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A amebíase é uma infecção causada pela Entamoeba histolytica. Entretanto, a diferenciação entre a E. histolytica e a Entamoeba dispar, espécies morfologicamente semelhantes, é fundamental para a conduta terapêutica, prevenção da doença invasiva e para a saúde pública. O propósito desde trabalho foi avaliar a Multiplex-PCR na detecção e diferenciação entre a E. histolytica e a E. dispar. Foi feita uma comparação entre os métodos de exame microscópico das fezes usando o conjunto diagnóstico Coprotest, a detecção de antígenos nas fezes usando um ensaio imunoenzimático comercial e o Multiplex-PCR padronizado no laboratório, para o diagnóstico da amebíase. A Multiplex-PCR foi padronizada usando amostras com parasitos obtidos de culturas padrões. Posteriormente, 127 amostras de fezes provenientes de duas comunidades do Estado do Rio de Janeiro, foram testadas e os resultados comparados. A análise de 127 amostras de fezes pelo exame parasitológico de fezes demonstrou que 27 (21%) amostras foram positivas para o complexo E. histolytica/E. dispar. Dessas amostras, 12 foram positivas pelo Multiplex-PCR, sendo que nove apresentaram o padrão de E. dispar (96 bp) e três o padrão E. histolytica (132 bp). Entre as amostras negativas detectadas pelo exame microscópico, três foram positivas para E. dispar e uma foi positiva para E. histolytica pelo Multiplex-PCR. Esse fato mostrou que a PCR foi mais eficiente do que o exame parasitológico realizado com uma amostra de fezes. Os resultados obtidos pelo ensaio de detecção de antígeno de E. histolytica foram concordantes com o do Multiplex-PCR. A análise estatística comparando os resultados do ELISA coproantígeno com o Multiplex-PCR, não pode ser feita devido ao baixo número de casos de E. histolytica. Os resultados acima indicam a necessidade do uso de métodos mais sensíveis para o diagnóstico da amebíase e a importância de se usar técnicas específicas na diferenciação entre E. histolytica e E. dispar. / Amebiasis is defined as an infection caused by Entamoeba histolytica. However, precise differentiation between E. histolytica and Entamoeba dispar, which are morphologically identical species, is essential for treatment decision, prevention of the invasive disease and public health. The purpose of the present study was to evaluate a Multiplex-PCR for detection and differentiation of E. histolytica from E. dispar. Microscopic examination of stools using the coprotest kit, detection of stool antigen using a commercial enzyme-linked immunosorbent assay kit and a home made Multiplex-PCR, were compared for the diagnosis of amebiasis infection. The Multiplex-PCR was standardized using stool samples with parasites obtained from standard cultures. Afterwards, 127 stool samples obtained from individuals living in two villages of the state of Rio de Janeiro were tested and the results were compared. Analysis of the 127 stools samples by microscopy examination demonstrated that only 27 (21%) samples were positive for E. histolytica /E. dispar complex. Among these stool samples, 12 were positive by Multiplex-PCR, with nine presenting the diagnostic fragment characteristic of E. dispar (96 bp) and three presenting diagnostic fragment of E. histolytica (132 bp). Among the negative samples detected by microscopic examination, three were positive for E. dispar and one was positive for E. histolytica by Multiplex-PCR. This denotes that Multiplex-PCR was more efficient than microscopic examination when single stool samples were analyzed. The results obtained by detection of E. histolytica antigen were in agreement with those obtained by the multiplex-PCR. Statistical analyses comparing coproantigen ELISA with Multiplex-PCR results were not done because of the low number of E. histolytica cases. The overall results indicate the need to use more sensitive methods for the diagnosis of amebiasis infection and the importance of using specific techniques for the differentiation between E. histolytica and E. dispar.

Submitted by Marlene Santos (marlene.bc.ufg@gmail.com) on 2018-08-01T17:33:51Z No. of bitstreams: 2 Dissertação - Maythsulene Inácio de Sousa Oliveira - 2015.pdf: 3135445 bytes, checksum: a08e11c7f2df635f3ff7726cfde58f49 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2018-08-02T11:09:15Z (GMT) No. of bitstreams: 2 Dissertação - Maythsulene Inácio de Sousa Oliveira - 2015.pdf: 3135445 bytes, checksum: a08e11c7f2df635f3ff7726cfde58f49 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-08-02T11:09:15Z (GMT). No. of bitstreams: 2 Dissertação - Maythsulene Inácio de Sousa Oliveira - 2015.pdf: 3135445 bytes, checksum: a08e11c7f2df635f3ff7726cfde58f49 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2015-04-11 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Common bean (Phaseolus vulgaris L.) is grown in Brazil in three different cropping seasons, and in diverse agroecosystems. In such different environments, the crop is exposed to several constraints responsible for yield losses, such as pathogenic organisms. Among common bean relevant diseases, fusarium wilt (Fusarium oxysporum f. sp. phaseoli), dry root-rot (Fusarium solani) and Curtobacterium wilt (Curtobacterium flaccumfaciens pv. flaccumfaciens) have similar symptoms, hindering diagnosis in the field, and whose identification in seed health testing is also limited. In both cases, identification at species level is an important step to manage this root pathogen complex, whose detection can be improved by molecular biology tools. Therefore, this study aimed to: 1) to develop and validate a multiplex PCR (m-PCR) method for simultaneous identification of three common bean pathogens, F. oxysporum f. sp. phaseoli, F. solani and C. flaccumfaciens pv. flaccumfaciens; and 2) develop an isothermal amplification of DNA (LAMP) method to detect of F. oxysporum f. sp. phaseoli on seeds. M-PCR method was developed for identification of isolated colonies, as well as infected seeds. In seeds, total DNA was obtained by alkaline lysis method, which inactivates nucleases during the extraction process. M-PCR allowed the identification of all pathogens, with detection of C. flaccumfaciens pv. flaccumfaciens, F. oxysporum f. sp. phaseoli and F. solani amplicons in agarose gel with respectively 306, 609 and 143 base pairs. Furthermore, m-PCR also reduced costs and time to detect Fusarium oxysporum f. sp. phaseoli from 10 days to three hours. It was not possible to develop an optimized protocol for detection of F. oxysporum f. sp. phaseoli by the LAMP method, using only the tf1 gene for design of primers, since such primers were functional only for amplifying large amounts of target DNA. Based on the negative results with LAMP, it is suggested that further studies should be performed using other DNA sequences available in GenBank database. / O feijoeiro-comum (Phaseolus vulgaris L.) é cultivado durante todo o ano no território brasileiro, em três épocas distintas e em vários agroecosistemas. Nestes ambientes distintos, a cultura está exposta a diversos fatores que causam perdas de rendimento, como o ataque de patógenos. Dentre as doenças do feijoeiro-comum encontram-se a murcha-de-fusarium (Fusarium oxysporum f. sp. phaseoli), a podridão-radicular-seca (Fusarium solani) e a murcha-de-curtobacterium (Curtobacterium flaccumfaciens pv. flaccumfaciens) que apresentam sintomas semelhantes, dificultando seu diagnóstico no campo, e cuja identificação em testes de sanidade de sementes também é limitada. Em ambos os casos, a identificação em nível de espécie é uma importante etapa do manejo deste complexo de patógenos, cuja detecção pode ser aperfeiçoada com a adoção de ferramentas de biologia molecular. Portanto, este estudo teve como objetivos: 1) Desenvolver e validar um método de multiplex PCR (m-PCR) para identificação simultânea de três espécies de patógenos do feijoeiro-comum, F. oxysporum f. sp. phaseoli, F. solani e C. flaccumfaciens pv. flaccumfaciens; e 2) desenvolver a técnica de amplificação isotérmica de DNA (LAMP) para detecção de F. oxysporum f. sp. phaseoli em sementes. O método de m-PCR foi desenvolvido para identificação de colônias isoladas bem como sementes infectadas. Nas sementes, o DNA total foi obtido pela lise alcalina, método que inativa nucleases durante o processo de extração. A m-PCR possibilitou a identificação de todos os patógenos, com detecção de C. flaccumfaciens pv. flaccumfaciens, F. oxysporum f. sp. phaseoli e F. solani em bandas formadas em gel de agarose respectivamente com 306, 609 e 143 pares de base. Além disso, a extração do DNA total das sementes pela lise alcalina em combinação com a m-PCR também possibilitou redução de custos e tempo de realização do diagnóstico de Fusarium oxysporum f. sp. phaseoli, de 10 dias para três horas. Não foi possível estabelecer um protocolo otimizado para detecção de F. oxysporum f. sp. phaseoli pelo método LAMP, utilizando somente o gene tf1 para desenho dos iniciadores, uma vez que, os iniciadores revelaram-se funcionais apenas para a amplificação com grandes quantidades de DNA alvo. Diante dos resultados obtidos com LAMP, sugere-se que estudos posteriores sejam realizados empregando outras sequências de DNA disponíveis no banco de dados GenBank.

Submitted by Sandra Pereira (srpereira@ufrrj.br) on 2016-08-03T15:45:32Z No. of bitstreams: 1 2010 - Raquel Silva Lisboa.pdf: 4230911 bytes, checksum: e7087ccd29d417f592ca09ec5f49c657 (MD5) / Made available in DSpace on 2016-08-03T15:45:32Z (GMT). No. of bitstreams: 1 2010 - Raquel Silva Lisboa.pdf: 4230911 bytes, checksum: e7087ccd29d417f592ca09ec5f49c657 (MD5) Previous issue date: 2010-04-14 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico, CNPq. / Dogs can be infected with various hemoparasites, and the occurrence of co-infections between Ehrlichia canis, Babesia canis, Anaplasma platys, and Hepatozoon canis species is very common, since they have the same tick vector. The objectives of this study were to delineate a multiplex PCR technique for the simultaneous diagnostic of microorganisms of Babesia and Ehrlichia genera in canine blood samples, and to realize the partial characterization of fragments of the 16S rRNA gene of the family Anaplasmataceae agents and, of 18S rRNA gene of Babesia detected in some samples PCR-positive, comparing the sequences obtained with sequences of other strains previously deposited in GenBank. Total DNA of 119 blood samples was extracted, of these, 40 were selected by showing cytoplasmatic inclusions in leukocytes and/or platelets suggesting infection by agents of Anaplasmataceae family (1E to 40E), 37 by showing piroplasms (1B to 37B), and two by presenting structures of both agents (M1 and M2), and finally, 40 samples with negative parasitological diagnostic and hematological exam without alterations. All these samples were tested by PCR to confirm the absence or presence of these hemoparasites, and them utilized in the multiplex PCR delineation. In multiplex PCR reactions the primers A17/EC3 were used to amplify an approximately 600bp region of the 16S rRNA gene of Ehrlichia species and the primers PIRO-A1/PIRO-B were used to amplify an approximately 450bp region of the 18S rRNA gene of Babesia species. Validation of multiplex PCR was performed by real time multiplex PCR. The multiplex PCR was able to simultaneously detect both agents in a DNA sample of a dog naturally co-infected and all the single infections by Babesia, but does not detected all the Ehrlichia infections. The real-time multiplex PCR was more sensitive in detect both single and also co-infections, as well as positive DNA mixtures for the two agents. The sequencing results confirmed the isolates identity, and that the primers PIRO-A1/PIRO-B also amplified the DNA of Hepatozoon canis. Phylogenetic analysis indicated that E. canis, A. platys, B. canis and H. canis species found in this study showed close similarities with sequences previously deposited in GenBank, forming monophyletic groups. / Os c?es podem se infectar com diversos hemoparasitos, sendo muito comum a ocorr?ncia de coinfec??es entre as esp?cies Ehrlichia canis, Babesia canis, Anaplasma platys e Hepatozoon canis, visto que possuem o mesmo carrapato vetor. Este estudo teve como objetivos delinear uma t?cnica de PCR multiplex para diagnosticar simultaneamente microrganismos dos g?neros Ehrlichia e Babesia em amostras de sangue de c?es e realizar a caracteriza??o parcial de fragmentos do gene 16S rRNA de agentes da fam?lia Anaplasmataceae e do gene 18S rRNA de Babesia detectados em algumas amostras positivas pela PCR, comparando as sequ?ncias obtidas com as sequ?ncias de outras cepas depositadas previamente no GenBank. O DNA total de 119 amostras de sangue foi extra?do. Destas, 40 foram selecionadas por apresentar inclus?es citoplasm?ticas em leuc?citos e/ou plaquetas sugestivas de infec??o por agentes da fam?lia Anaplasmataceae (1E a 40E), 37 por apresentar formas parasit?rias de piroplasm?deos (1B a 37B), duas por apresentar estruturas de ambos os agentes (M1 e M2) e, finalmente, 40 amostras com diagn?stico parasitol?gico negativo e exame hematol?gico sem altera??es. Todas estas amostras foram testadas por PCR, para a confirma??o da aus?ncia ou presen?a destes hemoparasitos, e depois utilizadas no delineamento da PCR multiplex. Nas rea??es de PCR multiplex utilizou-se os oligonucleot?deos iniciadores A17/EC3 que amplificam um produto de aproximadamente 600pb de uma por??o do gene 16S rRNA de esp?cies de Ehrlichia e os oligonucleot?deos iniciadores PIRO-A1/PIRO-B que amplificam um produto de aproximadamente 450pb de uma por??o do gene 18Sr RNA de esp?cies de Babesia. A valida??o da PCR multiplex foi realizada por PCR multiplex em tempo-real. A PCR multiplex foi capaz de detectar simultaneamente os dois agentes em uma amostra de DNA de um c?o naturalmente coinfectado e todas as infec??es individuais por Babesia, mas n?o detectou todas as infec??es por Ehrlichia. A PCR multiplex em tempo real foi mais sens?vel em detectar tanto infec??es ?nicas quanto coinfec??es, al?m de misturas de DNA positivo para os dois agentes. Os resultados dos sequenciamentos confirmaram a identidade dos isolados, e que os oligonucleot?deos PIRO-A1/PIRO-B amplificaram tamb?m, o DNA de Hepatozoon canis. As an?lises filogen?ticas indicaram que as esp?cies de E. canis, A. platys, B. canis e H. canis encontradas neste estudo possuem similaridades pr?ximas com sequ?ncias previamente depositadas no GenBank, formando grupos monofil?ticos.

Magister Scientiae - MSc / Initially, a pilot study was carried out in order to reconstruct the major paternal and maternal lineages of the Muslim population living in the Cape metropolitan area. The Study has shown the ability of molecular genetic tools to give insight into the origins and history of local communities. The study was also used as a point of reference for the Strand Muslim Community project. Genetic variations of the Y-chromosome and mitochondrial DNA for the pilot study were analyzed using the RFLP technique. The SNaPshot mini-sequencing technique was used to genotype single nucleotide polymorphisms (SNP) on the Y-chromosome and mitochondrial DNA in 115 males from the Strand Muslim community. / South Africa

Mycobacteriosis (fish tuberculosis) is a progressive disease of a wide range of wild and captive marine and freshwater fish species. While Mycobacterium marinum, M. fortuitum and M. chelonae are the most frequently reported species to be involved in the disease, several new mycobacteria species have also recently been implicated. Conventional detection / identification of fish mycobacteria is based on histopathology, culture and biochemical characteristics. In this study complementary molecular approaches were developed to assist in Mycobacterium identification. First, a highly specific and sensitive multiplex PCR-based assay, targeting two genes (hsp65 and 16S RNA), was established to simultaneously detect the genus Mycobacterium and identify M. marinum, M. fortuitum or M. chelonae from culture or infected fish tissue, based on presence / absence of specific amplicons. In addition, PCR-restriction enzyme analysis (PRA) and DNA sequence analysis of the 16S-23S internal transcribed spacer (ITS) region and a 441 bp fragment of the hsp65 gene demonstrated the limitations of multiplex PCR (and commercial line probe assays) to differentiate among the species of the M. fortuitum complex. However DNA sequence analysis of the hsp65 gene fragment was found to reliably identify M. fortuitum from closely related species, M. conceptionense and M. senegalense. Reliable identification of novel species (or very similar species) of aquatic mycobacteria requires more extensive DNA sequence comparisons. Thus, multigene (polygenetic) analyses, as used here, provide rapid, accurate and reliable species identification of aquatic mycobacteria. Furthermore, a number of novel species of aquatic mycobacteria, M. stomatepiae, ‘M. angelicum’, ‘M. aemonae’ and M. salmoniphilum were discovered using the polygenetic analysis approach. Correct identification of Mycobacterium species by DNA sequence comparisons relies on accurate database information. Difficulties in this study in assigning M. marine and M. gordonae to their correct taxa suggest errors in the current public sequence repositories. The above methods were successfully applied to detect and identify mycobacteria in field samples including formalin-fixed, paraffin-embedded (FFPE) fish tissue, water and frozen fish tissue.

Respiratorische Krankheitsbilder, an denen mehr als ein Pathogen ursächlich beteiligt ist, gewinnen in der Schweinepopulation zunehmend an Bedeutung. Diagnostische Methoden zum simultanen Nachweis mehrerer Erreger sind Bestandteil einer schnellen und effizienten Therapie und tragen zum ökonomischen Bestandsmanagement bei. Im Rahmen dieser Arbeit sollten Methoden für den Multiplex-Nachweis von Antikörpern und Nukleinsäuren viraler Erreger von respiratorischen Krankheitsgeschehen des Schweins entwickelt werden. Die Methode für den Multiplex-Nachweis von Antikörpern sollte auf Basis der xMAP® Flüssigchip-Technologie (Luminex Corporation, Austin, T, USA) an der LiquiChip®- Workstation (Qiagen, Hilden, D) etabliert werden. Da es sich um eine für den Antikörpernachweis im veterinärmedizinischen Bereich bislang nicht genutzte Methode handelte, erfolgte die Prüfung der Machbarkeit zunächst im Einfach-Format am Beispiel des Porzinen Circovirus Typ 2. Im Laufe der Arbeit wurde deutlich, daß die Kopplung des PCV2 ORF2-Proteins als Capture-Molekül sowie die Erstellung der Versuchsansätze mit akzeptablem Aufwand ohne Spezialtechniken durchführbar war. Aufgrund der Anordnung der Proben auf Platten im 96-well-Format und der vollautomatischen Messung war ein hoher Probendurchsatz möglich. Nach der Einführung von Waschschritten in die Versuchsansätze konnten hohe Fluoreszenzsignale erzeugt werden. Im Laufe der Optimierungsversuche wurde allerdings die fehlende Korrelation dieser Fluoreszenzsignale mit den Ergebnissen der Referenzmethode deutlich. Aufgrund der unbekannten Testeigenschaften sowie fehlender Kontrollmöglichkeiten wurden diese nicht sogleich als unspezifische bzw. falsch positive Signale erkannt. Erst durch die Testung von positiven und negativen Feldseren an verschiedensten Bead-Arten wurde ersichtlich, daß die Fluoreszenzen nicht ausschließlich durch die spezifische Bindung der PCV2-Antikörper an das Capture-Protein entstanden. Im Ausschlussverfahren konnte die Ursache eingegrenzt werden. Bestandteile aus dem Schweineserum führten vermutlich durch unspezifische Bindungen an die LiquiChip®-Beads zu einem Fluoreszenzereignis. Durch Vorinkubation der Beads in Pferdeserum und der Feldseren mit einem Block-Puffer wurde versucht, diese Serumbestandteile abzusättigen und so eine Bindung an die Beads zu verhindern. Die Inkubationsvarianten führten weder zu einer Minimierung der unspezifischen Bindung noch zu einer verbesserten Differenzierung PCV2-positiver und negativer Seren. Die in der vorliegenden Arbeit verwendeten Bead-Arten sind für den Nachweis von Antikörpern gegen das PCV2 ORF2-Protein nicht geeignet. Alternative Bead-Arten für einen vergleichbaren Versuchsansatz stehen derzeit nicht zur Verfügung. Ein weiteres Ziel der Arbeit bestand darin, eine bereits in der Diagnostik von Schweineviren etablierte Methode, die PCR, zu einer Multiplex-PCR zu erweitern. Als zu detektierende Parameter wurden die derzeit bedeutendsten viralen Erreger von respiratorischen Erkrankungen des Schweins, das PRRS-Virus (Typ 1 und Typ 2), das Porzine Influenzavirus mit den Subtypen H1N1, H3N2 und H1N2 und PCV2 gewählt. Es wurden die Primersequenzen von bereits etablierten Einfach-PCRs an die besonderen Ansprüche einer Multiplex-PCR angepasst und die Methode zunächst im Einzelansatz auf Funktionsfähigkeit überprüft. Im Anschluss wurden die Parameter zu einer Multiplex-PCR zusammengeführt, die Methode optimiert und auf Spezifität, Sensitivität und Verhalten in der Routinediagnostik überprüft. Aufgrund der im Gegensatz zur Einfach-PCR zum Teil herabgesetzten Sensitivität ist diese Methode für Ausschlussuntersuchungen weniger geeignet. Für die Untersuchung von Probenmaterial klinisch erkrankter Tiere ist sie jedoch gut geeignet und bietet die Möglichkeit einen schnellen Überblick über das Erregerspektrum zu erhalten. Es muss jedoch berücksichtigt werden, daß bestimmte Parameter, z.B. PCV2, die Sensitivität des Nachweises der anderen Parameter sehr deutlich herabsetzen kann. Dies ist insbesondere von Bedeutung, da PCV2-DNA in Probenmaterial von klinisch erkrankten Tieren in sehr hohen Mengen vorhanden ist und dadurch die weiteren Parameter noch zusätzlich beeinflusst werden können.

La paléoparasitologie est l’étude des restes de parasites préservés dans des échantillons archéologiques et permet de mieux comprendre l’état de santé des populations anciennes et d’obtenir des informations d’ordre anthropologique ou ethnologique, sur les régimes alimentaires ou les conditions d’hygiène au quotidien. Les restes de parasites peuvent être retrouvés sous forme de macro-restes (vers ou larves), d’antigènes, d’ADN ou d’œufs. Cesderniers peuvent être particulièrement bien préservés au cours du temps car ils sont composés en partie de chitine, les rendant résistants aux processus de dégradation. L’observation microscopique de leurs caractéristiques morphologiques et micrométriques permet d’identifier les taxons au niveau du genre ou de la famille. Dans le cadre de cette thèse, plusieurs helminthes gastro-intestinaux, dont les œufs sont fréquemment retrouvés dans des échantillons archéologiques, ont été ciblés par une approche génétique. Il s’agit des vers plats Tæniasaginata, T. solium, T. asiatica, Echinococcus granulosus, E. multilocularis, Diphyllobothriumlatum, D. dendriticum et D. nihonkaiense, des nématodes Trichuris trichiura, Enterobiusvermicularis et Ascaris sp. et des douves Fasciola hepatica, F. gigantica, Dicrocoeliumdendriticum et D. chinensis.La méthode « aMPlex Torrent » permet de détecter, dans un grand nombre d’échantillons archéologiques, une faible quantité d’ADN de parasites. Cette approche combine la spécificité et la sensibilité de la PCR au haut-débit du séquençage de nouvelle génération. Plusieurs vestiges, provenant de périodes et de régions géographiques diverses, ont été analysés. Des résultats génétiques ont été obtenus pour des échantillons aussi anciens que 7200 BP. Nous avons par ailleurs obtenus les premières séquences anciennes de Taenia sp., Diphyllobothriumsp., Echinococcus sp., et les premières séquences européennes d’Enterobius vermicularis. Auvu de ces résultats, notre approche apparait comme étant complémentaire à la microscopie. / Palaeoparasitogy, the study of parasite remains from archaeological samples, is adiscipline that can highlight questions about the health status of the ancient populations. It can give important anthropological or ethnological information such as the diet and the hygiene conditions of past societies. The remains can be preserved as macroremains (worms or larvae),antigens, DNA or eggs. Because they are partially made of chitin, eggs of gastrointestinalhelminths resist well over time to the taphonomic degradation process. It is possible to distinguish between different families or genera of parasites by looking at the morphological features of eggs. However, since several taxa share common features, the determination is rarelypossible at the species level. For this thesis, several parasite species for which eggs arecommonly observed in archeological samples have been studied by a genetic approach. Westudied the tapeworms Tænia saginata, T. solium, T. asiatica, Echinococcus granulosus, E.multilocularis, Diphyllobothrium latum, D. dendriticum, and D. nihonkaiense; the nematodesTrichuris trichiura, Enterobius vermicularis, and Ascaris sp.; and the flukes Fasciola hepatica,F. gigantica, Dicrocoelium dendriticum, and D. chinensis.The “aMPlex Torrent” approach has been set up to detect minute amounts of DNA from parasites in multiple archaeological samples. This approach combines the specificity andsensitivity of PCR to the throughput of Next-Generation sequencing. Several samples have been analyzed by this approach. We obtained genetic results for samples as old as 7200 BP and from various geographical and archeological contexts. We obtained the first ancient DNA sequences for Taenia sp., Diphyllobothrium sp., Echinococcus sp. and the first European sequences forEnterobius vermicularis. Genetic analyses and microscopic observations appear to be complementary. Indeed, at least one taxon per sample was detected by one of the two approaches.

Introdução: O gênero Acinetobacter sp apresenta considerável heterogeneidade possuindo inúmeras espécies. Atualmente, 23 espécies já foram nomeadas e nove outras espécies já foram descritas. Quatro destas espécies possuem contextos clínicos e epidemiológicos diferentes, no entanto são agrupadas em um complexo denominado Acinetobacter calcoaceticus-Acinetobacter baumannii (ABC) devido à sua similaridade genética e fenotípica. Diversos testes para diferenciação do complexo já foram descritos, porém a maioria não pode ser realizado na rotina laboratorial, pois são caros e laboriosos. Objetivos: Avaliar um método rápido e viável na rotina laboratorial, capaz de diferenciar as espécies do complexo ABC; Determinar a prevalência das diferentes espécies do complexo ABC; Avaliar o perfil de suscetibilidade aos antimicrobianos nas diferentes espécies. Métodos: Foram analisadas 118 amostras de dois hospitais de Porto Alegre-RS através do método Multiplex PCR para o gene gyrB e posteriormente confirmadas pelo padrão-ouro: sequenciamento do 16S-23S ITS. O perfil de suscetibilidade foi realizado através de microdiluição em caldo. Resultados: Das 118 amostras identificadas inicialmente como Acinetobacter sp., a grande maioria dos isolados (106 -89.9%) foram identificados como A. baumannii; mas doze isolados foram identificados como sendo das demais espécies do complexo ABC: 6 (5.1%) A. nosocomialis, 5 (4.2%) A. pittii, e 1 (0.8%) A. genoespécie 10, através da técnica de Multiplex PCR. Todos os resultados foram confirmados por sequenciamento. A. baumannii apresentou um elevado nível (72,6%) de resistência ao imipenem em comparação com as outras espécies seguido da espécie A. nosocomialis que apresentou metade de seus isolados resistentes. Todas as espécies apresentaram baixos índices (inferior a 7,5%) de resistência à Polimixina B e Tigeciclina. Conclusão: O Multiplex PCR para o gene gyrB apresentou resultados fidedignos quando comparados ao padrão-ouro, demonstrando, assim, ser um método confiável para a identificação das espécies do complexo ABC. Outras espécies, além de A. baumannii, ABC podem apresentar percentuais significativos de resistência ao imipenem. / Background: Introduction: The genus Acinetobacter sp presents considerable heterogeneity possessing numerous species. Currently, 23 species have been named and nine other species have been described. Four of these species have different clinical and epidemiological contexts, but are grouped in a complex called Acinetobacter calcoaceticus-Acinetobacter baumannii (ABC) due to their genetic and phenotypic similarity. Several tests for differentiation of the complex have been described, but most can not be performed routinely in the laboratory, they are expensive and laborious. Objectives: To evaluate a fast and feasible in routine laboratory able to differentiate the species of the complex ABC; determine the prevalence of different species of the complex ABC; evaluate the antimicrobial susceptibility profile of the different species. Methods: We analyzed 118 samples from two hospitals in Porto Alegre-RS by the method of Multiplex PCR for gene gyrB and subsequently confirmed by the gold standard: sequencing the 16S-23S ITS. The susceptibility profile was performed by microdilution. Results: Of the 118 samples initially identified as Acinetobacter sp. The great majority of isolates (106 -89.9%) were identified as A. baumannii, but twelve isolates were identified as being from other species of the complex ABC: 6 (5.1%) A. nosocomialis, 5 (4.2%) A. pittii, and 1 (0.8%) A. genospécie 10 by Multiplex PCR technique. All results were confirmed by sequencing. A. baumannii showed a high level (72.6%) of imipenem resistance in comparison with the other species followed by the species A. nosocomialis showed that half of his resistant isolates. All species showed low levels (less than 7.5%) of resistance to Polymyxin B and Tigecycline. Conclusion: Multiplex PCR for gene gyrB results presented totally reliable when compared to the gold standard, demonstrating thus be a safe method for the laboratory. Other species besides A. baumannii, ABC may have significant percentages of resistance to imipenem.

<p>Sexual assault is a significant problem facing the South African society. In this context, efficient but also affordable genotyping systems are needed for positive identification of criminals in incidences of sexual violence. The aim of this study was therefore to develop non-commercial Y-STR genotyping systems suitable for sexual assault cases in South Africa. Y-chromosome STR loci constituting the minimal haplotype are still the most widely used loci in investigating sexual assault cases despite the fact that DYS391 and DYS392 have shown low levels of polymorphism in Xhosa populations in Cape Town. The minimal haplotype was, therefore, further investigated in the Cape Muslim population. The Cape Muslim population generally exhibited high GD values among all the South African populations. These values were higher than 0.5 for most loci, and ranged from 0.447 for DYS391 to 0.957 for DYS385. The highest number of alleles in most loci was also recorded in this population. The overall assessment of the minimal haplotype has shown that this system is still a useful in investigating sexual assault case in many South African subpopulations. Therefore the exercise of internal validation of the minimal haplotype system was successfully carried out in the laboratory. iii The properties of additional novel and widely used STRs were also investigated in this study. Loci were successfully sequenced and allele nomenclature was assigned to them according to the ISFG guidelines.</p>