hypergammaglobulinaemia and transient lymphocytosis, as well as lymph node, liver and/or spleen enlargement. Nine weeks after infection, one primate showed pronounced weight loss, became moribund and was euthanized. The necropsy findings included granulomas composed of parasite-containing macrophages, lymphocytes and plasma cells in the liver, spleen and lymph nodes. The remaining macaques had a sustained course of infection but developed a mild-to-moderate illness that subsequently showed evidence of self-cure. Of note, pathological findings included a typical cell-mediated immunity-induced granulomatous reaction that had an effect on the control of parasite replication. All infected monkeys responded with increased production of anti-Leishmania-specific IgG antibodies. Despite the fact that clinical resistance to L. infantum was not consistently associated with a parasite-specific cell-mediated immune response, drug-cured macaques from the primary infection acquired immunity to homologous re-infection. These findings point to the feasibility of using the L. infantum macaque model for pre-clinical evaluation of novel chemotherapeutics or vaccine candidates for human VL.

Leishmaniose tegumentar na Região Metropolitana de Belo Horizonte: aspectos clínicos, laboratoriais, terapêuticos e evolutivos (1989-1995) / Cutaneous leishmaniasis in the Metropolitan Region of Belo Horizonte: clinical, laboratorial, therapeutic and prognosis features (1989-1995)

This study investigated clinical, laboratorial, therapeutic and prognostic aspects of American cutaneous leishmaniasis in Belo Horizonte in 358 patients with cutaneous leishmaniasis (CL) and 25 with mucocutaneous leishmaniasis (MCL). Compared to CL patients, the MCL patients reported longer duration of disease and higher frequency of other diseases, suggesting that debilitation caused by leishmaniasis or other conditions might contribute to activation and/or mucous dissemination of the parasite. The sensitivity of skin test, indirect immunofluorescence reactions and direct detection of parasites was 78.4, 79.3 and 68.3%, respectively. The treatment with meglumine antimoniate presented 100% efficacy, but 59% patients had side-effects. During two years of follow-up, there were 32/318 relapses after successful treatment. Most relapses (31/32) were of CL patients treated with 15 mg Sb5+/kg/day. The negative response to skin test was the only factor associated with a significant threefold increased risk of relapse. Higher dose or longer duration of treatment might improve the prognosis in these patients.

Foram investigados aspectos clínicos, laboratoriais, terapêuticos e evolutivos da leishmaniose tegumentar americana em Belo Horizonte. O estudo incluiu 358 pacientes com leishmaniose cutânea (LC) e 25 com leishmaniose mucosa (LM). Comparados aos pacientes com LC, aqueles com LM apresentaram maior tempo de doença e relato de outras doenças concomitantes, sugerindo que a debilitação pela leishmaniose e/ou outras doenças podem contribuir para a ativação e/ou disseminação mucosa do parasito. As sensibilidades das reações intradérmica, de imunofluorescência indireta e da pesquisa direta do parasito foram de 78,4, 79,3 e 68,3%, respectivamente. O tratamento com antimoniato de meglumina foi 100% eficaz, com 59% de efeitos colaterais ao longo do tratamento. A recidiva após tratamento ocorreu em 32 (10,1%) dos 318 casos seguidos por até dois anos. A maioria das recidivas (31 dos 32 casos) ocorreu em pacientes com LC tratados com 15mg Sb5+/kg/dia. Na investigação de critérios de cura, a reação intradérmica negativa foi o único fator associado a um risco três vezes maior de recidiva. Um aumento da dose ou do tempo de tratamento talvez melhore o prognóstico nestes pacientes.

Attempt to differentiate Leishmania (Leishmania) amazonensis, L. (L.) chagasi, Leishmania (Viannia) braziliensis and L. (V.) guyanensis using the SSR-PCR technique.

The ability to differentiate reference strains of Leishmania (Leishmania) amazonensis, L. (L.) chagasi, Leishmania (Viannia) braziliensis and L. (V.) guyanensis was evaluated using the simple sequence repeat polymerase chain reaction (SSR-PCR) technique. This technique differentiates the Leishmania species, generating distinct DNA amplicon profiles. The SSR-PCR profiles were similar to but more reproducible than those produced by RAPD. SSR-PCR is presented as an alternative to other molecular methods for the differentiation of Leishmania species or strains.

Simple form of clinical sample preservation and Leishmania DNA extraction from human lesions for diagnosis of American cutaneous leishmaniasis via polymerase chain reaction.

The diagnosis value of polymerase chain reaction (PCR) was assessed in patients from an area endemic for American cutaneous leishmaniasis (ACL) in Brazil. Different forms of clinical sample preservation and DNA extraction for PCR were tested. The 4 preservation forms of the skin biopsies from patients suspected to have ACL were as follows: imprinted on filter paper (FP); imprinted on nitrocellulose paper (NP); frozen at -20 degrees C (FB); or immersed in 70% ethanol (EB). The DNA was extracted by elution from FP and NP and by enzyme digestion from FB and EB. Clinical examinations and parasitological or immunological tests confirmed the cases of ACL. Of 164 patients suspected to have ACL, 133 patients (81.1%) were confirmed. The PCR was positive in 76.8% of the suspected cases and in 90.2% of the confirmed cases. Polymerase chain reaction alone showed nearly the same positivity of the parasitological and immunological tests together; positivity varied 73.3-82.2%, according to the means by which the samples were preserved or the way the DNA was extracted. This variation was not significantly different (P > 0.05). Therefore, we recommend that clinical samples from patients with ACL should be collected and preserved on FP and the DNA further extracted by elution. The samples can be mailed to reference laboratories for the definitive diagnosis of ACL. This alternative is simple, inexpensive, and adequate for field conditions in developing countries.

A reliable and specific enzyme-linked immunosorbent assay for the capture of IgM from human chagasic sera using fixed epimastigotes of Trypanosoma cruzi.

A rapid, sensitive, specific, and reliable enzyme-linked immunosorbent assay (ELISA) is proposed for determination of the levels of anti-Trypanosoma cruzi IgM in acute chagasic sera (ACD). The efficiency of this ELISA as a diagnostic method was compared with that of parasite DNA detection by polymerase chain reaction (PCR) and that of indirect immunofluorescence (iIF) anti-T. cruzi IgM detection. We tested whether this ELISA using fixed epimastigotes (epi) could detect anti-T. cruzi IgM in serum samples from two groups of children with acute Chagas' disease from a hyperendemic area in Bolivia. In a comparison of the ELISA method with other techniques, 95% and 71% of the results correlated with PCR and iIF findings, respectively. At the serum dilution applied (1:250), rheumatoid factor (RF) did not influence the results, and samples from patients carrying leishmaniasis or mixed Leishmania and T. cruzi infection could also be excluded from ACD. Highly specific and reliable results were obtained, a great number of the sera could be tested in only one assay, and a quantitative index of reactivity (IR) could be calculated without serial titration. Using test samples in triplicate, the method provides a useful tool for the detection of early acute-phase T. cruzi infection in humans.

American cutaneous leishmaniasis: use of a skin test as a predictor of relapse after treatment.

While relapses following clinical cure of American cutaneous leishmaniasis are frequent, no test has been described until now to predict such relapses. A cohort of 318 American cutaneous leishmaniasis patients was followed up for two years after treatment with meglumine antimoniate, during which time 32 relapses occurred, 30 in the first year and two in the second (accumulated risk: 10.5%). No association was found between these relapses and the parasite-specific antibody response before and after treatment, or between the relapses and stratification by sociodemographic and clinical characteristics. However when Leishmania was used as antigen, patients with a negative skin test at the time of diagnosis presented a 3.4-fold higher risk (hazard risk = 3.4; 95% confidence interval, 1.7-7.0) of American cutaneous leishmaniasis relapse, compared with patients with a positive response. This result shows that the skin test can be a predictor of American cutaneous leishmaniasis relapse after treatment.

Electrocardiographic changes during low-dose, short-term therapy of cutaneous leishmaniasis with the pentavalent antimonial meglumine.

The pentavalent antimonial (Sb5+) meglumine is the drug of choice for the treatment of cutaneous leishmaniasis (CL) in Brazil. Although the cardiotoxicity of high-dose, long-term Sb5+ therapy is well known, the use of low-dose, short-term meglumine has been considered to be safe and relatively free from significant cardiac effects. In order to investigate the cardiotoxicity of low-dose, short-term therapy with meglumine in cutaneous leishmaniasis, 62 CL patients treated with meglumine were studied. A standard ECG was obtained before and immediately after the first cycle of treatment (15 mg Sb5+ kg-1 day-1). The electrocardiographic interpretation was carried out blindly by two investigators using the Minnesota Code. There were no significant differences in qualitative ECG variables before and after meglumine treatment. However, the corrected QT interval was clearly prolonged after antimonial therapy (420.0 vs 429.3 ms, P < 10(-6)). QTc augmentation exceeded 40 ms in 12 patients, 7 of whom developed marked QTc interval enlargement (500 ms) after meglumine therapy. This previously unrecognized cardiac toxicity induced by short-term, low-dose antimonial therapy has potentially important clinical implications. Since sudden death has been related to QTc prolongation over 500 ms induced by high-dose antimonial therapy, routine electrocardiographic monitoring is probably indicated even in CL patients treated with short-term, low-dose meglumine schedules. Until further studies are conducted to establish the interactions between pentavalent antimonials and other drugs, special care is recommended when using meglumine in combination with other medications, in particular with drugs that also increase the QTc interval.

Leishmania (Viannia) braziliensis is the predominant species infecting patients with American cutaneous leishmaniasis in the State of Minas Gerais, Southeast Brazil.

Skin biopsies from 53 patients with American cutaneous leishmaniasis (ACL) from the State of Minas Gerais, Brazil, were used for a characterization of the Leishmania parasites. A pair of primers flanking the conserved region of the Leishmania minicircle kDNA was used to obtain amplified DNA via the polymerase chain reaction. The amplified products were subsequently hybridized with Leishmania subgenus-specific radiolabeled probes. Parasites from 49 out of 53 samples (92.5%) were characterized as belonging to the subgenus Viannia and four (7.5%) as belonging to the subgenus Leishmania. Clinical, epidemiological and molecular evidence allow us to conclude that Leishmania (V.) braziliensis and Leishmania (L.) amazonensis are the species present in the patients studied and that L. (V.) braziliensis is the predominant species in the State of Minas Gerais, Brazil.

Electrocardiographic changes during low-dose, short-term therapy of cutaneous leishmaniasis with the pentavalent antimonial meglumine

The pentavalent antimonial (Sb5+) meglumine is the drug of choice for the treatment of cutaneous leishmaniasis (CL) in Brazil. Although the cardiotoxicity of high-dose, long-term Sb5+ therapy is well known, the use of low-dose, short-term meglumine has been considered to be safe and relatively free from significant cardiac effects. In order to investigate the cardiotoxicity of low-dose, short-term therapy with meglumine in cutaneous leishmaniasis, 62 CL patients treated with meglumine were studied. A standard ECG was obtained before and immediately after the first cycle of treatment (15 mg Sb5+ kg-1 day-1). The electrocardiographic interpretation was carried out blindly by two investigators using the Minnesota Code. There were no significant differences in qualitative ECG variables before and after meglumine treatment. However, the corrected QT interval was clearly prolonged after antimonial therapy (420.0 vs 429.3 ms, P<10-6). QTc augmentation exceeded 40 ms in 12 patients, 7 of whom developed marked QTc interval enlargement (500 ms) after meglumine therapy. This previously unrecognized cardiac toxicity induced by short-term, low-dose antimonial therapy has potentially important clinical implications. Since sudden death has been related to QTc prolongation over 500 ms induced by high-dose antimonial therapy, routine electrocardiographic monitoring is probably indicated even in CL patients treated with short-term, low-dose meglumine schedules. Until further studies are conducted to establish the interactions between pentavalent antimonials and other drugs, special care is recommended when using meglumine in combination with other medications, in particular with drugs that also increase the QTc interval

Spontaneous obliteration of a carotid-cavernous fistula associated with fibromuscular dysplasia of the internal carotid artery.

The authors present a case of spontaneous recovery of a carotid-cavernous fistula associated with fibromuscular dysplasia of the aortocranial vessels in a 31-year-old woman. The pathogenesis of the carotid-cavernous fistula and the possible mechanism responsible for its spontaneous obliteration are both discussed.