El hemocultivo es el principal método de diagnóstico de bacteriemias, sin embargo el largo periodo del resultado del diagnóstico conlleva a una inadecuada o nula administración de tratamiento, provocando en muchos casos el fallecimiento del paciente. La implementación de nuevos métodos pueden contribuir con un diagnóstico rápido y seguro, para disminuir la mortalidad y resistencia antimicrobiana. Por ello, la implementación de un programa que adiciona mejoras en los métodos convencionales e integra métodos moleculares como FilmArray y GenXpert, pretende generar un impacto en el diagnóstico de bacteriemias verdaderas. Es por esto que el objetivo del siguiente estudio es evaluar la efectividad de un programa de evaluación y tipificación de cultivos positivos utilizando los nuevos métodos moleculares. Hasta el momento, son escasas las investigaciones y no existe un protocolo establecido que llegue a este objetivo, por lo que se pretende realizar un estudio observacional descriptivo de corte transversal que permita analizar las posibles mejoras en el tiempo y diagnóstico del programa que se está implementando actualmente. De manera que se pueda contribuir en un futuro a la generación de investigaciones posteriores que analicen el impacto del mismo en la salud pública.

Le phénomène émergent de la tuberculose multirésistante et ultrarésistante est un problème de santé publique à l’échelle mondiale. Dans les pays en développement, ce problème est accru du fait que les laboratoires de diagnostic de la tuberculose manquent d’équipement et d’outils de diagnostics pour identifier ces cas pour prescrire une chimiothérapie adaptée. La première partie de ce travail de doctorat a permis à travers le séquençage du locus pncA, de mettre en évidence que la résistance au Pyrazinamide survient généralement et de manière significative lorsque la souche est multirésistante, c’est-à-dire après l’acquisition de la résistance à la Rifampicine et à l’Isoniazide. Le pourcentage des souches résistantes au PZA est même plus élevé chez les souches MDR résistantes aux FQs. Dans la seconde partie de l’étude, nous proposons une méthode alternative à la culture de bacilles dans un environnement confiné de type P3. À partir d’échantillons cliniques non cultivés (expectoration) et grâce au GeneXpert MTB/RIF, au séquençage de gènes et au spoligotypage, nous avons pu identifier 19 souches multirésistantes, une transmission active de souches sensibles appartenant aux clades LAM10, T1, MANU, H3 et enfin une épidémie sous-jacente de 5 souches Beijing multirésistantes. / The emerging phenomenon of the MDR and XDR-TB is a worldwide public health issue. In developing countries, this problem is amplified due to the fact that TB diagnostic laboratories lack equipment and diagnostic tools to identify these cases and therefore prescribe appropriate chemotherapy. In the first part of this doctoral work, the sequencing of the pncA gene allowed us to show that the resistance to Pyrazinamide occurs significantly when the strain is MDR, corresponding to the acquisition of resistance to Rifampicin and Isoniazid; and that after the acquisition of Fluoroquinolones and to injectable antibiotics of second line (Amykacine, Kanamycine, Capreomycine) resistance by MDR strains, this rate increases even more. In the second part of the study, we propose an alternative method to the culture of bacilli in a BSL3 confined environment. From uncultivated clinical samples (sputum) and through GeneXpert MTB/RIF, sequencing of genes and spoligotyping, we identified 19 MDR strains, active transmission of sensitive strains belonging to clades LAM10, T1, MANU, H3 and finally as well as an underlying epidemic of 5 Beijing MDR strains.In the first study, 272 retrospective samples of Mycobacterium tuberculosis isolates were selected from two large cosmopolitan cities: Northern Paris (Bichat-Claude Bernard Hospital, 101 strains) and Southwest of Shanghai (Songjiang district, 171 Strains). These strains were selected according to their known phenotypic sensitivity to Rifampicin (RIF) and Isoniazid (INH). These phenotypic resistances were confirmed by the HAIN genotype analysis tools MTBDRplus and by the sequencing of the rpoB and katG/inhA genes. To determine the extensively drug resistance strains (XDR), we sequenced the gyrA/gyrB and rrs genes to identify genetic mutations associated with resistance to Fluoroquinolones (FQs) and second-line injectable antibiotics: Amikacin (AMK)-Kanamycin ( KAN)-Capreomycin (CAP), respectively. Finally, we sequenced the pncA gene of all isolates to identify the genetic mutations associated with resistance to Pyrazinamide (PZA). The strains were genotyped by spoligotyping and MIRU-VNTR.In the second study, from October 2014 to February 2015, 159 morning sputum samples with smear-positive smear after Ziehl-Neelsen staining were collected at the three main diagnostic laboratories for tuberculosis in Libreville, Gabon. These clinical samples were transported to the National Laboratory of Public Health in Libreville for analysis with the GeneXpert MTB/RIF automaton to confirm the microscopic diagnosis and to determine the resistance of bacilli to Rifampicin. Of the 159 samples, 29 samples had a sputum volume less than 1 ml, the minimum required according to the manufacturer's recommendations. For the 130 sputum samples analyzed by the GeneXpert automaton, 375 μl of the remaining GeneXpert solution not introduced into the cartridge was introduced into a 50 ml conical tube containing 25 ml of phosphate buffer (autoclaved solution) to neutralize the pH of the GeneXpert solution. The conical tube is centrifuged for 15 minutes at 4,500 rpm, the pellet is taken up in 100 μl of TE and then transferred to a 100 μl microtube which is subsequently heated for 30 minutes at 90°C. After a cycle of freezing (-40 ° C. for 1 h)-defrosting, the microtube is briefly centrifuged and the supernatant is transferred to a new microtube. From this new microtube we amplified by PCR and then sequenced the rpoB, katG/inhA, pncA, gyrA, rrs and rpsL genes to identify mutations associated with resistance to Rifampicin, Isoniazid, Pyrazinamide, Fluoroquinolones, Antibiotics in second lines: Amikacin-Kanamycin-Capreomycin and Streptomycin (SM), respectively. All the samples were genotyped by the multiplexed spoligotyping applied to the Luminex MagPix.

Anaerobe opportunist Clostridium difficile causes the majority of hospital-acquired antibiotic-associated diarrhea. Infections can be severe because of its ability to withstand many antibiotics, to sporulate and to produce toxins (A, B and binary).     In Sundsvall Hospital C. difficile is detected with real-time PCR, which targets the sequences of toxin B, binary toxin and a regulatory gene deletion seen in the virulent ribotype 027. All positive samples are stored frozen for one month, available for further analysis or outbreak investigation. The aim of this study was to investigate if temperature and transportation time may affect the viability of C. difficile, and the PCR-result.     Frozen feces samples were cultivated, identified with MALDI-TOF and analyzed with real-time PCR after at least one month of storage. To simulate the effect of transportation time, samples were stored at 4-8°C for three and seven days before cultivation and identification. Controls were cultivated after freezing for comparison.     Ninety percent of the frozen samples contained viable C. difficile. Discrepancies between PCR-results were found for two of the oldest samples collected (six months), which turned negative. Fresh samples showed lower amount of viable C. difficile after three days (50 %) than after seven days (60 %) of storage, perhaps because of competition with other bacteria and sporulation. The frozen control group contained a higher viable amount, 75 %. The results indicate that C. difficile tolerates to be stored at low temperatures as practiced today at the laboratory. Transportation time seem to affect the outcome of cultivation, but not the PCR-result.

En 10-plex GeneXpert analysmetod för diagnos av M. Tuberculosis infektion samt med kapacitet att särskilja aktiv och latent tuberkulos har utvecklats på Cepheid. Analysmetoden sker ex-vivo och kvantifierar uttryck av tuberkulos associerade mRNA biomarkörer i antigenstimulerat helblod. Ett original set av mRNA biomarkörer har nedselekterats till 9 biomarkörer som utgör mRNA signaturen i 10-plex analysmetoden. Urvalet av biomarkörer baserades på probescreening och optimeringstudier utfört med Cepheids ”in cartridge” qPCR teknologi. Den diagnostiska prestandan av mRNA signaturen utvärderades genom att analysera 380 antigenstimulerade kliniska prover klassificerade som M. Tuberculosis infekterad, aktiv tuberkulos, latent tuberkulos och icke infekterad. mRNA signaturens förmåga korrekt klassificera samt diskriminera aktiv och latent tuberkulos bestämdes genom att implementera ROC-kurvor vilket resulterade i ett optimalt AUC-värde på 0.784 vilket visar på diagnostisk kapacitet att separera aktiv och latent tuberkulos. / A 10-color prototype GeneXpert assay based on distinct mRNA expression profiles in ex-vivo antigen stimulated whole blood has been developed at Cepheid for diagnosis of M. Tuberculosis infection and separation of active and latent tuberculosis. An original larger set of mRNA biomarkers has been downselected to a set of 9 biomarkers constituting the mRNA signature in the 10-color assay. The downselection of biomarkers was based on probe screening and optimization studies performed with Cepheid’s “in cartridge” qPCR technology. The 10-color assay was evaluated for diagnostic performance by analyzing 380 banked antigen stimulated clinical samples classified as M. Tuberculosis infected, active tuberculosis, latent tuberculosis and not infected. The ability of the mRNA signature to correctly classify and separate active and latent tuberculosis was determined by implementing ROC curves using delta Ctvalues as test variables and resulted in an optimal AUC value of 0.784 indicating ability to separate active and latent tuberculosis.