Background: Malaria is a major world health issue and its continued burden is due, in part, to difficulties in the diagnosis of the illness. The World Health Organization recommends confirmatory testing using microscopy-based techniques or rapid diagnostic tests (RDT) for all cases of suspected malaria. In regions where Plasmodium species are indigenous, there are multiple etiologies of fever leading to misdiagnoses, especially in populations where HIV is prevalent and children. To determine the frequency of malaria infection in febrile patients over an 8-month period at the Regional Hospital in Bamenda, Cameroon, we evaluated the clinical efficacy of the Flourescence and Staining Technology (FAST) Malaria stain and ParaLens AdvanceTM microscopy system (FM) and compared it with conventional bright field microscopy and Giemsa stain (GS). Methods: Peripheral blood samples from 522 patients with a clinical diagnosis of "suspected malaria" were evaluated using GS and FM methods. A nested PCR assay was the gold standard to compare the two methods. PCR positivity, sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were determined. Results: Four hundred ninety nine samples were included in the final analysis. Of these, 30 were positive via PCR (6.01%) with a mean PPV of 19.62% and 27.99% for GS and FM, respectively. The mean NPV was 95.01% and 95.28% for GS and FM, respectively. Sensitivity was 26.67% in both groups and specificity was 92.78% and 96.21% for GS and FM, respectively. An increased level of diagnostic discrepancy was observed between technicians based upon skill level using GS, which was not seen with FM. Conclusions: The frequency of malarial infections confirmed via PCR among patients presenting with fever and other symptoms of malaria was dramatically lower than that anticipated based upon physicians' clinical suspicions. A correlation between technician skill and accuracy of malaria diagnosis using GS was observed that was less pronounced using FM. Additionally, FM increased the specificity and improved the PPV, suggesting this relatively low cost approach could be useful in resource-limited environments. Anecdotally, physicians were reluctant to not treat all patients symptomatically before results were known and in spite of a negative microscopic diagnosis, highlighting the need for further physician education to avoid this practice of overtreatment. A larger study in an area with a known high prevalence is being planned to compare the two microscopy methods against available RDTs.

La fertilidad masculina puede ser medida mediante un espermatograma convencional, sin embargo este examen no incluye la valoración de la integridad del ADN espermático. Esta variable ha sido correlacionada con las tasas de fertilización, viabilidad y desarrollo del embrión, convirtiéndose en una herramienta de importancia clínica tanto para los programas de reproducción animal como los tratamientos de fertilidad asistida. El bioensayo Cometa es capaz de determinar de una manera exacta el valor de la integridad del ADN espermático, lamentablemente este examen no es de uso rutinario por su elevado costo de implementación ya que utiliza microscopia especializada y tinciones fluorescentes para evidenciar la migración del ADN. El objetivo de esta investigación fue evaluar la compatibilidad de las tinciones no fluorescentes Diffquik, Giemsa, de Feulgen y FastBlast en el bioensayo Cometa usando un método visual y automatizado. Se utilizaron 15 eyaculados previamente seleccionados de acuerdo al manual OMS 2010, para luego ser capacitados en búsqueda de homogeneidad adecuada para la experimentación. Cada muestra fue expuesta a una gradiente de Peróxido de hidrogeno (0, 10, 30,60 y 100 mM) por 1 hora a 4°C para luego evaluar el coeficiente de daño mediante el método visual y porcentaje de ADN en la cola mediante el método automatizado. Las pendientes de la regresión lineal en el método visual indican que los valores obtenidos por la tinción control SybrGreen (m=3,69) difieren con Giemsa (m=3,45) y Diffquik (m=2,57). En el método automatizado de igual manera SybrGreen (m=0.83), Giemsa (m=0,79) y Diffquik (m=0,77). Sin embargo SybrGreen es 1,06 veces más efectivo que Giemsa en el visual y 1,05 veces en el automatizado, sugiriendo una compatibilidad con el bioensayo cometa. De igual manera SybrGreen es 1,07 veces más efectivo que Diffquik en el visual y 1,44 veces en el automatizado, concluyendo una compatibilidad solo en el método visual.Male fertility can be measured by a conventional semen analysis, however, this examination does not include the assessment of sperm DNA integrity. This variable has been correlated with fertilization rates, embryo viability and development, becoming a tool of clinical importance for both animal breeding programs and assisted fertility treatments. Comet bioassay is able to determine an exact way the value of sperm DNA integrity, unfortunately this test is not routinely used because of its high cost of implementation because it uses specialized microscopy and fluorescent dyes to demonstrate DNA migration. The objective of this research was to evaluate the compatibility of non-fluorescent dyes Diffquik, Giemsa, Feulgen and Comet FastBlast in the bioassay using a visual and automated method. 15 ejaculates were used previously manually selected according to WHO 2010 and then be trained in finding adequate homogeneity for experimentation. Each sample was exposed to a hydrogen peroxide gradient (0, 10, 30,60 and 100 mM) for 1 hour at 4 ° C and then assess the damage coefficient by visual method and percentage of DNA in the tail by automated method. The slopes of the linear regressions on the visual method indicate that the values obtained by the SybrGreen Control staining (m = 3.69) differ with Giemsa (m = 3.45) and Diffquik (m = 2.57). In the same way automated method SybrGreen (m = 0.83), Giemsa (m = 0.79) and Diffquik (m = 0.77). However SybrGreen is 1.06 times more effective than Giemsa visual and 1.05 times in the automated, suggesting a comet support bioassay. Similarly SybrGreen is 1.07 times more effective than Diffquik visual and 1.44 times in the automated, concluding compatibility only in the visual method. Keywords:

No description available.

VALIDERING AV MAY-GRÜNWALD GIEMSA VID FÄRGNING AV CYTOLOGIPROVEROPTIMERING AV IN VITRO-DIAGNOSTIK, MED ANLEDNING AV NYA EU-KRAVKARIN SVANTESSONSvantesson, K. Validering av May-Grünewald Giemsa vid färgning av cytologiprover. Optimering av in vitro-diagnostik, med anledning av nya EU-krav. Examensarbete i biomedicinsk laboratorievetenskap, 15 högskolepoäng. Malmö Universitet: Fakulteten för hälsa och samhälle, institutionen för Biomedicinsk vetenskap, 2023.Cancer uppstår vid onormal celldelning, men uppkommer även vid kronisk inflammation. För att kunna konstatera om patienten har cancer krävs olika typer av diagnostik, där cytologidiagnostik ingår. Med hjälp av olika färglösningar kan cancerceller i serösa vätskor färgas för att upptäckas, således kan den cancerdrabbade patienten behandlas. May-Grünwald Giemsa (MGG) ingår i Romanowsky-färgningsteknikerna och har använts sedan 1800-talet.Färglösningen ger en högkvalitativ visualisering av cellernas morfologi. Färgen används inom histologi-, hematologi- och cytologidiagnostiken. Laboratorier som arbetar med in vitro-diagnostik (IVD) måste arbeta med märkta produkter som är reglerade för IVD. År 2017 fastställdes att inom fem år skall alla laboratorier i Europeiska unionen (EU) som utför IVD, arbeta med in vitro-diagnostik reglerade (IVDR) produkter för att uppnå EU-direktiven. Syftet med studien var att optimera en ny färgblandning av MGG vid färgning av cytologiprover, så att IVDR-kraven uppfylls på Patologen i Skövde. Studien omfattade flera försök för att fastställa vilket av färgprotokollen som gav bästa kvalité på cellerna i cytologiska preparat. Under studiens gång har det även visats att olika faktorer, exempelvis färskheten av provet kan påverka infärgningens kvalité av preparatet. Gammalt eller felhanterat provmaterial kan leda till försämrad infärgning av cellerna. Resultat från studien visade att färgprotokollet Histo Lab (HL) gav goda resultat efter en modifiering av sammansättningen och tiden vid infärgning. / VALIDATION OF MAY-GRÜNWALD GIEMSA IN THE STAINING OF CYTOLOGY SAMPLESOPTIMIZATION OF IN VITRO-DIAGNOSTICS, DUE TO NEW EU REQUIREMENTSKARIN SVANTESSONSvantesson, K. Validation of May-Grünwald Giemsa in the staining of cytology samples. Optimization of in vitro-diagnostics, due to new EU requirements. Degree project in biomedical laboratory science, 15 higher education credits. Malmö University: Faculty of Health and Society, Department of Biomedical Sciences, 2023.Cancer is caused by abnormal cell division, but also occurs by chronic inflammation. In order to determine whether the patient has cancer, different types of diagnostics are required, where cytology diagnostics are included. With thehelp of different dye solutions, cancer cells in serous fluids can be stained and detected, allowing for diagnosis and treatment. May-Grünwald Giemsa (MGG) is part of the Romanowsky staining techniques and has been used since the 19th century. The dye solution provides a high-quality visualization of the cells' morphology and is used in histology-, hematology- and cytology diagnostics.Laboratories that work with in-vitro diagnostics (IVD) must use products that are in vitro-diagnostics regulated (IVDR). In 2017 it was determined that within five years all laboratories in the European Union (EU) performing IVD must work with IVDR labeled products to achieve EU directives. The aim of the study was to optimize a new dye stain of MGG for cytology samples, so that the IVDR requirements are achieved at the pathology laboratory in Skövde. The study included several attempts to determine which of the staining protocols produced the best quality of the cells in cytology preparations. During the study, it has also been shown that various factors can negatively affect the results. If sample material is too old or mishandled, it can lead to poor staining of the cells. Results from the study showed that the Histo Lab (HL) staining protocol gave goodresults after modification of the composition and time of staining.

Submitted by (edna.saturno@ufrpe.br) on 2016-06-29T13:52:35Z No. of bitstreams: 1 Maria Luiza da Silva.pdf: 1369807 bytes, checksum: 27a12fb618790d4807325a30dfc6fc12 (MD5) / Made available in DSpace on 2016-06-29T13:52:35Z (GMT). No. of bitstreams: 1 Maria Luiza da Silva.pdf: 1369807 bytes, checksum: 27a12fb618790d4807325a30dfc6fc12 (MD5) Previous issue date: 2014-02-10 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Eichhornia is a Neotropical genus belonging to the family Pontederiaceae. It occurs in aquatic environments with outstanding ecological importance. Chromosome studies in the genus are scarce, limited to the description chromosome numbers. This study aimed to analyze four species of Eichhornia, by conventional Giemsa staining, fluorochrome staining with CMA and DAPI and fluorescent in situ hybridization (FISH), to characterize the karyotypes and define numerical or structural karyotypic polymorphisms that could contribute to interspecific differentiation between these four species and the understanding of chromosome evolution of the genus Eichhornia. All species have small chromosomes and symmetrical karyotypes, from 0.73 μm in E. crassipes to 2.94 μm in E. heterosperma and predominantly metacentric. Chromosomal counts were 2n = 32 for E. crassipes, 2n = 30 for E. heterosperma, 2n = 28 for E. diversifolia and 2n = 16 for E. paniculata. The investigated species showed interphase nuclei ranging from areticulate to semi-reticulate and proximal early condensation in prophase chromosomes with most intense staining in pericentromeric regions. The CMA/DAPI staining revealed CMA+/DAPI- bands that were co-localized with 45S rDNA sites in terminal regions. Two signals were observed for E. heterosperma and E. diversifolia, and four signals in E. paniculata and E. crassipes, although six CMA+ signals were observed in the latter species. The 5S rDNA did not vary in the number of sites but in position. Two sites were observed in the terminal region in E. paniculata and in the pericentromeric region in the other species. Chromosomal inversion and dysploidy were suggested to explain the non-colinearity of 5S rDNA sites. / Eichhornia é um gênero Neotropical pertencente à família Pontederiaceae. Ocorre em ambientes aquáticos com destacada importância ecológica. Estudos cromossômicos em espécies do gênero são escassos, limitando-se a descrição da quantidade de cromossomos. O presente trabalho teve como finalidade analisar quatro espécies de Eichhornia através da coloração convencional com Giemsa, com os fluorocromos CMA e DAPI e pela hibridização in situ fluorescente (FISH), visando caracterizar os cariótipos e definir polimorfismos cariotípicos numéricos ou estruturais que contribuam para a diferenciação interespecífica entre essas quatro espécies e para a compreensão da evolução cromossômica do gênero Eichhornia. Todas as espécies apresentaram cromossomos pequenos e cariótipos simétricos, medindo desde 0.73 μm em E. crassipes a 2.94 μm em E. heterosperma e cromossomos predominantemente metacêntricos. As contagens cromossômicas foram 2n = 32 para E. crassipes, 2n = 30 para E. heterosperma, 2n = 28 para E. diversifolia e 2n = 16 para E. paniculata. As espécies investigadas apresentaram núcleos interfásicos variando de arreticulados a semi-reticulados e cromossomos com condensação profásica proximal com coloração mais intensa na região pericentromérica. A coloração CMA/DAPI revelou bandas do tipo CMA+/DAPI- que foram co-localizadas com os sítios de DNAr 45S nas regiões distais. Dois sinais foram observados para E. heterosperma e E. diversifolia e quatro sinais em E. paniculata e em E. crassipes, embora tenham sido observados seis sinais CMA+ nesta última espécie. O DNAr 5S não variou em número de sítios, mas em posição. Dois sítios foram observados na região terminal em E. paniculata e na região pericentromérica das demais espécies. Eventos de inversão e disploidia cromossômica foram sugeridos para explicar a não colinearidade dos sítios de DNAr 5S.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / CHAPTER II: Pequi, Caryocar brasiliense, is one of the species of highlight at the biome of the Brazilian savannah due to its utilization in culinary, popular medicine, industry in general, and iron and steel industry. It presents an elevated index of exploration, being capable of entering the list of the endangered species. In the region of São José do Xingu (MT), a tree of pequi without thorn at the mesocarp was found and this enables to improve pequi not only for consumption, taking advantage of the high appreciation it already has. To detect the existing genomic differences between the pequi with and without the thorn at the endocarp, RADP markers were utilized. The generated polymorphisms were cloned and sequenced in order to identify the sequences responsible for the fenotypical alteration. It was observed that the pequi without thorn is genetically isolated from the other populations of pequi with thorn at the endocarp, proving that this characteristic is related to the genetic divergence of the species. Analysis in BLASTn evidenced the similarity of the Dof1 genes of Zea mays, in the population with thorn, and in the population without thorn, with the gene of phosphinotricin acetyl transferase of Z. mays. In the analysis of BLASTx, the similarity was verified with the proteins responsible for the deficiency in ferric reductase 4, in the pequi without thorn and catalase, in the pequi without thorn. CHAPTER III:The pequi, Cayocar brasiliense Cambess. Is a feature of Brazilian cerrado plant suffering extractive action, mainly for food. It is a plant very appreciated in the regions of Minas Gerais, especially in the north of this state. In the region of Sao Jose do Xingu, Mato Grosso, was found in a plant which had no thorns in the core and this allowed further research to discover the lack of thorns as it is an unwanted feature of the plant. Thus, the cytogenetic study, using conventional Giemsa staining, NOR, banda C, DAPI and CMA3 were used for comparison between the pequi with and without spines in the putamen. It was found that these differences are not attributed to chromosomal differences between the two types of pequi. Both have the same chromosome number, the marking of NOR, C banding was not verified due to the size of chromosomes and differences in staining with DAPI and CMA3 could not be verified although related to these characteristics. CHAPTER IV: The pequi is a species that is gathered and today is seen as a source for the production of biodiesel. The objective of this study was to identify sequences from a cDNA library that are involved in the metabolic pathways for production of pequi fruit, to gain a better understanding of the species and its biological and genetic properties. To do so, a cDNA library was created and the clones were cloned and sequenced. These identified sequences were deposited in GenBank. Most of the sequences found are associated with protection against oxidative stress in plants, some are transcription factors and others provide structural and pathogenic resistance. / CAPÍTULO II: O pequi, Caryocar brasiliense, é uma das espécies de destaque no bioma do Cerrado devido a sua utilização na culinária, medicina popular, indústria e siderurgia. Apresenta índice de exploração elevado, podendo entrar na lista das espécies ameaçadas de extinção. Na região de São José do Xingu (MT) foi encontrada uma árvore produzindo pequi sem espinho no endocarpo, o que levantou a possibilidade de melhorar o pequi para consumo aproveitando a alta apreciação que já possui. Com o objetivo de analisar as diferenças genômicas entre o pequi com e o sem espinho no endocarpo, utilizou-se marcadores RADP (Random Amplified Polymorphism DNA). As bandas polimórficas geradas foram isoladas, clonadas e seqüenciadas, buscando identificar sequências responsáveis pela alteração fenotípica. Observou-se que o pequi sem espinho fica isolado geneticamente das demais populações de pequi com espinho no caroço, comprovando que essa característica está relacionada à divergência genética da espécie. Análises em BLASTn mostraram a similaridade aos genes Dof1 de Zea mays, na população com espinho e com o gene da Acetiltransferase Fosfinotricina de Z. mays, na população sem espinho. As análises em BLASTx revelaram similaridade com as proteínas responsáveis pela Deficiência em Redutase Férrica 4, no pequi sem espinho e com catalase, no pequi com espinho. CAPÍTULO III: O pequizeiro, Cayocar brasiliense Cambess., é uma planta característica do cerrado brasileiro que sofre ação extrativista, principalmente, para a alimentação. É uma planta bastante apreciada em regiões de Minas Gerais, especialmente no Norte desse estado. Na região de São José do Xingu, Mato Grosso, foi encontrada uma planta produzindo pequi sem espinhos no caroço. O estudo citogenético, por meio de coloração convencional de Giemsa, NOR, banda C, DAPI e CMA3 foi utilizado para a comparação entre plantas produtoras de pequi com e sem espinhos no caroço. Verificou-se que essas diferenças não podem ser atribuídas à diferenças cromossômicas entre os dois tipos de pequi. Ambas as plantas têm o mesmo número cromossômico, mesma marcação de NOR. Não foi verificado bandeamento C devido ao tamanho dos cromossomos. Diferenças de coloração com DAPI e CMA3 foram detectadas, porém não é possível afirmar que estejam relacionadas com a presença ou ausência de espinhos no fruto. CAPÍTULO IV: O pequizeiro (Caryocar brasiliense Cambess.) é uma espécie nativa do cerrado brasilieiro e que possui importância alimentar e social para a população que dela depende. É uma espécie que sofre ação extrativista e que, atualmente, é considerada fonte para a produção de biodiesel. Nesse trabalho o objetivo foi identificar, a partir de uma bilbioteca de cDNA, seqüências que estejam envolvidas em vias metabólicas para produção do fruto do pequi, para conhecimento da espécie, suas propriedades biológicas e genéticas. Dessa forma foi construída biblioteca de cDNA e os clones foram clonados e seqüenciados. As sequências identificadas foram depositadas no GenBank. Algumas das sequências encontradas estão relacionadas à proteção contra o estresse oxidativo em plantas, outras são fatores de transcrição e algumas sequências expressas têm funções estruturais e de resistência a patógenos. / Doutor em Genética e Bioquímica