Assessment of the sensitivity and specificity of serological (IFAT) and molecular (direct PCR) techniques for diagnosis of leishmaniasis in lagomorphs using a Bayesian approach

Leishmaniasis, caused by Leishmania infantum, is a vector-borne zoonotic disease that is endemic to the Mediterranean basin. The potential of rabbits and hares to serve as competent reservoirs for the disease has recently been demonstrated, although assessment of the importance of their role on disease dynamics is hampered by the absence of quantitative knowledge on the accuracy of diagnostic techniques in these species. A Bayesian latent-class model was used here to estimate the sensitivity and specificity of the Immuno-fluorescence antibody test (IFAT) in serum and a Leishmania-nested PCR (Ln-PCR) in skin for samples collected from 217 rabbits and 70 hares from two different populations in the region of Madrid, Spain. A two-population model, assuming conditional independence between test results and incorporating prior information on the performance of the tests in other animal species obtained from the literature, was used. Two alternative cut-off values were assumed for the interpretation of the IFAT results: 1/50 for conservative and 1/25 for sensitive interpretation. Results suggest that sensitivity and specificity of the IFAT were around 70–80%, whereas the Ln-PCR was highly specific (96%) but had a limited sensitivity (28.9% applying the conservative interpretation and 21.3% with the sensitive one). Prevalence was higher in the rabbit population (50.5% and 72.6%, for the conservative and sensitive interpretation, respectively) than in hares (6.7% and 13.2%). Our results demonstrate that the IFAT may be a useful screening tool for diagnosis of leishmaniasis in rabbits and hares. These results will help to design and implement surveillance programmes in wild species, with the ultimate objective of early detecting and preventing incursions of the disease into domestic and human populations.

The conundrum of human visceral leishmaniasis in Emilia-Romagna, Italy: are wild and peridomestic animals potential reservoirs?

Leishmaniasis is a complex parasitic disease caused by intracellular protozoans of the genus Leishmania mainly transmitted by the bite of sand flies. In Italy, leishmaniasis is caused by Leishmania infantum, responsible for the human visceral and canine leishmaniases (HVL and CanL, respectively). Within Emilia-Romagna region, Italy, recent molecular studies indicated that L. infantum strains circulating in dogs and humans are different. This suggests that an animal reservoir other than dog should be evaluated in the epidemiology of HVL in Emilia-Romagna. Therefore, the main aim of this PhD project was to investigate the role of wild and peridomestic mammals as potential animal reservoirs of L. infantum in the regional zones where HVL foci are still active, also evaluating the possible role of arthropod vectors other than phlebotomine sandflies as vectors of Leishmania spp. in the sylvatic cycle of the protozoa. Overall, 206 specimens of different animal species (roe deer, rats, mice, badgers, hares, polecats, foxes, beech martens, bank voles, hedgehogs, and shrews), collected in Emilia-Romagna were screened for Leishmania with a real-time PCR, revealing a prevalence of 33% for roe deer (first report in this species). Positivity was also found in brown rats (10.6%), black rats (13.1%), mice (10%), badgers (25%), hedgehogs (80%) and bank voles (11%). To distinguish the two strains of L. infantum circulating in Emilia-Romagna, a nested PCR protocol optimized for animal tissues was developed, demonstrating that over 90% of L. infantum infections in roe deer were due to the strain isolated from humans and suggesting their possible role as reservoirs in the study area. Furthermore, the presence of Leishmania kDNA was detected in unfed larvae, nymphs and males of questing Ixodes ricinus ticks collected in regional parks of Emilia-Romagna suggesting their possible role in the transmission of L. infantum in a sylvatic or rural cycle.

New chemical modalities for leishmaniasis drug discovery

Leishmaniasis is one of the major parasitic diseases among neglected tropical diseases with a high rate of morbidity and mortality. Human migration and climate change have spread the disease from limited endemic areas all over the world, also reaching regions in Southern Europe, and causing significant health and economic burden. The currently available treatments are far from ideal due to host toxicity, elevated cost, and increasing rates of drug resistance. Safer and more effective drugs are thus urgently required. Nevertheless, the identification of new chemical entities for leishmaniasis has proven to be incredibly hard and exacerbated by the scarcity of well-validated targets. Trypanothione reductase (TR) represents one robustly validated target in Leishmania that fulfils most of the requirements for a good drug target. However, due to the large and featureless active site, TR is considered extremely challenging and almost undruggable by small molecules. This scenario advocates the development of new chemical entities by unlocking new modalities for leishmaniasis drug discovery. The classical toolbox for drug discovery has enormously expanded in the last decade, and medicinal chemists can now strategize across a variety of new chemical modalities and a vast chemical space, to efficiently modulate challenging targets and provide effective treatments. Beyond others, Targeted p Protein Degradation (TPD) is an emerging strategy that uses small molecules to hijack endogenous proteolysis systems to degrade disease-relevant proteins and thus reduce their abundance in the cell. Based on these considerations, this thesis aimed to develop new strategies for leishmaniasis drug discovery while embracing novel chemical modalities and navigating the chemical space by chasing unprecedented chemotypes. This has been achieved by four complementary projects. We believe that these next-generation chemical modalities for leishmaniasis will play an important role in what was previously thought to be a drug discovery landscape dominated by small molecules.

Determination of microplastics in the digestive content of pelagic fish of commercial interest

The study aimed to determine the amount of microplastics (MPs) found in the digestive content of some pelagic tuna fish species of commercial interest caught along the Spanish coasts. In total, 601 individuals belonging to eight tuna species, and one species of the Corypheanidae family, were examined for the presence of MPs in their stomachs. Fish were collected from 9 different locations along the Spanish coasts by commercial fishers. A total of 170 MPs were extracted from the fish stomach of 75 individuals. The number of particles present in the stomach ranged from 1 to 20 (mean 0.3± 1.2 SD MPs per individual). The species with the highest amount of MPs was Auxix rochei whereas the species that did not present any MPs was Coryphaena hippurus, the only species not belonging to the tuna group. In terms of location, the highest ratio of MPs (71%) was found in the fish collected from Azohia, and the lowest in Mazagon (7%). In general, the fish that ingested more MPs were those caught from sites that were closer to the coasts (Canarias Islands stations and Azohia). No correlation between the number of ingested MPs and the size and weight of the fish were detected nor for the whole sample nor for each species separately. Analyzing the MPs in terms of colour, green colour MPs were the most dominant (52%) followed by blue colour (25%). Analyzing the MPs in terms of size, 11 categories were considered and the most dominant size was 0-0.5 micron. Fibers were excluded from the analysis because no protocol was adopted to prevent their contamination. Furthermore, 100% of the MPs found were fragments. The data did not show a high presence of MPs in the analyzed study areas. A bias could probably be due to the method used, allowing to loose a certain quantity of very small microplastics, and also to ethological and biological factors of the species, as documented by other studies: very often MPs do not accumulate in the stomach and are expelled from the individual.

Hemorrhagic Shock Secondary To Button Battery Ingestion.

Button battery ingestion is a frequent pediatric complaint. The serious complications resulting from accidental ingestion have increased significantly over the last two decades due to easy access to gadgets and electronic toys. Over recent years, the increasing use of lithium batteries of diameter 20 mm has brought new challenges, because these are more detrimental to the mucosa, compared with other types, with high morbidity and mortality. The clinical complaints, which are often nonspecific, may lead to delayed diagnosis, thereby increasing the risk of severe complications. A five-year-old boy who had been complaining of abdominal pain for ten days, was brought to the emergency service with a clinical condition of hematemesis that started two hours earlier. On admission, he presented pallor, tachycardia and hypotension. A plain abdominal x-ray produced an image suggestive of a button battery. Digestive endoscopy showed a deep ulcerated lesion in the esophagus without active bleeding. After this procedure, the patient presented profuse hematemesis and severe hypotension, followed by cardiorespiratory arrest, which was reversed. He then underwent emergency exploratory laparotomy and presented a new episode of cardiorespiratory arrest, which he did not survive. The battery was removed through rectal exploration. This case describes a fatal evolution of button battery ingestion with late diagnosis and severe associated injury of the digestive mucosa. A high level of clinical suspicion is essential for preventing this evolution. Preventive strategies are required, as well as health education, with warnings to parents, caregivers and healthcare professionals.

Neutralisation Of The Pharmacological Activities Of Bothrops Alternatus Venom By Anti-pla2 Iggs.

Basic phospholipases A2 (PLA2) are toxic and induce a wide spectrum of pharmacological effects, although the acidic enzyme types are not lethal or cause low lethality. Therefore, it is challenging to elucidate the mechanism of action of acidic phospholipases. This study used the acidic non-toxic Ba SpII RP4 PLA2 from Bothrops alternatus as an antigen to develop anti-PLA2 IgG antibodies in rabbits and used in vivo assays to examine the changes in crude venom when pre-incubated with these antibodies. Using Ouchterlony and western blot analyses on B. alternatus venom, we examined the specificity and sensitivity of phospholipase A2 recognition by the specific antibodies (anti-PLA2 IgG). Neutralisation assays using a non-toxic PLA2 antigen revealed unexpected results. The (indirect) haemolytic activity of whole venom was completely inhibited, and all catalytically active phospholipases A2 were blocked. Myotoxicity and lethality were reduced when the crude venom was pre-incubated with anti-PLA2 immunoglobulins. CK levels in the skeletal muscle were significantly reduced at 6 h, and the muscular damage was more significant at this time-point compared to 3 and 12 h. When four times the LD50 was used (224 μg), half the animals treated with the venom-anti PLA2 IgG mixture survived after 48 h. All assays performed with the specific antibodies revealed that Ba SpII RP4 PLA2 had a synergistic effect on whole-venom toxicity. IgG antibodies against the venom of the Argentinean species B. alternatus represent a valuable tool for elucidation of the roles of acidic PLA2 that appear to have purely digestive roles and for further studies on immunotherapy and snake envenoming in affected areas in Argentina and Brazil.

S-nitrosoglutathione (gsno) Is Cytotoxic To Intracellular Amastigotes And Promotes Healing Of Topically Treated Leishmania Major Or Leishmania Braziliensis Skin Lesions-authors' Response.

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Laparoscopic Antireflux Surgery In Patients With Extra Esophageal Symptoms Related To Asthma.

Asthma, laryngitis and chronic cough are atypical symptoms of the gastroesophageal reflux disease. To analyze the efficacy of laparoscopic surgery in the remission of extra-esophageal symptoms in patients with gastroesophageal reflux, related to asthma. Were reviewed the medical records of 400 patients with gastroesophageal reflux disease submitted to laparoscopic Nissen fundoplication from 1994 to 2006, and identified 30 patients with extra-esophageal symptoms related to asthma. The variables considered were: gender, age, gastroesophageal symptoms (heartburn, acid reflux and dysphagia), time of reflux disease, treatment with proton pump inhibitor, use of specific medications, treatment and evolution, number of attacks and degree of esophagitis. Data were subjected to statistical analysis, comparing the pre- and post-surgical findings. The comparative analysis before surgery (T1) and six months after surgery (T2) showed a significant reduction on heartburn and reflux symptoms. Apart from that, there was a significant difference between the patients with daily crises of asthma (T1 versus T2, 45.83% to 16.67%, p=0.0002) and continuous crises (T1, 41.67% versus T2, 8.33%, p=0.0002). Laparoscopic Nissen fundoplication was effective in improving symptoms that are typical of reflux disease and clinical manifestations of asthma.

The Putative Leishmania Telomerase Rna (leishter) Undergoes Trans-splicing And Contains A Conserved Template Sequence.

Telomerase RNAs (TERs) are highly divergent between species, varying in size and sequence composition. Here, we identify a candidate for the telomerase RNA component of Leishmania genus, which includes species that cause leishmaniasis, a neglected tropical disease. Merging a thorough computational screening combined with RNA-seq evidence, we mapped a non-coding RNA gene localized in a syntenic locus on chromosome 25 of five Leishmania species that shares partial synteny with both Trypanosoma brucei TER locus and a putative TER candidate-containing locus of Crithidia fasciculata. Using target-driven molecular biology approaches, we detected a ∼2,100 nt transcript (LeishTER) that contains a 5' spliced leader (SL) cap, a putative 3' polyA tail and a predicted C/D box snoRNA domain. LeishTER is expressed at similar levels in the logarithmic and stationary growth phases of promastigote forms. A 5'SL capped LeishTER co-immunoprecipitated and co-localized with the telomerase protein component (TERT) in a cell cycle-dependent manner. Prediction of its secondary structure strongly suggests the existence of a bona fide single-stranded template sequence and a conserved C[U/C]GUCA motif-containing helix II, representing the template boundary element. This study paves the way for further investigations on the biogenesis of parasite TERT ribonucleoproteins (RNPs) and its role in parasite telomere biology.

Dysphagia After Antireflux Fundoplication: Endoscopic, Radiological And Manometric Evaluation.

The transient dysphagia after fundoplication is common and most often disappears until six weeks postoperatively. Analyze a group of patients who presented late and persistent dysphagia postoperatively. Forty-one patients after Nissen fundoplication, 14 male and 27 female, mean age 48 year, were evaluated based on medical history, esophagogastroduodenoscopy, contrast radiographic examination and esophageal manometry. The results were compared with another 19 asymptomatic individuals. Contrast radiographic examination of the esophagus revealed in six cases delayed emptying, characterizing that four patients had achalasia and two diffuse spasm of the esophagus. Esophageal manometry showed that maximal expiratory pressure of the lower sphincter ranged from 10 to 38 mmHg and mean respiratory pressure from 14 to 47 mmHg, values similar to controls. Residual pressure ranged from 5 to 31 mmHg, and 17 patients had the same values as the control group. The residual pressure of the lower sphincter was higher and statistically significant in patients with dysphagia compared with those operated without dysphagia. Future studies individualizing and categorizing each motility disorder, employing other techniques of manometry, and the analysis of the residual pressure may contribute to understand of persistent dysphagia in the postoperative fundoplication.

Omentum For Mammary Disorders: A 30-year Systematic Review.

Although the safety of applying omentum to the female breast for total breast reconstruction is controversial, it has recently been used to treat certain mammary disorders as well. A systematic review was therefore conducted to analyze and establish the suitability and safety of applying omentum to the breast. Covereing the interval from January 1984 to December 2013, we performed searches in MEDLINE, Embase, SciELO, and Google-Scholar for original articles describing the applicability of greater omentum to the breast and its clinical complications. Sixty observational articles with 985 women were chosen. The main clinical indications were total breast reconstruction after mastectomy due to breast cancer (45 studies), radiation damage (23 studies), and congenital Poland syndrome (4 studies). Altogether, 273 complications were identified among the 985 women treated. The most frequent was flap necrosis (26.74 %). The most serious was injury to the digestive system (1.10 %). There was a 35.48 % incidence of local breast cancer recurrence in eight observational studies on oncological risk. Seven of the eight included only women with advanced cancer. One of these studies reported the incidence and relapse time predominantly according to the primary tumor size. Although the oncological risk remains unclear, there was a high volume of complications that affected the digestive system. These findings suggest that omentum has well established applicability, but only for total breast reconstruction of huge defects, where muscular/myocutaneous or perforator flaps may be unsuitable.

Distribution Of Lutzomyia Longipalpis Chemotype Populations In São Paulo State, Brazil.

American visceral leishmaniasis (AVL) is an emerging disease in the state of São Paulo, Brazil. Its geographical expansion and the increase in the number of human cases has been linked to dispersion of Lutzomyia longipalpis into urban areas. To produce more accurate risk maps we investigated the geographic distribution and routes of expansion of the disease as well as chemotype populations of the vector. A database, containing the annual records of municipalities which had notified human and canine AVL cases as well as the presence of the vector, was compiled. The chemotypes of L. longipalpis populations from municipalities in different regions of São Paulo State were determined by Coupled Gas Chromatography - Mass Spectrometry. From 1997 to June 2014, L. longipalpis has been reported in 166 municipalities, 148 of them in the Western region. A total of 106 municipalities were identified with transmission and 99 were located in the Western region, where all 2,204 autochthonous human cases occurred. Both the vector and the occurrence of human cases have expanded in a South-easterly direction, from the Western to central region, and from there, a further expansion to the North and the South. The (S)-9-methylgermacrene-B population of L. longipalpis is widely distributed in the Western region and the cembrene-1 population is restricted to the Eastern region. The maps in the present study show that there are two distinct epidemiological patterns of AVL in São Paulo State and that the expansion of human and canine AVL cases through the Western region has followed the same dispersion route of only one of the two species of the L. longipalpis complex, (S)-9-methylgermacrene-B. Entomological vigilance based on the routes of dispersion and identification of the chemotype population could be used to identify at-risk areas and consequently define the priorities for control measures.

A questão da licenciatura em Educação Fisica : a transição a pratica profissional

Universidade Estadual de Campinas. Faculdade de Educação Física

Aspectos epidemiológicos da fluorose dentária no Brasil: pesquisas no período 1993-2006

A epidemiologia da fluorose dentária resulta de inquéritos realizados recentemente, em decorrência da melhor compreensão de aspectos metabólicos dos fluoretos no organismo humano e de preocupações no âmbito da saúde coletiva. Objetiva-se apresentar os estudos realizados sobre a fluorose entre 1993 e 2006. O período 1993-2004 demarca o intervalo entre a 2ªe a 3ª Conferência Nacional de Saúde Bucal. Em 2005-2006, a busca de dados primários apresentados em reuniões científicas confirmou os achados existentes na literatura, mostrando que a agenda científica brasileira não foi substancialmente influenciada pelas discussões travadas durante a 3ª Conferência. A maioria dos estudos concentra-se em áreas urbanas e a predominância no Brasil dos graus "muito leve" e "leve" de fluorose mostra não haver comprometimentos de ordem funcional. A baixa percepção da fluorose pela população, aliada à sua baixa prevalência, evoca o necessário debate sobre as questões de saúde coletiva no país. Visto que a produção científica nacional constitui uma importante fonte de conhecimentos para subsidiar a elaboração de políticas públicas para o setor da saúde, os sucessivos e pontuais estudos analisados mostram que, no que diz respeito à fluorose, o diagnóstico epidemiológico reafirma a necessidade, a importância e a segurança da fluoretação das águas de abastecimento público como medida de saúde coletiva.

Avaliação cefalométrica das alterações dentoesqueléticas de jovens com má oclusão de Classe II dentária tratados com distalizadores Jones jig

OBJETIVO: esta pesquisa objetivou avaliar cefalometricamente as alterações dentoesqueléticas de jovens com Classe II dentária tratados com o distalizador Jones jig. METODOLOGIA: foram avaliados 30 pacientes, sendo 15 de cada gênero, com média de idades iniciais de 13,63 anos; brasileiros, naturais da cidade de Bauru/SP, caracterizados por má oclusão de Classe II, 1ª e 2ª divisões de Angle sem comprometimento esquelético. Os jovens foram tratados com aparelho Jones jig a fim de distalizar os molares superiores a uma relação molar de "super Classe I"; sendo que esse dispositivo permaneceu, em média, por 0,55 anos. Ao final da sobrecorreção, os molares distalizados receberam um botão de Nance e, como ancoragem extrabucal, o aparelho extrabucal (AEB) com tração média-alta, com o intuito de verticalizar e corrigir a angulação radicular dos molares distalizados. Foram realizadas telerradiografias em normal lateral inicial (T1) e pós-distalização (T2). As medidas cefalométricas foram submetidas ao teste t dependente de Student para avaliar as alterações de T1 para T2. RESULTADOS: com base nos resultados obtidos e a partir da metodologia empregada, observou-se alterações dentárias significativas, como a movimentação distal linear e angular, assim como a intrusão dos segundos e primeiros molares superiores no sentido vertical. Também se confirmou efeitos indesejáveis, como a perda de ancoragem refletida em mesialização, extrusão e angulação mesial dos segundos pré-molares, a protrusão dos incisivos superiores e o aumento do trespasse vertical e horizontal. Pode-se confirmar que certas movimentações dentárias promovem significativas alterações esqueléticas de estruturas localizadas à distância, ou seja, observou-se extrusão significativa dos segundos pré-molares superiores, o que resultou em rotação mandibular, aumento significativo da altura facial anteroinferior e protrusão do lábio inferior. CONCLUSÃO: pode-se concluir que o distalizador Jones jig promove, basicamente, alterações dentárias.