Introduction: Bordetella pertussis is an important human pathogen that causes whooping cough (pertussis), an endemic illness responsible of significant morbidity and mortality, especially in infants and children. Worldwide, there are an estimated of 16 million cases of pertussis, resulting in about 195,000 child deaths per year. In Peru, pertussis is a major health problem that has been on the increase despite immunization efforts. The objective of this study was to determine the prevalence of B. pertussis among children under five years of age suspected to have whopping cough in Cajamarca, Peru. Methodology: Children diagnosed with whooping cough admitted to the Hospital Regional de Cajamarca from August 2010 to July 2013 were included. Nasopharyngeal samples were obtained for B. pertussis culture and polymerase chain reaction (PCR) detection. Results: In 133 children, the pertussis toxin and IS481 gene were detected in 38.35% (51/133) of the cases by PCR, while only 9.02% (12/133) of the Bordetella cultures were positive. The most frequent symptoms in patients with positive B. pertussis were paroxysm of coughing 68.63% (35/51), cyanosis 56.86% (29/51), respiratory distress 43.14% (22/51), and fever 39.22% (20/51). Pneumonia and acute bronchial obstructive syndrome were present in 17.65% (9/51) and 13.72% (7/51) of the cases, respectively. Conclusions: B. pertussis is responsible for an important proportion of whooping cough in hospitalized children in Cajamarca. Epidemiologic surveillance programs for B. pertussis are essential in Peru, especially in children who could most benefit from the vaccine.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Trichomonads are anaerobic flagellated protists that are either parasites or commensals. They frequently inhabit digestive, respiratory, and urogenital tracts of vertebrates, including domestic cats and dogs. In these hosts, four trichomonad species has been described: Tetratrichomonas canistomae and Tetratrichomonas felistomae that are commensals of the host oral cavity; Pentatrichomonas hominis, a commensal of intestinal tract that could be found in dogs and cats but also in other mammals including humans; and pathogenic Tritrichomonas foetus that causes, in addition to cattle infection, feline intestinal trichomonosis. Although, trichomonads in dogs and cats are probably of cosmopolitan distribution we have no information about their presence in Czech Republic. The first aim of this study was to distinguish types of trichomonads present in the oral cavity of dogs and cats and to get preliminary epidemiological data. The second aim was to demonstrate the presence of Tritrichomonas foetus in cats and dogs in the Czech Republic and to identify potential risk factors for the disease. Cultivation and nested PCR were used to determine the presence of trichomonads in dogs and cats. Sequencing and phylogenetic analysis based on ITS1-5.8rRNA-ITS2 gene sequence was used to identify species of isolated...

Clostridium difficile is a major cause of infectious diarrhea in hospitalized patients. Clostridium difficile-associated disease (CDAD) is of gaining importance now due to its increasing incidence and severity. However, little is known about the C. difficile infections in the Czech Republic. The aim of the study was to characterize C. difficile strains recently isolated (2008 to 2011) from patients hospitalized with gastrointestinal disease in four Prague health care institutions using molecular typing methods; PCR toxinotyping, PCR ribotyping and MLVA (multilocus variable number tandem repeat analysis). Among 273 C. difficile strains, we identified 8 toxinotypes (0, III, IV, V, VI, VIII, IX a XXIII) and 63 ribotypes, of which ribotypes 596 (23,4 % patient), 017 (13,9 %) and 176 (7 %) were the most frequent. According to PCR ribotyping, the situation in the Czech Republic is the most similar to the situation in Poland. Within ribotypes 017, 017/1 and 017/2 and ribotypes 596 and 596/1, 5 and 4 distinct clusters were identified by MLVA, none of which was institution-specific. Additionally, pathogenic C.difficile were isolated from piglet faeces (63,3 %) in a single piglet farm, evaluating the role of C. difficile as an emerging animal pathogen. All piglet isolates belonged to the toxinotype 0 and the ribotype...

Las E. coli productoras de tipo shiga (STEC) son patógenas en el hombre y pueden provocarle colitis hemorrágica y síndrome urémico hemolítico. Dentro de este grupo, el serotipo E. coli O157:H7 es el que más se ha reportado como problema de salud pública. Las infecciones por este agente generalmente se producen por consumir alimento contaminado, siendo la carne de bovino el principal vehículo de este patógeno, en el Perú se ha reportado que es causante de diarreas y hospitalizaciones en niños. El objetivo del estudio es identificar la presencia de E. coli O157:H7 productora de toxinas shiga en canales de bovinos faenados en Lima. Para ello se recolectaron 180 muestras de 2 mataderos de Lima en 2 momentos del oreo; momento lavado (final del lavado e inicio de oreo) y momento oreo ( 2-3 h. en la playa de oreo). Se emplearon 2 metodologías, una técnica estándar para E. coli que requiere de medios tripticasa de soya (37°C por 24 h) y agar Mac conkey (37°C por 24 h) y una técnica microbiológica específica para la detección de E. coli no fermentadoras de sorbitol (característica de la E. coli O157:H7) usando los medios específicos EC con novobiocina (37°C por 24 h) y agar Mac conkey sorbitol (37°C por 24 h). Las colonias compatibles con E. coli se confirmaron con pruebas bioquímicas, luego se guardó a 2 °C una cepa de cada placa positiva para posteriormente y en conjunto realizar un PCR múltiple con la finalidad de identificar los factores de virulencia Stx1, Stx2, eaeA, rfbO157, hlyA y fliCH7. No se detectó E. coli O157:H7. Se encontró 29.4% (53/180) E. coli positivos, 5.6% (10/180) STEC, 1.1% (2/180) E. coli O157 y 0.5% (1/180) STEC O157. No se encontró diferencia significativa (p>0.05) entre la presencia de agentes (E. coli y STEC) y el momento de muestreo; ni con algunas de las técnicas microbiológicas. Los resultados obtenidos muestran la presencia de E. coli en las canales bovina desde su procesamiento primario, en los mataderos evaluados. Palabras claves: cepas, diarrogénicas, PCR, oreo, enfermedades transmitidas por alimentos / --- Shiga toxin producing E. coli (STEC) are pathogenic in humans and it can cause them hemorrhagic colitis and hemolytic uremic syndrome. Within this group, E. coli serotype O157: H7 is the most widely reported as a public health problem. Infections by this agent are generally produced by consuming contaminated food, being beef the main vehicle for this pathogen. In Peru it has been reported to be causing diarrhea and hospitalizations in children. The aim of the study is to identify the presence of E. coli O157: H7 shiga toxinproducing in cattle carcasses of slaughtered in Lima. For this purpose, 180 samples were collected of 2 slaughterhouses in Lima in 2 times of oreo; washing time (end of washing and beginning of oreo) and oreo time (2-3 h. in oreo). 2 methodologies were used, a standard technique for E. coli, which requires trypticase soy media (37°C for 24 h) and Mac Conkey agar (37°C for 24 h) and a specific microbiological technique for detecting E . coli non fermenting sorbitol (characteristic of E. coli O157: H7) using EC medium with specific novobiocina (37°C for 24 h) and Mac Conkey sorbitol agar (37°C for 24 h). Compatible colonies with E. coli were confirmed by biochemical tests, then stored at 2 °C one isolate from each positive plate, later and together make a multiple PCR for the purpose of identify virulence factors Stx1, Stx2, eaeA, rfbO157, hlyA and fliCH7. E. coli O157: H7 wasn´t detected. It was detected 29.4% (53/180) positive E. coli, 5.6% (10/180) STEC, 1.1% (2/180) E. coli O157 and 0.5% (1/ 180) STEC O157. No significant difference (p>0.05) was found between the presence of agents (E. coli y STEC) and the sampling time neither with some microbiological techniques. The results show the presence of E. coli in cattle from its primary processing, in slaughterhouses evaluated. Keywords : strains, diarrheagenic, oreo, foodborne diseases

Der Wilms Tumor (WT), auch Nephroblastom genannt, zählt zu den im Kindesalter am häufigsten auftretenden malignen Tumoren und entsteht meist unilateral (90 – 95 %) und sporadisch (98 – 99 %). Leider sind bis heute die molekularen Ursachen, die zur Entwicklung dieser Tumoren führen nur unzureichend aufgeklärt. So werden bisher nur drei Gene mit dem Auftreten von WT in Verbindung gebracht: WT1, CTNNB1 und WTx. Während WT1 und CTNNB1 jeweils Mutationsraten von etwa 10 – 15 % aufweisen, die zudem häufig gemeinsam vorliegen, werden für WTx Mutationsraten von etwa 30 % beobachtet. Die genetischen Alterationen der anderen Tumoren sind noch immer komplett unbekannt. Ziel dieser Arbeit war aus diesem Grund die Identifikation von relevanten Regionen und Genen, die an der Entstehung bzw. dem klinischen Fortschreiten von Wilms Tumoren beteiligt sind. Zusätzlich sollten weitere Untersuchungen zur Einschätzung ihres prognostischen Potenzials dienen. In einem ersten Ansatz wurden die Chromosomenbereiche 11q und 16q in einer großen Anzahl von Wilms Tumoren auf LOH (=loss of heterozygosity), d.h. den (partiellen) Verlust von genetischem Material, untersucht. In beiden Fällen wurden erhöhte LOH-Raten von etwa 20 % beobachtet, jedoch war keine Eingrenzung der relevanten Regionen möglich, da Allelverluste nicht stets ab einem bestimmten Marker beobachtet wurden. Ein Vergleich mit der Histologie ergab signifikante Assoziationen der Allelverluste mit anaplastischen und Mischtyp-Tumoren (nur für LOH 11q), wohingegen kaum LOHs in epithelialen und stromareichen Tumoren festgestellt wurden. Somit scheinen auf 11q und 16q Gene vorzuliegen, die einerseits die Differenzierung in Epithel und Stroma begünstigen oder andererseits ein blastemreiches und anaplastisches Erscheinungsbild verhindern. Jedoch könnte auch die Assoziation von bestimmten Subtypen mit LOH 11q und 16q auf eine Entstehung aus unterschiedlichen Zellen hindeuten. Weiterhin war das Auftreten von LOH, v.a. wenn jeweils der komplette Chromosomenarm betroffen war, mit einem erhöhten Rezidiv- und Sterberisiko (nur LOH 11q) verbunden. Somit konnte gezeigt werden, dass LOH-Untersuchungen auf 11q und 16q zur Identifikation von Hochrisikopatienten für die Entwicklung von Rezidiven bzw. erhöhter Mortalität eingesetzt werden können, wodurch eine individuelle Anpassung der Therapiemaßnahmen ermöglicht wird. In einem zweiten Ansatz wurden eine Reihe von bereits publizierten potenziellen Markergenen in einer großen Anzahl von Wilms Tumoren mit Hilfe der Realtime RT-PCR auf ihre Relevanz überprüft. Allen diesen Genen wurde zuvor eine Funktion bei der histologischen Klassifikation der Tumoren bzw. bei der Vorhersage bestimmter klinischer Verläufe zugeschrieben. Die univariate Analyse diente der Beurteilung der Relevanz einzelner Gene, wohingegen die multivariate Analyse zur Bestimmung von prognostischen Genkombinationen eingesetzt wurde. Anschließend erfolgte die Validierung mittels eines zweiten und unabhängigen Tumorsatzes. Auch wenn viele der bereits publizierten Marker und in der ersten Analyse erhaltenen Assoziationen in einem weiteren und unabhängigen Tumorsatz nicht verifizierbar waren, konnten dennoch einige frühere Ergebnisse repliziert und die Relevanz der entsprechenden Gene nachgewiesen werden. Neben der Verbindung der Repression von HEY2 und TRIM22 mit Hochrisikotumoren bzw. einer höheren Sterbewahrscheinlichkeit fanden sich schwach signifikante Assoziationen auch für die verminderte Expression von TRIM22 und VEGF mit der Histologie. Ebenso waren erhöhte Level von TERT und die Repression von TRIM22 mit der Entwicklung eines Rezidivs verbunden. Vor allem aber die Korrelation der Repression von HEY2 und VEGF sowie einer Überexpression von CA9 mit Rezidiven, Tumoren hoher Malignität oder primären Metastasen verweisen auf die Notwendigkeit, besonders die Hypoxie- und Angiogenese-Signalkaskaden in Wilms Tumoren zu untersuchen, um deren Einfluss v.a. auf das Fortschreiten und die Ausbreitung der Tumoren zu evaluieren. Auch wenn die multivariate Analyse nicht zu relevanten Genkombinationen führte, konnte hier dennoch eine schwache Assoziation der verminderten Expression von TOP2A und TRIM22 mit primären Metastasen oder einer erhöhten Mortalität, sowie der Überexpression von TERT mit der Rezidivbildung bestätigt werden. Interessanterweise stellte sich die Histologie, die derzeit das Hauptkriterium für die Risikoklassifikation darstellt, weder als geeigneter prognostischer Marker für die Beurteilung des Rezidiv- noch des Sterberisikos heraus. Somit sollten Realtime RT-PCR Analysen in Zukunft als weiterer Faktor zur Beurteilung des Rezidiv- und Sterberisikos eingesetzt werden, um eine individuelle Anpassung der Therapie zu ermöglichen. Basierend auf den Ergebnissen der Realtime RT-PCR Analyse wurde der Einfluss der Expression ausgewählter Gene auf Primärkulturen, die aus nativem Wilms Tumormaterial gewonnen wurden, untersucht. Nach der Überexpression von HEY2, EGR1, MYCN und TRIM22 wurden bei allen Zellen hohe Sterberaten beobachtet, v.a. bei HEY2 und EGR1. Leider konnte weder für HEY2 noch für EGR1 der Grund hierfür aufgeklärt werden, allerdings war bei EGR1 weder die Apoptose noch die Seneszenz beteiligt. Im Gegensatz hierzu wurde die Apoptose als entscheidender Mechanismus bei MYCN und v.a. TRIM22 ermittelt. Außerdem scheint bei MYCN ein großer Anteil an Zellen in die Seneszenz einzutreten. Auch wenn diese ersten Untersuchungen an Primärkulturen von Wilms Tumoren eindeutig die Relevanz dieser Gene für die Entwicklung bzw. das Fortschreiten der Tumoren bestätigten, so sind trotz alledem weitere Experimente v.a. in einer größeren Anzahl genetisch unterschiedlicher Primärkulturen nötig, um das endgültige Potenzial dieser Gene aufzuklären. / Wilms tumor (WT), also called Nephroblastoma, belongs to the most common malignant tumors occurring in childhood. Most of these Wilms tumors develop unilaterally (90 – 95 %) and sporadically (98 – 99 %). Unfortunately, only little is known about the molecular background underlying their development with only three genes known so far: WT1, CTNNB1 and WTx. Mutations in WT1 and CTNNB1 occur only in a minor fraction of Wilms tumors of about 10 - 15 % and are often associated with each other, while mutations in WTx can be found in about 30 % of tumors. The genetic alterations of the other tumors are completely unknown. Hence, the aim of this thesis was to identify important regions and genes that are involved in Wilms tumor formation and/or progression and to further characterize their potential as markers for the prediction of certain clinical outcomes. First, chromosome arms 11q and 16q were screened for LOH (= loss of heterozygosity), which means the (partial) loss of the genome in a cell, in a large cohort of Wilms tumors. In both regions LOH rates of about 20 % were detected, but since allele losses did not always start at the same marker in the different tumors it was not possible to delimit any relevant subregions. Since there were significantly higher rates of allele loss in anaplastic and mixed-type (only 11q) tumors and almost no allele loss in epithelial and stromal tumors, 11q and 16q must contain genes that either facilitate the epithelial and stromal differentiation of cells or hamper the appearance of blastemal and anaplastic phenotypes. Otherwise, the obvious possibility to discriminate these histological subtypes by allele loss on 11q and 16q might be explained by a development from different precursor cells. Higher rates of LOH could also be linked to higher risks for relapse and death (11q only), especially when the whole chromosome arms were involved. Therefore, investigation of LOH on 11q and 16q may help to adjust the therapeutic regimens by identifying high-risk patients for relapse and death. A second approach was to reinvestigate the expression of a number of published marker genes in a larger set of Wilms tumors with a uniform method, the realtime RT-PCR. All of the genes were suggested to facilitate classification and/or prediction of certain clinical outcomes. Univariate analysis was performed to screen for relevant genes, followed by multivariate analysis to search for predictive gene combinations. Finally, validation of associations found in the first cohort was performed in a second and independent tumor set. Unfortunately, many of the previously published markers as well as associations of the first tumor set could not be verified in a new and independent tumor set. Nevertheless, it was possible to replicate the results of a number of genes and evidence their prognostic relevance. These included the repression of HEY2 and TRIM22 for high-risk tumors or mortality. Weaker correlations were verified for the repression of TRIM22 and VEGF with the histological risk and for overexpression of TERT and repression of TRIM22 with later relapse. Since the weaker expression of HEY2 and VEGF as well as the overexpression of CA9 was significantly linked to relapse, high malignant tumors or metastasis the hypoxia / angiogenesis pathways should be investigated in Wilms tumors especially with regard to the progression and spreading of tumors. Finally, multivariate analysis substantiated a weak association of repression of TOP2A and TRIM22 with metastasis or death and of overexpression of TERT with subsequent relapse. Most interestingly, histology, the current gold standard used for prediction of risks for relapse and death, could not be verified as potent prognostic factor for neither of them. Hence, realtime RT-PCR analyses can aid in stratification of tumors and prediction of relapse and death risks to intensify therapy for high-risk patients on one hand and to reduce therapy and side-effects in low-risk patients on the other hand. Based on the results of the realtime RT-PCR analyses the expression of several genes was ascertained in primary cell cultures cultivated from native Wilms tumor material. Overexpression of MYCN, TRIM22 and especially HEY2 and EGR1 by viral transduction resulted in high rates of cell death. Unfortunately, the underlying mechanism of death could be determined neither for HEY2 nor for EGR1, though for EGR1 the involvement of apoptosis and senescence could be excluded. In contrast, death in MYCN and especially in TRIM22 overexpressing cells could be attributed to high rates of apoptosis. Furthermore, a large fraction of MYCN cells seem to enter cell senescence and stop to proliferate. These results clearly corroborate the proposed relevance of the investigated genes in the development and/or progression of Wilms tumors. Nevertheless, further experiments in different primary cell cultures of Wilms tumors are necessary to clarify the real potential of these genes.

Faculty of Health Sciences, Master of Science in Medicine, 9803855e / Approximately 5 million individuals are infected with HIV/AIDS in South Africa. The South African government has initiated a National Anti-retroviral therapy (ARV) Program to manage this disease. The emergence of drug resistance to ARV therapy is of great concern. Commercial gold standard sequence-based genotyping assays for monitoring resistance are unaffordable. This project aimed at developing affordable methods to detect specific point mutations relevant to HIV-1 subtype C. The Oligonucleotide Ligation assay (OLA), a real-time PCR assay and a Restriction Fragment Length Polymorphism (RFLP) assay were explored. Results were compared to the Viroseq genotyping assay. OLA performed poorly on HIV-1 subtype C samples and needs modification. The real-time PCR assay using short Minor Groove Binding probes, accurately detected the K65R mutation. The Mae III RFLP assay detected all V106M mutations accurately. Longitudinal cohort studies are required to confirm relevant mutations, appropriate assays and algorithms for resistance monitoring in HIV-1 subtype C.

O objetivo do trabalho foi avaliar a qualidade, a quantidade e a capacidade de amplificação por PCR de DNA extraído de grãos de soja e milho, seus derivados e produtos acabados contendo como ingredientes obtidos desses grãos, com vistas à detecção de resíduos de organismos geneticamente modificados em alimentos. Para a amplificação de DNA pela PCR convencional, não houve melhor adequação de um protocolo de extração. Ambos métodos, CTAB e coluna de sílica tiveram desempenho comparável para as 32 matrizes avaliadas. A técnica de PCR em tempo real se mostrou mais sensível à qualidade do DNA testado e nesse contexto, o método CTAB se mostrou mais eficiente do que o método de coluna de sílica. Independentemente do método de extração utilizado não foi possível detectar DNA em óleos de soja e milho e em alguns derivados de amido, sugerindo que a aplicabilidade da lei de rotulagem pode esbarrar num entrave técnico no caso de algumas matrizes alimentares altamente processadas. / The aim of the study was to evaluate the quality, the quantity and the amplifiability by PCR of DNA isolated from soybean and maize grains and their by-products targeting the detection of genetically modified organisms in food. PCR amplification of DNA samples isolated either from CTAB and silica-column extraction methods achieved comparable performances. Both extraction methods showed similar results for the 32 tested matrices. The DNA amplification by real time PCR appeared to be affected by the quality of the isolated DNA. In this context, the CTAB extraction method showed to be more suitable when compared to the silica-column method. No DNA was amplified from soy and maize oils, as well as from some starch by-products, regardless the DNA extraction method used. It suggests that, the labeling requirement may rely on technical issues considering some high processed foodstuffs.

O objetivo deste estudo foi investigar longitudinalmente a transmissão de Streptococcus mutans em um grupo de famílias brasileiras de baixa renda. Um critério de inclusão importante foi o de todos os adultos conviverem na mesma casa com a criança. Participaram da pesquisa 14 mães, pais e crianças e 8 avós. Amostras de saliva das crianças foram coletadas em quatro visitas durante 22 meses, para pesquisa de S.mutans. Foram positivas apenas 8 crianças, que tiveram os seus isolados e os isolados de suas famílias identificados pelo método de hibridização DNA-DNA. Um total de 506 isolados de S.mutans foi genotipado pelo método de AP-PCR, usando o primer OPA-02. Foram detectados 20 genótipos diferentes nas 8 famílias, variando de 1 a 5 nos adultos e 1-2 nas crianças. Todas as mães e alguns pais e avós compartilharam genótipos com as crianças. Em todas as famílias foram encontrados genótipos homólogos nos adultos. Alguns genótipos foram estáveis, e outros, se perderam, mas o compartilhamento pode favorecer a contínua reinfecção. Três crianças desenvolveram cárie no período. O encontro de genótipos de cada membro da família na criança e o compartilhar de genótipos nos adultos, sugerem uma reavaliação de modelos preventivos antimicrobianos focalizados apenas na figura materna. / The objective of this study was to investigate in a longitudinal study the transmission of Streptococcus mutans in Brazilian families of a low socioeconomic status. An important entry criterion for the study was to include all members of a household in the study. The study cohort was comprised of 14 mothers, fathers and children and 8 grandmothers. Saliva samples were collected for S. mutans analysis in 4 visits during 22 months. Only eight children were positive for S. mutans by employing DNA-DNA hybridization that was also applied to household members. A total of 506 isolates of S. mutans were genotyped by AP-PCR with the primer OPA-02. Twenty genotypes were detected in 8 families ranging from 1 to 5 in the adults and 1-2 in the children. All mothers and some fathers and grandmothers shared similar genotypes with the children. In all families homologous genotypes were encountered among adults. Some genotypes were stable, and others were lost although sharing a similar environment may favor additional transmission episodes. Three children developed decay during the study period. The fact that children shared genotypes from all household members suggest that reevaluation of preventive methods aimed at suppressing S. mutans infections should include additional family members and not only the mothers.

O objetivo deste estudo foi avaliar a taxa de detecção, quantidade e atividade metabólica de Propionibacterium acnes, antes e após os procedimentos endodônticos de desinfecção, utilizando métodos moleculares baseados em rRNA e rDNA. Foram selecionados 22 pacientes com necrose pulpar e periodontite apical assintomática. Amostras microbiológicas foram coletadas dos canais radiculares após a cirurgia de acesso (S1), após o preparo químico-cirúrgico realizado com Sistema Reciproc e NaClO- 2,5% (S2) e após medicação intracanal com Ca(OH)2 por 14 dias (S3). As amostras dos canais radiculares foram submetidas à extração de DNA e RNA. O RNA foi submetido à reação de transcrição reversa (RT) para confecção de DNA complementar (cDNA). DNA e cDNA foram submetidos a reações de qPCR utilizando iniciadores complementares à sequência de 16S rRNA de P. acnes. O efeito dos procedimentos endodônticos na redução bacteriana foi determinado por qPCR baseada em rDNA. A atividade metabólica bacteriana foi calculada pela razão rRNA/rDNA baseados nos dados dos ensaios de qPCR. Os dados foram analisados pelo teste de Wilcoxon para análise entre as amostras e teste de McNemar para comparação da taxa de detecção dos métodos baseados em rDNA e rRNA, com nível de significância de 5%. A taxa de detecção de P. acnes nas amostras endodônticas foi maior pelo método baseado em rRNA do que pelo método baseado em rDNA (P < 0,0001). P. acnes foi detectado em 36,4% (8/22) e 90,9% (20/22) das amostras S1 utilizando qPCR baseado em rDNA e rRNA, respectivamente. Nas amostras S2, P. acnes foi detectado em 36,4% (8/22) das amostras utilizando o método baseado em rDNA e em 86,4% (19/22) pelo método baseado em rRNA. Nas amostras S3, P. acnes persistiu em níveis detectáveis em todas as amostras positivas em S2. Na análise quantitativa, o nível médio de P. acnes foi 1,28 x 103 cópias de rDNA nas amostras S1. Não houve uma alteração significante dos níveis bacterianos nas amostras S2 e S3 quando comparadas às amostras S1 (P > 0,05). O número de cópias de rRNA foi maior do que o de rDNA em todas as amostras (P < 0,0001). Em S1, o valor mediano da razão rRNA/rDNA foi 7,93 (intervalo de 2,03 a 2,04 x 102). Essa razão permaneceu positiva em S2 (mediana 16,40, intervalo de 2,21 a 4,87 x 102) e S3 (mediana 25,34, intervalo de 0,58 a 1,05 x 103), sem diferença estatística na comparação entre as amostras S1-S2 e S2-S3 (ambos P > 0,05). Baseados nesses achados, concluiu-se que o ensaio de qPCR baseado em rRNA revelou uma alta prevalência de P. acnes nas infecções endodônticas primárias e que este permaneceu metabolicamente ativo nos canais radiculares após o preparo químico-cirúrgico e medicação intracanal. / This study aimed to evaluate the detection rate, quantity and metabolic activity of Propionibacterium acnes before and after endodontic disinfection procedures using RNA- and DNA-based molecular methods. Twenty-two patients with pulp necrosis and asymptomatic apical periodontitis were selected. The microbiological samples were collected from the root canals after access cavity (S1), after the chemo-mechanical preparation performed with the Reciproc System and NaClO- 2.5% (S2) and after intracanal medication with Ca(OH)2 for 14 days (S3). The root canal samples were submitted to DNA and RNA extraction. Complementary DNA (cDNA) was synthetized using the reverse transcription reaction. cDNA and genomic DNA were subjected to qPCR with primers complementary for P. acnes 16S rRNA sequence. The effect of endodontic procedures on bacterial reduction was determined by rDNA-based qPCR. The bacterial metabolic activity was calculate by the rRNA / rDNA ratio based on qPCR assays. Data were analysed by the Wilcoxon test for analysis between samples and McNemar test for detection rate of RNA- and DNA-based molecular methods, with a significance level of 5%. The detection rate of P. acnes in endodontic samples was higher by the rRNA-based method than by the rDNA-based method (P < 0.0001). P. acnes was detected in 36.4% (8/22) e 90.9% (20/22) of the S1 samples using on rDNA- and rRNA- based qPCR, respectively. In S2 samples, P. acnes was detected in 36.4% (8/22) of the samples using the rDNA based method and 86.4% (19/22) by the rRNA based method. In S3 samples, detectable levels of P. acnes persisted in all S2-positive samples. Quantitative analysis of S1 samples revealed that the mean level of P. acnes was 1.28 x 103 rDNA copies per sample. There was no significant change in bacterial levels in S2 and S3 samples when compared to S1 samples (P> 0.05). The number of rRNA copies was greater than the rDNA ones in all samples (P < 0.0001). In S1, the median value of the rRNA / rDNA ratio was 7.93 (range: 2.03 - 2,04 x 102). This ratio remained positive in S2 samples (median 16.40, range: 2.21 - 4.87 x 102) and in S3 (median 25.34, range: 0.58 - 1.05 x 103), with no statistical difference in the comparisons between S1-S2 and S2-S3 samples (both P> 0.05). Based on these findings, it was concluded that the rRNA-based qPCR assay revealed a high prevalence of P. acnes in primary endodontic infections and that it remained metabolically active in the root canals after chemical-surgical preparation and intracanal medication.