A leishmaniose visceral canina (LVC) é uma grave zoonose, causada pela Leishmania infantum (syn. chagasi). O ciclo deste parasito é heteroxeno e a transmissão acontece principalmente pela picada da fêmea do vetor, dípteros da espécie Lutzomyia longipapis. O cão doméstico é o principal alvo das campanhas de controle da doença por ser a principal fonte de infecção para o vetor no ambiente urbano. O Ministério da Saúde adota testes sorológicos para a detecção de animais positivos; no entanto a sensibilidade e especificidade desses testes são questionáveis. Além das falhas inerentes a qualquer teste diagnóstico, no caso da LVC existem alguns entraves, especialmente pela distribuição geográfica, comum a outras doenças causadas por tripanossomas, e pela similaridade genética com os outros parasitas da mesma família. Nessas áreas de sobreposição pode haver tanto reação cruzada quanto co-infecção, dificultando a interpretação dos testes. No presente estudo, foram coletadas amostras de suabe conjuntival e sangue de cães em inquérito soroepidemiológico realizado no município de Ilha Solteira - SP. A presença de Leishmania spp. e Leishmania infantum foram testadas por PCR convencional e PCR em tempo real com primers direcionados ao kDNA de Leishmania spp. A avaliação sorológica foi realizada através da RIFI, e a identificação e caracterização dos tripanossomatídeos foi realizada através da PCR com primers ITS1. A SC-qPCR foi o teste que detectou o maior número de animais. De 204 cães utilizados no estudo, 19,12% (30/204) foram positivos na SC-qPCR. Na SG-qPCR foram 12,74% (26/204) de animais positivos. O teste que detectou o menor número de animais foi a SC-cPCR, com 10,78% (22/204). Enquanto na SG-cPCR obtivemos 13,23% (27/204) animais positivos. De 28 amostras selecionadas para o sequenciamento do gene ITS1, 19 (67,85%) foram 100 ou 99% similares à L. infantum, sugerindo que a maioria dos cães positivos para LVC estavam realmente infectados com esta espécie. Entretanto, 2 cães (7,14%), que tiveram suas amostras sequenciadas para tal gene, revelaram 99% de similaridade com Crithidia fasciculata. Dos testes avaliados, esses cães foram positivos apenas na SG-cPCR para Leishmania spp. Os resultados indicam que a SC-qPCR foi o teste mais eficaz em detectar amostras realmente positivas para L. infantum, e que se deve atentar ao fato de existirem outros tripanossomatídeos infectando os cães em área endêmica de LVC, podendo dificultar o diagnóstico adequado dos animais infectados por L. infantum. / Canine visceral leishmaniasis (CVL) is a serious zoonosis caused by Leishmania infantum (syn. chagasi). The life cycle of this parasite is heteroxenous and the transmission occurs through the bite of the female sandfly, diptera species Lutzomyia longipalpis. The domestic dog is the main focus disease control campaigns, since it is the most important source of infection for the vector in urban environment. For positive dogs detection the health ministry uses serological tests, however the sensitivity and specificity of these tests are questionable. In addition to flaws inherent in any diagnostic test, in case of CVL there are some obstacles, especially by geographic distribution, common to other diseases caused by trypanosomes, and also by genetic similarity with other parasites of the same family. In areas of disease overlap, cross-reaction or co-infection may occur, making it difficult to interpret the results. In this study, conjunctival swab samples and whole blood of dogs were collected in seroepidemiological survey conducted in Ilha Solteira - SP. The presence of Leishmania spp. and Leishmania infantum were tested by conventional PCR and real-time PCR with Leishmania spp. kDNA-targeted primers. The serological evaluation was carried out by RIFI, and the identification and characterization of trypanosomatids was performed by PCR with ITS1 primers. SC-qPCR was the test that detected the largest number of animals. Of 204 dogs used in this study, 19.12% (30/204) were positive in SC-qPCR. In SG-qPCR 12.74% (26/204) animals were positive. The test that detected the lowest number of animals was SC-cPCR, with 10.78% (22/204). While in the SG-cPCR we obtained 13.23% (27/204) positive animals. From 28 samples selected for ITS1 gene sequencing, 19 (67.85%) were 100 or 99% similar to L. infantum, suggesting that most CVL positive dogs were infected with this species. However, two dogs (7.14%), which had their samples sequenced for the same gene, showed 99% similarity with Crithidia fasciculata. From the evaluated tests, these dogs were only positive in SG-cPCR for Leishmania spp. The results indicate that SC-qPCR was the most effective test to detect L. infantum positive samples , and it should be noted that there are other trypanosomatids infecting dogs in an endemic CVL area, which can difficult to diagnose animals properly infected by L. infantum.

In the first study, three different transport systems for bacteria were evaluated. The CLSI M40-A guideline was used to monitor the maintenance of both mono- and polymicrobial samples during a simulated transportation at room temperature that lasted 0-48 h. All systems were able to maintain the viability of all organisms for 24 h, but none of them could support all tested species after 48 h.  The most difficult species to recover was Neisseria gonorrhoeae, and in polymicrobial samples overgrowth was an observed problem. The aim of the second study was to study the presence of TSST-1 and three other important toxin genes in invasive isolates of Staphylococcus aureus collected during the years 2000-2012 at two tertiary hospitals. The genes encoding the staphylococcal toxins were detected by PCR, and whole-genome sequencing was used for analyzing the genetic relatedness between isolates. The results showed that the most common toxin was TSST-1, and isolates positive for this toxin exhibited a clear clonality independent of year and hospital. The typical patient was a male aged 55-74 years and with a bone or a joint infection. The third study was a clinical study of the effect of silver-based wound dressings on the bacterial flora in chronic leg ulcers. Phenotypic and genetic silver-resistance were investigated before and after topical silver treatment, by determining the silver nitrate MICs and by detecting sil genes with PCR. The silver-based dressings had a limited effect on primary wound pathogens, and the activity of silver nitrate on S. aureus was mainly bacteriostatic. A silver-resistant Enterobacter cloacae strain was identified after only three weeks of treatment, and cephalosporin-resistant members of the Enterobacteriaceae family were relatively prone to developed silver-resistance after silver exposure in vitro. The last study was undertaken in order to develop an easy-to-use method for simulating the laundering process of hospital textiles, and apply the method when evaluating the decontaminating efficacy of two different washing temperatures. The laundering process took place at professional laundries, and Enterococcus faecium was used as a bioindicator. The results showed that a lowering of the washing temperature from 70°C to 60°C did not affect the decontamination efficacy; the washing cycle alone reduced the number of bacteria with 3-5 log10 CFU, whereas the following tumble drying reduced the bacterial numbers with another 3-4 log10 CFU, yielding the same final result independent of the washing temperature. To ensure that sufficient textile hygiene is maintained, the whole laundering process needs to be monitored. The general conclusion is that all developmental work in the bacterial field requires time and a large strain collection.

Underdiagnosis of intestinal parasites in patients with diarrhea A compilation from the Swedish public health authority indicates that infections caused by Cryptosporidium spp. increased in Sweden from 47 cases in 2004 to 594 cases in 2016 and Giardia intestinalis causes around 1300 infections per year. The primary aim of this study was to investigate the prevalence of parasites in patients with diarrhea. Furthermore, the study investigated whether samples taken with E-swab could be analyzed with real-time polymerase chain reaction (PCR) for detection of Cryptosporidium spp., Dientamoeba fragilis, Entamoeba histolytica/dispar and G. intestinalis rather than Sodium acetate-acetic acid-formaline fixative (SAF-fixative). Prevalence of parasites in fecal samples was collected from 200 samples from patients with bacterial issue ordered. For evaluation of E-swab, 22 frozen, unfixed samples that were positive for intestinal parasites was used. Twelve positive E-swab samples was used as comparative positive controls. This was analyzed using real-time PCR. Bacteria was counted for 9.5% of the infections whilst parasites counted for 14% of the infections. The conclusion was that E-swab could replace SAF-fixative in the diagnosis of intestinal parasites and that there is that an underdiagnosis of intestinal parasites. Keywords: Cryptosporidium spp, Dientamoeba fragilis, Entamoeba histolytica, Entamoeba dispar, Giardia intestinalis, real-time PCR, E-swab, prevalence.

No description available.

Made available in DSpace on 2016-06-02T20:20:34Z (GMT). No. of bitstreams: 1 4932.pdf: 1990172 bytes, checksum: 7025c9dd589a8c2a6771bc24539895b5 (MD5) Previous issue date: 2013-03-05 / Universidade Federal de Sao Carlos / The genetic diversity of the cattle egret (Bubulcus ibis) was analyzed to investigate reproductive behavior and characterize Brazilian populations of the species. Genotypes at seven microsatellite loci were used to investigate the occurrence of more than one female laying eggs in the same nest, characterizing the occurrence of multiple maternity. DNA was extracted from swabs collected from the outer surface of eggs and sexed; males were excluded. Forty-eight clutches from two breeding seasons (2010 and 2011) were genetically analyzed. Thirty-nine eggs laid by a second or third female were recorded. In five nests, the first egg of the clutch was from a different female, the laying happening prior to that of the incubating female. Suggesting nest takeover by another pair of egrets that kept the pre-existing eggs together with its own clutch. In the other 43 nests, the hypothesis of brood parasitism was posed to explain why one or two additional females were found laying eggs in a nest. A 463-bp fragment of the mitochondrial DNA control region Domain I was amplified for 148 individuals from seven Brazilian populations to investigate genetic-population and demographic parameters. Genetic diversity indices, the population structure tests Fst and AMOVA, a haplotype network, mismatch distribution and neutrality tests (Tajima s D, Fu s Fs, Fu and Li s D* and F*, Ramos-Onsins and Rozas R2) revealed the following: i) a high level of diversity was recorded for the cattle egret in Brazil in comparison to other closely related species studied in the country; ii) genetic diversity levels determined for the Brazilian regions are similar; iii) genetic structuring was not observed between the seven studied populations; and iv) the different tests performed to determine demographic expansion revealed no significant results. This is the first genetic characterization study for Bubulcus ibis to date and the findings indicate a high degree of plasticity in reproductive behavior and confirm a marked dispersion behavior of the species, leading to the homogenization of Brazilian populations. / A diversidade genética da garça-vaqueira (Bubulcus ibis) foi utilizada para se investigar o comportamento reprodutivo e para se caracterizar populações brasileiras da espécie. Genótipos em locos de microssatélites foram utilizados na detecção da presença de mais de uma fêmea ovipositando em um mesmo ninho, o que pode caracterizar a ocorrência de maternidade múltipla. O DNA extraído dos swabs coletados na superfície externa dos ovos foi sexado e eventuais amostras de machos foram excluídas. Quarenta e oito ninhadas, de duas temporadas reprodutivas (2010 e 2011) foram analisadas geneticamente. Foram registrados 39 ovos ovipositados por uma segunda ou terceira fêmea. Em 33 ninhos foram encontrados genótipos distintos de duas fêmeas e em seis ninhos genótipos de três fêmeas. Em cinco ninhos o primeiro ovo na sequência de oviposição mostrou-se ser de uma fêmea diferente, tendo a oviposição acontecido previamente àquela da fêmea incubante. Esse achado foi explicado supondo a tomada de ninho por um segundo casal de garças, com a manutenção do ovo pré-existente juntamente com os da própria ninhada. Nos outros 43 ninhos, a presença das fêmeas extras foi explicada hipotetizando a ocorrência de parasitismo de ninho intraespecífico. Um fragmento de 463 pb do Domínio I da região controladora do DNA mitocondrial foi amplificado para 148 indivíduos para a investigação de parâmetros genéticopopulacionais e processos demográficos nas sete populações estudadas. A estimativa de índices de diversidade genética, testes de estruturação populacional Fst e AMOVA, a construção de uma rede de relação entre haplótipos, a análise de mudanças no tamanho populacional pela mismatch distribution e a realização de testes de neutralidade (D de Tajima, Fs de Fu, D* e F* de Fu e Li, R2 de Ramos-Onsins e Rozas) permitiram identificar: i) um nível alto de diversidade genética para B. ibis no Brasil, quando comparado a espécies proximamente relacionadas estudadas no país; ii) níveis semelhantes de diversidade genética determinados para as regiões brasileiras; iii) ausência de estruturação genética entre as sete populações estudadas; e iv) ausência de sinais de expansão demográfica pelos testes realizados. Os resultados aqui apresentados são os primeiros resultados genéticos na espécie até o momento e apontam para uma alta plasticidade no comportamento reprodutivo e confirmam a dispersão bastante acentuada da espécie, levando a homogeneização das populações brasileiras.

<p>Ambient ionization mass spectrometry enables rapid <i>in-situ</i> analysis of a plethora of analytes that are relevant to the forensic and defense communities. As the arsenal of ambient ionization techniques, aimed at solving specific targeted problems, continues to expand, the adoption of these techniques into non-academic settings has been relatively slow. At times, although the technique can provide answers in a more rapid and cheaper manner, the technique does not pass all of the required legal rules for a particular analysis when dealing with forensic evidence. This can be demonstrated with the rapid detection of drugs by paper spray ionization mass spectrometry. Paper spray ionization mass spectrometry can have drugs deposited onto the paper substrate, the paper can wipe a surface for trace analytes, and there are commercial and automated ionization sources for this process. While analysis by paper spray is rapid, the Scientific Working Group for the Analysis of Seized Drugs (SWGDRUG) states that a minimum of two instrumental techniques need to be utilized. Utilizing paper substrates that have nanoparticles embedded for surface enhanced Raman spectroscopy, that can also be utilized for paper spray ionization mass spectrometry, makes ambient ionization more appealing as it completes that first legal requirement. </p> <p>Other times, the slow adoption of these new ambient ionization techniques is due to specific communities not being aware of ambient ionization, and specific applications have not yet been demonstrated. Swab touch spray ionization mass spectrometry follows similar processes as paper spray ionization, as the swab acts both as the sampling substrate and the ionization source and can swab for analytes in a manner where the paper substrate may be damaged and unable to perform the ionization for analysis. This can be seen for the swabbing of organic gunshot residues and explosives, both of which current methods already use a swab for sampling but then need lengthy extraction techniques. The applicability of paper spray ionization and swab touch spray ionization for these forensic and defense analyses is only furthered by the fact that they both couple extremely well with portable mass spectrometers for analysis in the field.</p> <p>There are also many fields that ambient ionization is just starting to take its place in the analytical toolbox. Two such defense fields that are just beginning to expand into ambient ionization are the analysis of pyrotechnics and microelectronics. Pyrolysis gas-chromatography mass spectrometry methods have been developed and utilized for environmental tests for pyrotechnic formulation, but they are slow and there is an abundance of cleaning steps between analyses to prevent carry over and contamination. Using paper and swabs as the collection device and ionization source for environmental analysis of these pyrotechnics allow for them to be functioned at ambient conditions at the scale at which will be utilized in the field by the Warfighter. Similarly, authenticating microelectronics by desorption electrospray ionization mass spectrometry removes the subjectivity of the current methods, while rendering the integrated circuit intact enabling future use if deemed as a genuine part. By taking slower or more subjective tests, in a field that has not utilized ambient ionization heavily in the past and adding these new capabilities to their tool chest expands the acceptance and future applications of the technique.</p> <p>As acceptance and utilization of ambient ionization grows, the next generation of scientists need to have hands on training in these techniques. Through the development of new teaching laboratories that couple both the fundamentals of the technique at hand, while also examining an interesting application to better engage the students, a number of laboratory exercises have been developed. The creation of new laboratory exercise utilizing the next generation of instrumentation and analytical techniques is vital for the future and rapid application of these techniques. The work discussed herein chronicles the utilization and demonstration of ambient ionization mass spectrometry in monitoring clandestine activities, supporting the Warfighter, and redeveloping chemical laboratory education. </p>