The medical laboratory science (MLS) profession is in need for published molecular diagnostics competency-based standards and curriculum. To assess their expectations of new MLS graduates, professionals performing and supervising performance of clinical molecular assays were surveyed to rate the importance of relevant cognitive and psychomotor learning objectives. A modified, asynchronous, iterative online Delphi process was utilized for assessment of consensus on the importance of the objectives. The survey was delivered through online REDCap application. Program directors of 221 MLS programs accredited by the National Accrediting Agency for Clinical Laboratory Science (NAACLS) were asked to forward the first Delphi survey to target participants at their affiliated clinical sites. Ninety-four experts submitted complete surveys, including 88 who provided email addresses, indicating agreement to participate in future Delphi rounds. Most of the participants were certified by ASCP or NCA (81.9%), had over 10 years of laboratory experience (76.6%), and worked in a hospital setting (43.6%). The reliability of the surveys, assessed using Cronbach’s alpha, was 0.96 and 0.97. In the second survey, the objectives assigned low importance by the majority were removed; and others, assigned high importance were expanded. Respondents were given the opportunity to confirm or change their opinion on the objectives after reviewing quantitative results and narrative comments collected in the preceding survey. Upon completion of the Delphi process, 25 essential items were identified as necessary for inclusion in the entry-level MLS curriculum. These concepts and objectives focused on basic molecular biology principles and general molecular laboratory operations, including practical knowledge of techniques designed to maintain specimen integrity and intense theoretical background of the polymerase chain reaction, as well as comprehension of the principles of laboratory assays designed for pathogens most commonly tested for using molecular methods. In this study, the investigator also provided information on the preferred number of contact hours devoted to each group of the identified essential items. The goal of creating the list of essential concepts and objectives was to share it with MLS educators, the NAACLS and the provider of MLS certification exam, the American Society for Clinical Pathology Board of Certification (ASCP-BOC), to contribute to the existing exam content guidelines.

Made available in DSpace on 2016-08-10T10:38:39Z (GMT). No. of bitstreams: 1 ANDREIA PIRES AMANCIO.pdf: 1468482 bytes, checksum: cfd035e6b0a287fb6f6c412088f7aa20 (MD5) Previous issue date: 2013-01-31 / The Intellectual Disability (DM) is defined as an incomplete level of intellectual development, and its manifestation usually occurs before 18 years of age. It is one of the most common neuropsychiatric disorders, with a prevalence of 5% in our population. The Fragile X syndrome (FXS) is the most common cause of inherited mental retardation worldwide, and the second most common genetic cause of mental retardation. The phenotype of FXS is associated with mutations in FMR1 (Fragile X Mental Retardation-linked type 1), which is caused by the expansion of repetition of a trinucleotide sequence (CGG) in the regulatory region of the FMR1 gene, located on chromosome X (Xq27 .3). The importance of clinical recognition and specific diagnosis of FXS comes from the fact that theoretically all cases are hereditary and familial. In this context, this study aimed to validate the molecular genetic diagnosis simplified and cost, for patients suspected of FXS in the Laboratory of Cytogenetics and Molecular Genetics (LaGene) of the Department of Health of the State of Goiás in Goiânia using the PCR technique. We selected 35 patients referred by medical service of public health to LaGene with clinical indication for diagnosis of FXS. The patients' DNA was extracted and subjected to two PCR methods, here called PCR Screening (PCR-T) and PCR for Pre-mutation (PCR-P). The new methodology, PCRT, 88% produced conclusive results against 100% conclusiveness of the standard technique (PCR-P) and getting a p> 0.11. The alleles amplified by PCR-P allowed the diagnosis of pre-mutation in a sample. From these observations we propose a strategy for the diagnosis of the syndrome using PCR-P research in pre-mutation individuals in parents with inconclusive results. Given the successful results and improved the PCR, including the new and easy diagnosis of pre-mutation samples not completed by the technique of drawing, we suggest the implementation of both PCR genetics laboratory in the State of Goiás (LaGene ) for the diagnosis of FXS. / A Deficiência Mental (DM) é definida como sendo um nível incompleto do desenvolvimento intelectual, e sua manifestação ocorre normalmente antes dos 18 anos de idade. É um dos transtornos neuropsiquiátricos mais comuns, com uma prevalência de 5% na população brasileira. A síndrome do X-Frágil (SXF) é a causa mais comum de retardo mental hereditário em todo o mundo, e a segunda causa genética mais frequente de deficiência mental. O fenótipo da SXF está associado a mutações no gene FMR1 (Fragile X-linked Mental Retardation type 1), que é causada pela expansão da repetição de uma sequência de trinucleotídeos (CGG) na região reguladora do gene FMR1, localizado no cromossomo X (Xq27.3). A importância do reconhecimento clínico e diagnóstico específico da SXF vem do fato de que teoricamente todos os casos são hereditários e familiais. Nesse contexto, este estudo teve como objetivo validar o diagnóstico genético-molecular simplificado e de baixo custo, para pacientes com suspeita da SXF no Laboratório de Citogenética e Genética Molecular (LaGene) da Secretaria Estadual de Saúde do Estado de Goiás, na cidade de Goiânia, utilizando a técnica de PCR. Foram selecionados 35 pacientes encaminhados pelo serviço médico da rede pública de saúde ao LaGene com indicação clínica de diagnóstico da SXF. O DNA desses pacientes foi extraído e submetido a dois métodos de PCR, aqui denominados PCR de Triagem (PCR-T) e PCR para Pré-mutação (PCR-P). A nova metodologia, PCRT, produziu 88% de resultados conclusivos contra 100% de conclusividade pela técnica padrão (PCR-P) e obtendo um p>0,11. Os alelos amplificados pela PCR-P possibilitou o diagnóstico de pré-mutação em uma amostra. A partir destas observações propõe-se uma estratégia para o diagnóstico da síndrome utilizando a PCR-P na pesquisa de pré-mutação em pais de indivíduos com resultados inconclusivos. Considerando os resultados bem sucedidos e aprimorados da técnica de PCR, incluindo o novo e fácill diagnóstico da pré-mutação de amostras não concluídas pela técnica de triagem, sugere-se a implementação de ambas as PCR no laboratório de genética do Estado de Goiás (LaGene) para o diagnóstico da SXF.

The aim of this study is to improve our understanding of the ecology, spatiotemporal patterns, and risk of infectious pathogens of migratory waterfowl, using the blue-winged teal (Anas discors, BWTE), as a model. From 2007-2010, 1,869 BWTE were sampled in the prairie provinces (Alberta, Saskatchewan and Manitoba, Canada) to examine infection status and/or evidence of previous exposure to avian influenza virus (AIV), West Nile virus (WNV), and avian paramyxovirus-1 (APMV-1), in relation to host demographic variables (age, sex, body condition, exposure to other pathogens), other ecological variables such as local waterfowl breeding population density and local pond density, and year. The probability of AIV infection depended on an interaction between age and AIV antibody status. Hatch year birds with antibodies to AIV were more likely to be infected, suggesting an antibody response to an active infection. After hatch year birds with antibodies to AIV were less likely to be infected, suggesting immunity resulting from previous exposure. AIV infection was positively associated with local BWTE density, supporting the hypothesis of density dependent transmission. Exposure to WNV and APMV-1 were also associated with age and year. Furthermore, the probability of WNV exposure was positively associated with local pond density rather than host population density, likely because ponds provide suitable breeding habitat for mosquitoes, the primary vectors for transmission. We also investigated large-scale spatiotemporal trends in apparent prevalence of AIV across Canada and the United States throughout the year, using data from national avian influenza surveillance programs in Canada and the US in 2007-2010. Our analyses revealed that age, sex, year of sampling, flyway, latitude, and season (categorized by stages of the BWTE annual life cycle) were all important variables in predicting probability of AIV infection. There was an interaction between age and season. During late summer staging (August) and fall migration (September-October), hatch year birds were more likely to be infected than after hatch year birds, however there was no difference between age categories for the remainder of the year (winter, spring migration, and breeding season). Probability of infection increased non-linearly with latitude, and was highest in summer, corresponding to the beginning of fall migration when densities of birds and the proportion of susceptible hatch year birds in the population are highest. Birds in the Pacific, Central and Mississippi flyways were significantly more likely to be infected compared to those in the Atlantic flyway. Observed trends in seasonal, annual, and geographic patterns of AIV infection in BWTE across Canada and the US were primarily driven by the dynamics of AIV infection in hatch year birds. Our results demonstrate demographic as well as seasonal, latitudinal and flyway trends across Canada and the US. This research provided further evidence for the role of wild dabbling ducks, particularly BWTE, in the maintenance and ecology of AIV. This improved understanding of the role of BWTE as natural hosts, and the geographic, demographic and temporal variables that affect infection and transmission parameters, moves us closer to deciphering the overall ecology of the virus and its transmission and transportation pathways at the individual, population and continental levels. This knowledge, in turn, will permit development of better tools to predict and perhaps to prevent possible outbreaks in domestic animals as well as in humans.

Yaws is a skin debilitating disease caused by Treponema pallidum subspecies pertenue with most cases reported in children. World Health Organization (WHO) aims at total eradication of this disease through mass treatment of suspected cases followed by an intensive follow-up program. However, effective diagnosis is pivotal in the successful implementation of this control program. Recombinase polymerase amplification (RPA), an isothermal nucleic acid amplification technique offers a wider range of differentiation of pathogens including those isolated from chronic skin ulcers with similar characteristics such as Haemophilus ducreyi (H. ducreyi). We have developed a RPA assay for the simultaneous detection of Treponema pallidum (T. pallidum) and H. ducreyi (TPHD-RPA). The assay demonstrated no cross-reaction with other pathogens and enable detection of T. pallidum and H. ducreyi within 15 min at 42 °C. The RPA assay was validated with 49 clinical samples from individuals confirmed to have yaws by serological tests. Comparing the developed assay with commercial multiplex real-time PCR, the assay demonstrated 94% and 95% sensitivity for T. pallidum and H. ducreyi, respectively and 100% specificity. This simple novel TPHD-RPA assay enables the rapid detection of both T. pallidum and H. ducreyi in yaws-like lesions. This test could support the yaws eradication efforts by ensuring reliable diagnosis, to enable monitoring of program success and planning of follow-up interventions at the community level.

Die Störung von Apoptose-Signalwegen spielt eine zentrale Rolle sowohl bei der Tumorentstehung als auch bei der Entwicklung von Therapieresistenz in malignen Tumoren. Von besonderer Bedeutung für das Ansprechen auf zytotoxische Tumortherapien sind die Komponenten des mitochondrialen Apoptosesignalwegs und dessen übergeordneten Regulatoren. Durch die Analyse solch zentraler Regulatoren der Apoptose, wie den Mitgliedern der Bcl-2 Genfamilie, des p53- und des Rb-Signalwegs, konnten Patienten mit guter bzw. schlechter Prognose identifiziert werden. Hierbei zeigte sich, dass die kombinierte Analyse von einander nachgeschalteten Signalwegkomponenenten, wie z.B. p53 und Bax, der Analyse einzelner Markergene überlegen ist. Solche Signalweganalysen konnten bei akuten und chronischen Leukämien, Kolon-, Ösophagus-, und Mammakarzinomen erfolgreich durchgeführt werden. Neben diesen deskriptiven genetischen Analysen an Tumorproben ermöglichte die funktionelle Manipulation dieser Signalwege die Sensibilisierung von Tumorzellen für Chemo-, Radio- und auch biologische Therapien. Mittels nicht-viraler, retro- und adenoviraler Gentransfervektoren wurden Regulatoren der Apoptose, wie z.B. Apoptose-fördernde Mitglieder der Bcl-2 Genfamilie, das Tumorsuppressorgen p14ARF oder auch Procaspase-3 in Tumorzellen eingebracht, um Resistenzen zu überwinden bzw. um direkt Zelltodsignalwege in den malignen Zellen zu aktivieren. Signalweganalysen sowohl in primärem Tumorgewebe von Patienten als auch in Zellinienmodellen identifizierten die hierfür notwendigen Komponenten der betreffenden Signalwege. Von besonderem Interesse war hierbei, dass durch die genetische Manipulation von Apoptose- und Zellzyklus-Regulation Signaldefekte in resistenten Tumoren umgangen und überwunden wurden. Dies könnte in Zukunft als mögliche Basis für neue, molekulare Therapieansätze in der Tumortherapie dienen. / Disruption of apoptosis signaling pathways plays a key role in both tumorigenesis and the development of resistant phenotypes in malignant tumors. Components of the mitochondrial apoptosis signaling pathway and its upstream regulators are of special importance regarding the response to cytotoxic anticancer therapies. The analysis of such central regulators, e.g. members of the Bcl-2 gene family, or the p53 and Rb pathways, identifies patients with good or poor prognosis. In this context, the combined analysis of consecutively involved signaling components, e.g. p53 and Bax, was shown to be superior as compared with analysis of single marker genes. Such signaling pathway analyses were successfully performed in acute and chronic leukemia, colon, esophageal and breast carcinoma. Apart from these descriptive genetic analyses in tumor specimen, the functional manipulation of these signaling events permitted to sensitize tumor cells for chemotherapy and irradiation as well as biological therapeutic modalities. To overcome resistance or to directly promote cell death signaling in the malignant cells, apoptosis regulators such as apoptosis promoting Bcl-2 homologs, the p14ARF tumor suppressor gene or procaspase-3 were introduced in cancer cells by means of non-viral, retro- and adenoviral gene transfer vectors. Analysis of signaling pathways identified the critical components in the respective model system. Interestingly, the genetic manipulation of cell cycle and apoptosis components was capable to circumvent signaling defects in resistant tumors and to overcome resistance to therapy. Such strategies could therefore serve as basis for novel, molecular therapeutic strategies in future cancer therapy.

Made available in DSpace on 2016-08-16T18:18:41Z (GMT). No. of bitstreams: 1 Tese Andressa Almeida Santana.pdf: 1466363 bytes, checksum: ae0eefc75fb09cd9f96594f4e5656ed5 (MD5) Previous issue date: 2011-07-29 / FUNDAÇÃO DE AMPARO À PESQUISA E AO DESENVOLVIMENTO CIENTIFICO E TECNOLÓGICO DO MARANHÃO / Canine babesiosis and canine visceral leishmaniasis (CVL) are vector borne diseases, where dogs exert a play as reservoir or source for arthropods responsible by the transmission of these protozoosis. Babesia canis vogeli, is transmited by tick Rhipicephalus sanguineus while Leishmania infantum (sin. Leishmania chagasi) is transmited by sand fly (Lutzomyia longipalpis). The results showed that CVL remains endemic in São Luís Municipality. Despite that the coinfection between Leishmania and Babesia was low considering that both diseases are endemic in this tropical area. Beside that was observed that Yorkshire terrier presented higher predisposition to acquire the infection by B. canis vogeli. A remarkable result was the occurrence of ocular lesions associated to L. infantum infection. / A babesiose canina e a leishmaniose visceral canina são doenças transmitidas por vetores, sendo os cães competentes reservatórios e fonte alimentar dos artrópodes envolvidos. Babesia canis vogeli, é um parasita intraeritrocitário transmitido pelo carrapato Rhipicephalus sanguineus. Leishmania infantum (sin. Leishmania chagasi) transmitidos a mamíferos pela picada dos flebotomíneos (Lutzomyia longipalpis), infectando macrófagos do Sistema Fagocítico Mononuclear do hospedeiro. O presente trabalho é divido em capítulos e os resultados encontrados mostraram que a leishmaniose visceral canina ainda é endêmica no município de São Luís, e que apesar disso a taxa de coinfecção com Babesia foi baixa. Também foi observado que a raça Yorkshire terrier, dentre as raças estudadas, apresentou maior predisposição para contrair a infecção por B. canis vogeli. Outro resultado significativo foi a ocorrência de lesões oculares associadas à infecção por L. infantum.

Made available in DSpace on 2016-08-16T18:18:42Z (GMT). No. of bitstreams: 1 Tese PATRICIA RIBEIRO AZEVEDO.pdf: 3836248 bytes, checksum: 69781a9e30881bb327b2148598b3fe8e (MD5) Previous issue date: 2013-06-24 / FUNDAÇÃO DE AMPARO À PESQUISA E AO DESENVOLVIMENTO CIENTIFICO E TECNOLÓGICO DO MARANHÃO / The von Hippel Lindau syndrome (VHL) is an autosomal dominant disorder with an incidence of 1:36.000 to 1:53.000 individuals, characterized by multiple tumors, affecting individuals of twenty and forty years, with life expectancy of 60 years. The objective of this study was to characterize the molecular changes in the VHL gene associated with von Hippel Lindau syndrome in a family of African descent with hemangioblastoma of the central nervous system in the state of Maranhão. For investigation of the family history, all family members were interviewed, and performed molecular analysis of seven patients with clinical diagnosis of VHL and 89 family members at risk. For DNA extraction was used peripheral blood. The technique used was the amplification of multiplex ligation probes dependent binding (MLPA). After PCR- MLPA, was performed sequencing and the software GeneMaker was used for screening of mutations. The research followed the ethical precepts. We investigated the family history of the eight individuals of African descent carriers hemangioblastomas of the central nervous system. In addition to this injury, these individuals had retinal hemangioblastoma, pancreatic cyst and bilateral kidney tumor. The average age of first presentation of the lesion was 29 years, not having occurred predominantly in relation to gender. In the screening protocol was not evidenced pheochromocytoma. No injuries were surveyed in the reproductive system because of the absence of symptoms. The molecular test detected the deletion c.1-? _340 +? in all symptomatic individuals and 14 family members. The MLPA, technique has proven to be fast and reliable for diagnosis of large deletions. A positive family history, the absence of pheochromocytoma and detection of genomic deletion of exon 1, allowed the clinical diagnosis and molecular VHL syndrome and classification as type 1. In this study it was possible to evaluate, in addition to the proband, other family members. This approach decreases the morbidity of the disease and provides a better quality of life for families. / A síndrome de von Hippel Lindau (VHL) é uma doença autossômica dominante, com incidência de 1:36.000 a 1:53.000 indivíduos, caracterizada por múltiplos tumores, acometendo os indivíduos entre vinte e quarenta anos, sendo a expectativa de vida de 60 anos O objetivo deste estudo foi caracterizar a base molecular do gene VHL associado à Síndrome von Hippel Lindau em uma família com hemangioblastoma de sistema nervoso central no Estado do Maranhão. Para investigação da história familiar, foram entrevistados todos os familiares, sendo realizada análise molecular de sete indivíduos com diagnóstico clínico de VHL e 89 familiares em risco. Para extração de DNA foi utilizado sangue periférico. A técnica utilizada foi a amplificação de múltiplas sondas dependente de ligação (MLPA). Após a PCR-MLPA foi realizado sequenciamento e utilizado o programa GeneMaker para a triagem de mutações. A pesquisa obedeceu aos preceitos éticos. Foi investigada a história familiar dos oito indivíduos afrodescendentes portadores de hemangioblastomas de sistema nervoso central. Além dessa lesão, um indivíduo apresentou hemangioblastoma de retina, um apresentou cisto no pâncreas e outro apresentou massa tumoral bilateral no rim. A média de idade da primeira apresentação da lesão foi de 29 anos, não tendo ocorrido predominância em relação ao sexo. No protocolo de rastreamento não foi evidenciado feocromocitoma. Não foram pesquisadas lesões no sistema reprodutor devido ausência de sintomatologia. No teste molecular foi detectada a deleção c.1-?_340+? em sete indivíduos sintomáticos e em 14 familiares. A técnica MLPA demonstrou ser rápida e segura para diagnóstico de grandes deleções. A história familiar positiva, ausência de feocromocitoma e a detecção da deleção genômica do exon 1, permitiu o diagnóstico clínico e molecular da síndrome VHL e a classificação como tipo 1. Neste estudo foi possível avaliar, além do probando, os outros familiares. Essa conduta poderá diminuir a morbimortalidade da doença e proporciona uma melhor qualidade de vida aos familiares.

[ES] La enfermedad de Wilson (EW) es un trastorno hereditario del metabolismo del cobre causado por mutaciones en ATP7B, que codifica una proteína transportadora de cobre en el hígado. Su mal funcionamiento provoca un fallo en la excreción biliar de cobre y una acumulación progresiva de este metal en el organismo, especialmente en hígado y cerebro. En este trabajo, se explora la posible utilidad de miRNAs circulantes en plasma para identificar biomarcadores que sirvan para controlar la progresión de la enfermedad en pacientes con EW bajo tratamiento. Los modelos desarrollados para cada miRNA mostraron un buen rendimiento al clasificar a los pacientes con factores de evolución desfavorable, por lo que estos tres miRNAs se proponen como candidatos para mejorar el seguimiento clínico o para respaldar la eficacia de nuevas terapias en la EW. / [CA] La malaltia de Wilson és un trastorn hereditari del metabolisme del coure causat per mutacions en ATP7B, que codifica per a una proteïna transportadora del coure al fetge. El seu mal funcionament produeix alteracions en l'excreció biliar i l'acumulació progressiva de coure, especialment en fetge i cervell. Es va explorar la possible utilitat del perfil de miRNAs circulants com biomarcadors de progressió de la patologia hepàtica. L'avaluació dels models obtinguts per a cadascun dels tres miRNAs va mostrar un bon rendiment per a classificar al grup de pacients amb factors d’evolució desfavorable, en conseqüència, es proposen com a candidats per tal de millorar el seguiment clínic o comprovar l’efectivitat de noves teràpies en la malaltia de Wilson. / [EN] Wilson disease (WD) is an inherited disorder of copper metabolism caused by mutations in ATP7B, which encodes for a liver copper-transporting protein. Its dysfunction causes a deficit in biliary copper excretion and a progressive accumulation of this metal in the organism, mainly in liver and brain. In this work, circulating miRNAs profiling in plasma has been accomplished to identify biomarkers that could serve to monitor disease progression in WD patients under chelation therapy. Developed models for each miRNA exhibited good performance classifying patients with poor outcome factors, consequently, these three miRNAs are proposed as candidates to improve clinical follow-up or to support efficacy of novel therapies in WD. / Esta Tesis Doctoral ha sido financiada por los siguientes proyectos de investigación: “Avanzar en el diagnóstico y la prognosis de la enfermedad de Wilson” Duración: 2016-2019, Fundació Per Amor a l’Art (FPAA) IP: C. Espinós; “Bases genéticas y biomarcadores pronóstico de la enfermedad de Wilson y Wilson-like” 2020-2022, Fundació Per Amor a l’Art (FPAA) IP: C. Espinós; “Estudios clínicos, bases genéticas y biomarcadores pronóstico en enfermedades raras neurodegenerativas” 2019-2021, Instituto de Salud Carlos III (Expediente: PI18/00147) IP: C. Espinós; “De genes a terapia en enfermedades neurodegenerativas y neuromusculares” 2018-2021, Generalitat Valenciana, Programa Prometeo para grupos de investigación de excelencia (Expediente: PROMETEO/2018/135) Consorcio de investigadores formado por F. V. Pallardó (coordinador), J.M. Millán, I. Galindo, P. Sanz, T. Sevilla y C. Espinós. / Sánchez Monteagudo, A. (2021). Genética molecular y biomarcadores de la enfermedad de Wilson [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/171454

Thesis (PhD )--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Diagnostic delay is regarded as a major contributor to the continuous rise in tuberculosis (TB) cases and the emergence and transmission of multidrug-resistant tuberculosis (MDR-TB) and extensively drug resistant tuberculosis (XDR-TB). It is therefore essential that more rapid diagnostic methods are developed. Molecular-based assays have the potential for the rapid species-specific diagnosis of TB and associated drug-resistances directly from clinical specimens. We investigated whether high resolution melting analysis (HRM) could enable the rapid diagnosis of TB and associated drug resistance, since the HRM apparatus and reagents are relatively inexpensive and the methodology can easily be implemented in high incidence, low income regions. Application of this methodology allowed for the rapid identification of mycobacterial lymphadenitis from fine-needle aspiration biopsy (FNAB) samples in 2 studies. This was done by targeting the region of deletion 9 (RD9), present in M. tuberculosis and M. canettii, but absent from all other members of the complex. However, the sensitivity of the method was low (51.9% and 46.3%, respectively) when compared to the reference standard (positive cytology and/or positive culture). Despite this limitation our method was able to provide a rapid diagnosis in more than half of the infected patients with a relatively high specificity (94.0% and 83.3%, respectively). We therefore proposed a diagnostic algorithm allowing the early treatment of patients with both HRM and cytology results indicative of mycobacterial disease. We developed the Fluorometric Assay for Susceptibility Testing of Rifampicin (FAST-Rif) which allowed the rapid diagnosis of MDR-TB by detecting rifampicin (RIF) resistance mutations in the rpoB gene with a sensitivity and specificity of 98% and 100%, respectively. The FAST-Rif method was easily adapted to detect ethambutol (EMB) resistance due to mutations in the embB gene with a sensitivity and specificity of 94.4% and 98.4% respectively, as compared to DNA sequencing. The FAST-EMB method was a significant improvement over the inaccurate culture based method. We identified a strong association between EMB resistance (and pyrazinamide resistance) and MDR-TB and subsequently advised modifications to the current (2008) South African National TB Control Programme draft policy guidelines. Due to the potential for amplicon release, we adapted the FAST-Rif and FAST-EMB methods to a closed-tube one-step method using the detection of inhA promoter mutations conferring isoniazid (INH) resistance as a model. The method (FASTest-inhA) was able to identify inhA promoter mutations with a sensitivity and specificity of 100% and 83.3%. These mutations are of particular interest as they confer low level INH resistance and cross-resistance to ethionamide (Eto). Since inhA promoter mutations are strongly associated with XDR-TB in the Western and Eastern Cape Provinces of South Africa, data generated by the recently implemented GenoType® MDRTBPlus assay may allow individualised treatment regimens to be designed for a patient depending on their INH mutation profile. Our proposed treatment algorithm may be particularly useful in XDR-TB cases, for which only few active drugs remain available. Since current diagnostic methods all carry advantages and disadvantages, a combination of phenotypic and genotypic-based methodologies may be the best scenario while awaiting superior methods. / AFRIKAANSE OPSOMMING: Die onvermoë om tuberkulose (TB), multi-weerstandige tuberkulose (MDR-TB) en uiters weerstandige tuberkulose (XDR-TB) vinnig te diagnoseer, is ‘n belangrike oorsaak vir die volgehoue toename en verspreiding daarvan. Dit is noodsaaklik dat diagnostiese toetse wat vinniger resultate oplewer, ontwikkel word. Molukulêre toetsing het die potensiaal om vinnig spesie-spesifieke diagnoses van TB en die weerstandigheid teen TB-medikasie te lewer. Hierdie studie wil vasstel of hoë-resolusie smeltingsanalise (HRS) ‘n vinnige diagnose van TB en die weerstandigheid teen TB-medikasie kan oplewer aangesien die relatiewe lae koste van reagense en apparaat, asook die minimale infrastruktuur en vaardighede wat vir dié toets benodig word, dit uiters geskik maak vir pasiënte in gebiede met ‘n hoë TB-insidensie en lae inkomste. Die toepassing van die HRS-metode op fynnaald-aspiraatbiopsies in twee afsonderlike studies, het gelei tot die vinnige identifisering van mikrobakteriële-limfadenitis. Dit is bemiddel deur die gebied van delesie 9 (RD9) teenwoordig in Mycobacterium tuberculosis en M. canettii, maar afwesig in al die ander lede van die kompleks, te teiken. Die sensitiwiteit van die metode was (51.9% en 46.3%, vir die twee studies onderskeidelik) in vergelyking met die verwysingstandaard (positiewe sitologie en/of positiewe kultuur). Ten spyte van dié beperking was ‘n vinnige diagnose in meer as die helfte van geïnfekteerde pasiënte met ‘n redelike hoë spesifisiteit (94.0% en 83.3%, onderskeidelik) moontlik. ‘n Diagnostiese algoritme wat gebaseer is op die resultate van die HRS en sitologie-toetse, is voorgestel om pasiënte vroeër te behandel. ‘n Fluorometriese toets (FAST-Rif) is ontwikkel vir die vinnige diagnose van MDR-TB deur mutasies in die rpoB-geen op te spoor met ‘n hoë sensitiwiteit en spesifisiteit (98% en 100%, onderskeidelik). Hierdie mutasies is verantwoordelik vir weerstandigheid teen die antibiotikum rifampicin (FAST-Rif) en word beskou as ‘n vinnige diagnose vir MDR-TB. Die FAST-Rif metode kon maklik aangepas word om mutasies in die embB-gene, verantwoordelik vir weerstandigheid teen die antibiotikum ethambutol (EMB), op te spoor. Die FAST-EMB-metode het ‘n sensitiwiteit en spesifisiteit van 94.4% en 98.4% onderskeidelik getoon in vergelyking met DNS volgordebepaling. Die FAST-EMB-metode was ‘n betekenisvolle verbetering op die onakkurate kultuurgebaseerde metodes. ‘n Sterk korrelasie tussen EMB-weerstandigheid (en weerstandigheid teen pyrazinamide) en MDR-TB is geïdentifiseer. Vervolgens is veranderinge aan die Suid-Afrikaanse Nasionale TB-beheerprogram se Konsepbeleidsgids (2008) voorgestel. Om die potensiële vrylating van amplikone te verhoed, is die FAST-Rif en FAST-EMB aangepas tot ‘n enkelstap geslote buissisteem deur gebruik te maak van die opsporing van inhA promotormutasies wat weerstandigheid teen isoniazid (INH) veroorsaak. Die metode het ‘n sensitiwiteit en spesifisiteit van 100% en 83.3% onderskeidelik, getoon. Hierdie mutasies veroorsaak laevlak weerstandigheid teen INH, maar ook kruisweerstandigheid teen ethionamide (Eto). Aangesien daar ‘n sterk verbintenis tussen inhA-promotormutasies en XDR-TB in die Oos en Wes-Kaapprovinsies van Suid-Afrika is, kan data van die GenoType® MDRTBPlus-toets moontlik gebruik word om ‘n meer geïndividualiseerde behandeling te ontwerp afhangende van die pasiënt se INH-mutasieprofiel. Ons behandelingsalgoritme is veral geskik vir XDR-TB pasiënte vir wie daar weinig aktiewe antibiotika beskikbaar is. Huidige diagnostiese metodes het almal voor- en nadele, dus bied ‘n kombinasie van fenotipiese en genotipiese metodes moontlik die beste oplossing totdat beter metodes ontwikkel word.

Introdução: A cardiomiopatia hipertrófica (CH) é uma doença cardíaca estrutural primária, caracterizada por hipertrofia do ventrículo esquerdo, sem dilatação, geralmente assimétrica e predominantemente septal. Na população geral a prevalência estimada da CH é de 0,2% (1:500), correspondendo a 0,5% de todas as cardiopatias. Atualmente estão descritas mais de 1400 mutações associadas à CH em 20 genes relacionados com os miofilamentos do sarcômero, o disco-Z e o transporte de cálcio, sendo que os três mais associados são os genes MYH7, MYBPC3 e TNNT2, responsáveis por 50% do casos com diagnóstico molecular positivo no Brasil. Dessa forma, o advento de novas tecnologias de sequenciamento de DNA de alta performance promete revolucionar o diagnóstico molecular, tornando mais rápida e barata a identificação de alterações genéticas, impactando positivamente na custo-efetividade do manejo diagnóstico e terapêutico de pacientes e famílias com o diagnóstico de CH. Materiais e Métodos: Noventa e uma amostras de uma casuística de pacientes não relacionados, portadores de CH com diagnóstico molecular prévio para os 3 genes mais associados (19 positivas e 72 negativas) foram utilizadas juntamente com uma amostra referência do HapMap (NA12878) na validação de um pipeline proposto para a identificação de alterações genéticas em um painel com 74 genes associados à cardiomiopatias hereditárias, utilizando a plataforma Ion Torrent PGM. A etapa de chamada de variantes foi testada em dois limiares diferentes de cobertura de sequenciamento (30x e 10x) e três limiares de frequência de alelo variante (35%, 25% e 20%). A amostra NA12878 foi utilizada na aferição de valores de reprodutibilidade intra e inter-ensaio. As amostras da casuística de CH com diagnóstico molecular prévio negativo foram utilizadas na análise de ganho diagnóstico. Eram consideradas alterações potencialmente patogênicas aquelas que apresentassem associação prévia com CH ou classificação deletéria em dois de três algoritmos de predição de impacto funcional (PROVEAN, SIFT, PolyPhen2) e MAF<0,01, se disponível. Resultados: A plataforma de sequenciamento utilizada apresentou desempenho aceitável, gerando em média 165,9 ±13,1 Mb, com um valor médio de 146,9 ± 11,54 Mb acima de PhredQ>=20, por amostra. O valor médio de cobertura de sequenciamento por amostra foi de 250 ± 23,94x, com 95,2% das regiões alvo cobertas pelo menos 10x. A sensibilidade máxima observada para SNVs foi de 96,7% enquanto que para InDels foi 28,5%. Os valores de reprodutibilidade inter e intra-ensaio de 89,5% e 87,3%, respectivamente. Das 72 amostras negativas, 35 puderam ser reclassificadas como positivas, sendo que os dois genes com mais ocorrências de alterações genéticas foram FLNC e TRIM63, ambos já relacionados com CH. Vinte e duas amostras foram reclassificadas como inconclusivas e 15 permaneceram negativas. O ganho diagnóstico foi de 21,5%. Conclusões: A plataforma Ion Torrent PGM apresenta potencial no sequenciamento de genes relacionados à cardiomiopatias hereditárias e o pipeline validado mostrou valores analíticos praticáveis em uma rotina diagnóstica. A utilização do painel genético ampliado se mostrou viável na detecção de alterações genéticas, propiciando uma boa margem de ganho diagnóstico em comparação com o sequenciamento apenas dos três genes mais associados à CH / Introduction: Hypertrophic cardiomyopathy (HCM) is a primary cardiac disease, mainly characterized by unexplained left ventricle hypertrophy, in the absence of dilatation, usually asymmetric and predominantly septal. The estimated prevalence is 1:500 individuals in the general population, corresponding for 0.5% of all cardiac diseases. Up to now, more than 1400 mutations are associated with HCM in 20 genes related with sarcomeric myofibrils, Z-disc and calcium homeostasis, wherein the 3 most associated genes are MYH7, MYBPC3 and TNNT2, accounting for 50% of cases with positive molecular diagnostics in Brazil. Thus, the advent of new high throughput DNA sequencing technologies promise to revolutionize the use of molecular diagnostics, turning the identification of genetic mutations in a fast and cheap practice, increasing the cost-effectiveness of diagnostic and treatment of patients and families with HCM. Materials and Methods: Ninety one samples from an HCM casuistic of unrelated individuals with previous molecular diagnostics for the three most HCM-associated genes (19 positives and 72 negatives) were processed along with a reference HapMap sample (NA12878) in the validation process of a pipeline proposed for the detection of genetic alterations in a genetic panel composed of 74 genes associated with inherited cardiomyopathies, using Ion Torrent PGM platform. The variant call step was tested for two cutoffs of sequencing coverage (30x and 10x) and three cutoffs of variant allele frequency (35%, 25% and 20%). The sample NA12878 was used in the assessment of intra and inter-assay reproducibility. Negative samples from the HCM casuistic were used in the assessment of diagnostic yield. Variants were considered potentially pathogenic if previously described as associated with HCM or if presenting a deleterious score in at least two of three impact prediction algorithms tested (PROVEAN, SIFT and PolyPhen-2) and MAF < 0.01, if available. Results: The chosen next-generation sequencing platform presented an acceptable performance, with a mean throughput of 165,9 ±13,1 Mb, with a mean value of 146,9 ± 11,54 Mb above PhredQ >= 20. Mean sequencing coverage was 250 ± 23,94x, wherein 95.2% of target bases were covered at least 10x. Maximum achieved sensitivity for SNVs was 96.7% while for InDels was 28.5%. Both values of inter and intra-assay reproducibility were 89.5% and 87.3%, respectively. Of all 72 negative samples, 35 were reclassified as positive with the two most frequently mutated genes being FLNC and TRIM63, both already associated with HCM. Twenty two samples were reclassified as inconclusive and 15 remained negatives. Diagnostic yield was 21.5%. Conclusions: Ion Torrent PGM platform presented a feasible potential for the sequencing of inherited cardiomyopathies-associated genes and the designed pipeline presented reliable analytical values for diagnostic use. The expanded panel proved to be a good strategy for the detection of genetic alteration providing a good value of diagnostic yield in comparison with the sequencing of the three most HCM-associated genes alone