No description available.

Yes / We compared cetylpyridinium chloride (CPC), ethanol (ETOH), and OMNIgene.SPUTUM (OMNI) for 28-day storage of sputum at ambient temperature before molecular tuberculosis diagnostics. Three sputum samples were collected from each of 133 smear-positive tuberculosis (TB) patients (399 sputum samples). Each patient's sputum was stored with either CPC, ETOH, or OMNI for 28 days at ambient temperature, with subsequent rpoB amplification targeting a short fragment (81 bp, GeneXpert MTB/RIF [Xpert]) or a long fragment (1,764 bp, in-house nested PCR). For 36 patients, Xpert was also performed at baseline on all 108 fresh sputum samples. After the 28-day storage (D28), Xpert positivity did not significantly differ between storage methods. In contrast, higher positivity for rpoB nested PCR was obtained with OMNI (n = 125, 94%) than with ETOH (n = 114, 85.7%; P = 0.001). Smears with scanty acid-fast bacilli (AFB) had lower rpoB PCR positivity with ETOH storage (n = 10, 41.7%) than with CPC (n = 16, 66.7%; difference, 25%; 95% confidence interval [CI], 3.5 to 46.5; P = 0.031) or OMNI (n = 16, 69.6%; difference, 26.1%; 95% CI, 3.8 to 48.4; P = 0.031), with no difference between CPC and OMNI. Poststorage, the threshold cycle (CT ) values significantly decreased compared to those prestorage with ETOH (difference, -1.1; 95% CI, -1.6 to -0.6; P = 0.0001) but not with CPC (P = 0.915) or OMNI (P = 0.33). For one patient's ETOH- and CPC-stored specimens with a CT of <10, Xpert gave results of rifampin false resistant at D28, which was resolved by repeating Xpert on a 1/100 diluted specimen. In conclusion, 28-day storage of sputum in OMNI, CPC, or ETOH at ambient temperature does not impact short-fragment PCR (Xpert), including for low smear grades. However, for long-fragment PCR, ETOH yielded a lower PCR positivity for low smear grades, while the performance of OMNI and CPC was excellent for all smear grades. (The study has been registered at ClinicalTrials.gov under registration number NCT02744469.). / Directorate General for Development (DGD), Belgium (FA4 to C.N.S., B.C.D.J., D.A., and L.R.), and the European Research Council-INTERRUPTB starting grant (number 311725 to B.C.D.J., C.J.M., and L.R.)

Global prevalence of allergic diseases has been on the rise for the last 30 years. In Canada, this upward trend in allergic diseases has resulted in over 3 million Canadians being affected by allergic asthma. Allergic asthma is triggered by inhalation of environmental allergens resulting in bronchial constriction and inflammation, which leads to clinical symptoms such as wheezing, coughing and difficulty breathing. Asthmatic airway inflammation is initiated by the release of inflammatory mediators (-eg- histamine) released by granulocytic cells (-eg- mast cells and basophils). However, immunoglobulin E (IgE) antibody is also necessary for the initiation of the allergic cascade, and IgE is produced and released exclusively by memory B cells and plasma cells. Allergen crosslinking of IgE:FcεRI complexes on the surface of mast cells and basophils causes degranulation of pro-inflammatory mediators. Acute allergen exposure has also been shown to increase IgE levels in the airways of patients diagnosed with allergic asthma; however, more studies are needed to better understand local airway inflammation. Our group's work, in accordance with the literature, has shown an increase of IgE in the airways of subjects with mild allergic asthma following allergen inhalation challenge. Although regulatory B cells (Bregs) have been shown to modulate IgE-mediated inflammatory processes in allergic asthma pathogenesis, particularly in mouse models of allergic airway disease, the levels and function of these IgE+ B cells and Bregs remain to be elucidated in human models of asthma. The overall objective for this dissertation was to investigate the biology of B cells in allergic asthma pathogenesis, specifically investigating the frequency of IgE+ B cells and Bregs in allergic asthma, and the kinetics of these cells after allergen exposure. First, we characterized IgE+ B cells in the blood and sputum of allergic asthmatics and healthy controls with and without allergies (Chapter 2). We showed that IgE+ B cell levels were higher in sputum, but not blood, of allergic asthmatics compared to controls. We further demonstrated that these findings were consistent across airway IgE+ B cell subsets, which include IgE+ memory B cells and IgE+ plasma cells. Additionally, IgE+ B cells in sputum positively correlated with sputum eosinophils, total IgE and B cell activating factor (BAFF) measured in sputum fluid phase. These findings highlight the association of airway IgE+ B cells with allergic asthma, and suggest that local IgE+ B cell functions contribute to the pathogenesis of asthma. Second, we measured the trafficking of IgE+ B cells in periphery (blood, bone marrow and tonsil) and locally (sputum) in allergic asthmatics following whole lung allergen challenge (Chapter 3). IgE+ B cells only increased in the airways of allergic asthmatics following allergen inhalation challenge; there were no allergen-induced changes in IgE+ B cell levels in blood, bone marrow and tonsil. In addition, we showed allergen-induced increases in BAFF and total IgE, but not allergen-specific IgE in sputum fluid phase. Taken together, chapters 2 and 3 show that allergic asthmatics have elevated levels of IgE+ B cells in the airways, that can be further increased after allergen exposure. Therefore, local B cell production of IgE in the lungs may be an important source of IgE for initiation of acute inflammatory responses in allergic airways. Third, we evaluated the levels of Bregs in allergic asthmatics compared to controls, and examined the kinetics, function and distribution (bone marrow, blood and sputum) of Bregs following allergen inhalation challenge (Chapter 4). We showed that Bregs were 2-fold lower in the blood of allergic asthmatics compared to controls, highlighting a possible dysregulation of this regulatory cell type in allergic asthmatics, which may contribute to disease pathology. Furthermore, after whole lung allergen challenge Bregs decreased in the bone marrow with a co-incident increase in the blood and sputum of allergic asthmatics. This pattern reflects potential trafficking of these cells from bone marrow to the airways after exposure to allergic stimuli. Lastly, we stimulated CD19+ B cells purified from blood of allergic asthmatic with IL-4 in vitro. IL-4 is a type 2 cytokine known to isotype-switch B cells to IgE+ B cells, as well as differentiates naïve T cells into Th2 cells, thus propagating the allergic cascade. We found that IL-4 promoted higher proportions of IL-10+ and FoxP3+ Bregs, which demonstrates that Bregs may have a role in dampening IgE-mediated inflammation in a type 2 environment. However, further functional studies are warranted. Taken together, the findings of this dissertation highlight the local compartmental changes in IgE+ B cells and Bregs following allergen challenge of allergic airways. Better understanding the temporal and compartmental shifts in B cell subpopulations, particularly IgE+ B cells and Bregs, may aid in future development of therapeutics. / Thesis / Doctor of Philosophy (PhD)

<p>Uvod: Povezanost dijabetesa melitusa i&nbsp; tuberkuloze je odavno primećena i bila je predmet ispitivanja mnogih studija. Dijagnoza &scaron;ećerne bolesti pre otkrića insulina značila je smrtni ishod u roku od pet godina, a najče&scaron;ći uzrok smrti su bile infekcije, uključujući tuberkulozu. Poslednjih godina incidenca tuberkuloze je u padu, ali je i dalje prisutan značajan broj obolelih od tuberkuloze u zemljama u razvoju. Sa druge strane incidenca dijabetes melitusa je u porastu, pre svega zbog tendencije porasta broja gojaznih osoba. Procenjeno je da će prevalencija obolelih od dijabetes melitusa dostići 438 miliona obolelih do 2030, a 80% svih slučajeva će biti stanovnici zemalja u razvoju gde je i dalje visoka prevalencija tuberkuloze. Kao rezultat ovakve epidemiolo&scaron;ke situacije ove dve bolesti će se sve če&scaron;će javljati uporedo, modifikujući tok jedna drugoj. Preduslov za uspe&scaron;no lečenje dijabetičara obolelih od tuberkuloze je postizanje zadovoljavajuće metaboličke regulisanosti &scaron;ećerne bolesti. smrtni ishod u roku od pet godina, a najče&scaron;ći uzrok smrti su bile infekcije, uključujući tuberkulozu. Poslednjih godina incidenca tuberkuloze je u padu, ali je i dalje prisutan značajan broj obolelih od tuberkuloze u zemljama u razvoju. Sa druge strane incidenca dijabetes melitusa je u porastu, pre svega zbog tendencije porasta broja gojaznih osoba. Procenjeno je da će prevalencija obolelih od dijabetes melitusa dostići 438 miliona obolelih do 2030, a 80% svih slučajeva će biti stanovnici zemalja u razvoju gde je i dalje visoka prevalencija tuberkuloze. Kao rezultat ovakve epidemiolo&scaron;ke situacije ove dve bolesti će se sve če&scaron;će javljati uporedo, modifikujući tok jedna drugoj. Preduslov za uspe&scaron;no lečenje dijabetičara obolelih od tuberkuloze je postizanje zadovoljavajuće metaboličke regulisanosti &scaron;ećerne bolesti. Cilj istraživanja: Cilj rada je bilo ispitivanje uticaja dijabetesa melitusa na tok plućne tuberkuloze, prvenstveno na bakteriolo&scaron;ki status, radiolo&scaron;ku prezentaciju bolesti, dužinu terapijskog režima i učestalost recidiva bolesti. Materijal i metode: Ispitivanjem su obuhvaćene dve grupe od po pedeset bolesnika koji su hospitalizovani u Institutu za plućne bolesti Vojvodine. Prvu grupu činili su bolesnici sa plućnom tuberkulozom i pridruženom &scaron;ećernom bole&scaron;ću, a drugu grupu bolesnici sa plućnom tuberkulozom bez pridružene &scaron;ećerne bolesti. Svi bolesnici su analizirani po sledećim karakteristikama: starost, pol, klinička slika, bakteriolo&scaron;ki status, radiolo&scaron;ka prezentacija, prisustvo neželjenih efekata antituberkulotika, prisustvo rezistencije M. tuberculosis na lekove, trajanje terapijskog režima, ishod lečenja, pojava recidiva i dužina hospitalizacije. Oboleli od &scaron;ećerne bolesti bili su dodatno analizirani prema: tipu bolesti, dužini trajanja bolesti, metaboličkoj regulisanosti bolesti i prisustvu komplikacija. Svi bolesnici obuhvaćeni ispitivanjem bili su podvrgnuti standardnom dijagnostičkom algoritmu koji obuhvata: anamnezu i fizikalni pregled, direktnu mikroskopiju sputuma, kultivaciju sputuma, radiogram grudnog ko&scaron;a, CT grudnog ko&scaron;a u slučaju postavljenih kliničkih indikacija. Invazivna dijagnostika će se sprovesti kod bolesnika kod kojih dijagnoza nije mogla biti postavljena prethodno sprovedenom neinvazivnom dijagnostikom. Terapijski režim će biti započet tokom hospitalizacije u Institutu za plućne bolesti Vojvodine, a nastavljen ambulantno pod kontrolom Dispanzera za plućne bolesti. Po zavr&scaron;etku terapijskog režima predviđena je kontrola u Institutu za plućne bolesti Vojvodine koja obuhvata procenu kliničke slike, bakteriolo&scaron;kog statusa, radiolo&scaron;kog nalaza i eventualnu potrebu za produženjem terapijskog režima. Rezultati: Istraživanje je pokazalo da je u grupi obolelih od tuberkuloze bez pridruženog dijabetes melitusa bio sličan broj bolesnika mu&scaron;kog i ženskog pola, a veći broj ispitanika se nalazio u starosnim kategorijama do 50 godina starosti, dok je u grupi obolelih od tuberkuloze sa dijabetes melitusom bio značajno vi&scaron;e zastupljen mu&scaron;ki pol i značajno vi&scaron;e ispitanika se nalazilo u starosnim kategorijama preko 50 godina starosti. Beleži se statistički značajno veći broj recidiva u grupi obolelih od tuberkuloze sa dijabetes melitusom (p=0,001). Između ispitvanih grupa se ne beleži statistički značajna razlika u kliničkoj prezentaciji bolesti. U grupi obolelih od tuberkuloze sa dijabetes melitusom, statistički značajno je veći broj direktno pozitivnih nalaza sputuma (p=0,046). Utvrđeno je postojanje statistički značajne razlike u prosečnoj dužini vremena potrebnoj za direktnu konverziju sputuma (p=0,000) i prosečnoj dužini vremena potrebnoj za konverziju kulture sputuma (p=0,000). U oba slučaja je grupa obolelih od tuberkuloze sa pridruženim dijabetes melitusom imala duže prosečno vreme potrebno za konverziju. U grupi obolelih od tuberkuloze sa pridruženim dijabetes melitusom bilo je statistički značajno vi&scaron;e bolesnika sa prisustvom kaverne (p=0,006) i lokalizacijom promena u sva tri režnja (p=0,000). Nije zapažena statistički značajna razlika u trajanju terapijskog režima, ispoljavanju neželjenih efekata lekova, pojavi rezistencije na lekove i ishodu lečenja između dve ispitivane grupe. Grupa obolelih od tuberkuloze sa pridruženim dijabetesom imala je statistički značajno veći broj bolničkih dana (p=0,000). Poređenjem grupa obolelih od tuberkuloze sa pridruženim zadovoljavajuće regulisanim dijabetesom i grupe obolelih od tuberkuloze sa lo&scaron;e regulisanim dijabetesom uočeno je statistički značajno duže trajanje terapijskog režima kod dijabetičara sa lo&scaron;e regulisanom bole&scaron;ću (p=0,018). Nije bilo statistički značajne razlike u zastupljenosti recidiva, kliničke prezentacije bolesti, bakteriolo&scaron;kog i radiolo&scaron;kog statusa, ispoljavanju neželjenih efekata lekova, pojavi rezistencije na lekove, ishoda lečenja i broja bolničkih dana između dve ispitivane grupe. Dodatnim poređenjem grupa (oboleli od tuberkuloze bez pridruženog dijabetesa, oboleli od tuberkuloze sa pridruženim zadovoljavajuće regulisanim dijabetesom i oboleli od tuberkuloze sa pridruženim lo&scaron;e regulisanim dijabetesom) primećeno je da je grupi bolesnika obolelih od tuberkuloze sa lo&scaron;e regulisanim dijabetesom potrebno najduže vreme za direktnu konverziju i konverziju kultura sputuma na MT i da imaju najveći broj bolničkih dana. U grupi obolelih od tuberkuloze sa pridruženim lo&scaron;e regulisanim dijabetesom je bilo statistički značajno veći broj bolesnika koji su lečeni osam meseci u odnosu na druge dve grupe (p=0,011). Poređenjem grupa obolelih od tuberkuloze sa tipom 1 dijabetesa tipom 2 dijabetesa nije uočena statističk značajna razlika između grupa po svim ispitivanim varijablama. U grupi dijabetičara sa dobro regulisanim dijabetesom nalazi se veći broj onih koji imaju tip 2 bolesti, u odnosu na grupu bolesnika sa lo&scaron;e regulisanim dijabetesom. Grupa dijabetičara sa lo&scaron;e regulisanom bolesti ima statistički značajno veći broj komplikacija &scaron;ećerne bolesti. Zaključak: Dokazano je da &scaron;ećerna bolest značajno utiče na bakteriolo&scaron;ki status, radiolo&scaron;ku prezentaciju, dužinu terapijskog režima, učestalost recidiva tuberkuloze i broj bolničkih dana obolelih od tuberkuloze,kao i da je regulisanost &scaron;ećerne bolesti imala značajan uticaj na dužinu terapijskog režima.</p> / <p>Introduction: The association of diabetes mellitus and tuberculosis has long been observed and has been the subject of many studies. The diagnosis of diabetes before the discovery of insulin meant death within five years, a leading cause of death were infections, including tuberculosis. Last years the incidence of tuberculosis has declined, but there is still a significant number of TB patients in developing countries. On the other hand, the incidence of diabetes is on the rise, primarily due to the tendency of an increasing number of obese people. It is estimated that the prevalence of patients with diabetes will reach 438 million sufferers by 2030, and 80% of all cases will be people in developing countries where it is still a high prevalence of tuberculosis. As a result of the epidemiological situation, these two diseases will increasingly occur in parallel, modifying the current one another. Aim: The aim of this study was to investigate the influence of diabetes mellitus on the course of pulmonary tuberculosis, primarily in the bacteriological status, radiological presentation of disease, duration of the treatment regimen and the frequency of disease relapse. Materials and Methods: The study included two groups of fifty patients who were hospitalized at the Institute for pulmonary diseases. The first group consisted of patients with pulmonary tuberculosis and concomitant diabetes mellitus, a second group consisted of patients with pulmonary tuberculosis without associated diabetes. All patients were analyzed by the following characteristics: age, gender, clinical picture, bacteriological status, radiological presentation, the presence of side effects of antituberculosis drugs, the presence of M. tuberculosis resistant to the drugs, the duration of the therapeutic regimen, treatment outcome, recurrence and length of hospitalization. Diabetics were further analyzed with respect to: the type of disease, duration of disease, a metabolic disease and the regulation for the presence of a complication. All patients completed this study were subjected to a diagnostic algorithm comprising: history and physical examination, direct microscopy of sputum, cultivation of sputum, radiographs of the chest, chest CT scan in case positioned on clinical indications. Invasive diagnostic will be performed in patients in whom the diagnosis could not be set previously conducted noninvasive diagnostics. The treatment regimen will be started during the hospitalization in the Institute of pulmonary diseases and is set under the control of ambulatory pulmonary dispensaries. Results: The study showed that in the group of TB patients without concomitant diabetes mellitus was a similar number of patients male and female, a greater number of respondents was in the age groups up to the age of 50, while in the group of TB patients with diabetes mellitus was significantly more frequent male half and significantly more respondents were in the age groups over 50 years of age. Significantly higher number of relapses is recorded in a group of TB patients with diabetes mellitus (p = 0,001). Between the two study groups was not significant difference in the clinical presentation of the disease. In the group of TB patients with diabetes mellitus, is statistically significant higher number of smear positive findings (p = 0,046). There is a statisticaly significant difference in the average length of time required for the smear conversion (p = 0,000) and average length of time needed for the conversion of sputum cultures (p = 0,000). In both cases, the group of TB patients with associated diabetes mellitus had a longer average time needed for the conversion. In the group of patients with tuberculosis associated with diabetes mellitus was statistically significantly more patients with the presence of the cavern (p = 0,006), and the localization of the pulmonary changes in all three lobes (p = 0,000). Between the two study groups was not observed a statistically significant difference in duration of the treatment regimen, the expression of adverse drug effects, develop resistance to the drugs, and the outcome of the treatment. Group of patients with tuberculosis associated with diabetes had a statistically significantly greater number of hospital days (p = 0,000). Between the groups of patients with tuberculosis associated with satisfactory controlled diabetes and the group of TB patients with poorly controlled diabetes was statistically significantly longer duration of the therapeutic regimen in diabetic patients with poor regulation of the disease (p = 0,018). There was no significant difference in the appearance of relapses, the clinical presentation of disease, the bacteriological status, radiology, the expression of adverse drug effects, develop resistance to the drugs, outcome of the treatment, number of hospital days between the two study groups. Comparing the three groups (tuberculosis without associated diabetes mellitus, TB patients with associated satisfactorily controlled diabetes and TB patients with associated poorly controlled diabetes), it was observed that the group of patients suffering from tuberculosis with poorly controlled diabetes takes the longest time to smear conversion and conversion of sputum culture and to have the highest number of hospital days. In the group of patients with tuberculosis associated with poorly controlled diabetes was significantly greater number of patients who were treated for eight months compared to the other two groups (p = 0,011). Comparing the group of TB patients with type 1 diabetes and type 2 diabetes is not a statistically significant difference between the groups in all variables. In the group of diabetic patients with satisfactorily controlled diabetes, there are a large number of those with type 2 disease, in comparison to the group of patients with poorly controlled diabetes. Group of diabetics with poorly regulated disease has a significantly greater number of diabetes comlications. Conclusion: It has been shown that diabetes mellitus has a significant effect on the bacteriological status, radiological presentation, the length of the treatment regimen, the frequency of recurrence of tuberculosis and the number of hospital days of patients with tuberculosis, and that the adjustment of diabetes had a significant effect on the length of the treatment regimen.</p>

Introdução: Os mecanismos fisiopatológicos da asma grave resistente ao tratamento (STRA) em crianças não está totalmente elucidado e parece diferir do observado em adultos, justificando investigações específicas neste grupo de pacientes. O escarro induzido é método útil para identificar fenótipos e endotipos de asma grave através de marcadores inflamatórios. O objetivo deste estudo foi investigar os padrões inflamatórios de crianças com STRA através escarro induzido e comparar com um grupo de crianças com asma grave que atingiram o controle. Métodos: Crianças (6-18 anos) com diagnóstico de asma grave (critério GINA) em tratamento a pelo menos 6 meses em um centro de referência foram avaliadas em um coorte prospectivo por 3 meses (3 visitas consecutivas). Foi averiguada técnica inalatória, adesão ao tratamento e investigado as principais comorbidades. Realizado coleta de escarro induzido para análise citológica e avaliação quantitativa de citocinas do sobrenadante, espirometria, pletismografia e medidas da FeNO. Após período de seguimento, os pacientes foram classificados em dois grupos: asma grave controlada e asma grave resistente ao tratamento conforme critérios da ATS/ERS. Resultados: Foram incluídos 40 pacientes (idade média 12,8 anos; 62,5% sexo masculino), sendo 13 (32,5%) classificados como STRA após o período de seguimento. A mediana do número de exacerbações foi maior e do escore de ACT menor nos pacientes STRA e esta diferença foi significativa. Não foram encontradas diferenças significativas: nos dados demográficos, nos parâmetros funcionais espirométricos e de pletismografia (CVF, VEF1, VEF/CV, FEF 25-75%, LTC, RV, RV/LTC, resistência e condutância das vias aéreas) e nos valores de FeNO quando comparado o grupo de pacientes controlados com o de STRA. O padrão inflamatório eosinofílico foi predominante nos dois grupos de pacientes, entretanto, o grupo STRA apresentou porcentagem proporcionalmente maior de neutrófilos no escarro comparados com o grupo de asma grave controlada, na visita 3 e também na visita 1 quando analisados retrospectivamente (p < 0,05). As medianas nos níveis das citocinas IL10, GM-CSF, INFy e TNFalfa no escarro foram significativamente maiores no grupo STRA quando comparado ao grupo controlado (p < 0,05) e o GM-CSF e TNF-alfa apresentaram correlação inversa com escore de ACT. Conclusão: Nesta coorte prospectiva, os parâmetros funcionais e a FeNO não discriminaram crianças com STRA dos que atingiram o controle. A presença de neutrófilos no escarro e das citocinas IL10, INFy e, particularmente, GM-CSF e TNFalfa podem ter para um papel na resistência ao tratamento da asma grave em crianças e adolescentes. Antagonistas específicos dessas citocinas podem no futuro representar uma estratégia na terapêutica / Background: The pathophysiological mechanisms of severe therapyresistant asthma (STRA) in children are not fully elucidated and seem to differ from findings in adults, thus justifying specific research on children. Induced sputum is useful for detecting phenotypes and endotypes of severe asthma via inflammatory markers. The aim of the present study was to investigate the inflammatory patterns of children with STRA by the induced sputum method and to compare them with a group of children who achieved control of severe asthma. Methods: A prospective cohort of children (6-18 years old) diagnosed with severe asthma (Global Initiative for Asthma - GINA criteria) and in treatment for at least 6 months at a reference center was assessed for 3 months (3 consecutive visits). Inhalation technique, adherence to treatment and main comorbidities were assessed. Induced sputum samples were collected for cytology analysis and quantitative assessment of cytokines in the supernatant; the participants were also subjected to spirometry, plethysmography and fractional exhaled nitric oxide (FeNO) measurements. At the end of follow-up, the patients were classified into two groups: controlled severe asthma and STRA according to the European Respiratory Society and American Thoracic Society (ERS/ATS) criteria. Results: Forty patients were included (average age 12.8 years old; 62.5% male); 13 (32.5%) were classified as STRA at the end of follow up. The median number of exacerbations was higher and the Asthma Control Test (ACT) score was lower in the STRA group; these differences were significant. Significant differences were not found relative to demographic data, spirometry and plethysmography function parameters [forced vital capacity (FVC), forced expiratory volume in the first second (FEV1), ratio of FEV1 to slow vital capacity (FEV1/SCV), forced expiratory flow at 25-75% of FVC (FEF 25-75%), total lung capacity (TLC), residual volume (RV), RV/TLC, airway resistance and conductance] and FeNO after comparison of the STRA and controlled asthma groups. The eosinophilic inflammatory pattern predominated in both groups; however, the STRA group showed a proportionally higher percentage of sputum neutrophils compared with the controlled asthma group at visit 3 and visit 1 upon retrospective analysis (p<0.05). The median sputum levels of the cytokines IL-10, GM-CSF, IFN-y and TNF-alpha were significantly higher in the STRA group compared with the controlled asthma group (p < 0.05); GM-CSF and TNF-? showed inverse correlations with ACT scores. Conclusion: In the analyzed prospective cohort, functional parameters and FeNO did not discriminate between children with STRA and children with controlled asthma. The presence of neutrophils and the cytokines IL-10, IFN-y and, more particularly, TNF-alpha and GM-CSF in the sputum might have a role in resistance to treatment for severe asthma among children and adolescents. Antagonists specific for these cytokines might represent a therapeutic strategy in the future

Introdução: Os mecanismos fisiopatológicos da asma grave resistente ao tratamento (STRA) em crianças não está totalmente elucidado e parece diferir do observado em adultos, justificando investigações específicas neste grupo de pacientes. O escarro induzido é método útil para identificar fenótipos e endotipos de asma grave através de marcadores inflamatórios. O objetivo deste estudo foi investigar os padrões inflamatórios de crianças com STRA através escarro induzido e comparar com um grupo de crianças com asma grave que atingiram o controle. Métodos: Crianças (6-18 anos) com diagnóstico de asma grave (critério GINA) em tratamento a pelo menos 6 meses em um centro de referência foram avaliadas em um coorte prospectivo por 3 meses (3 visitas consecutivas). Foi averiguada técnica inalatória, adesão ao tratamento e investigado as principais comorbidades. Realizado coleta de escarro induzido para análise citológica e avaliação quantitativa de citocinas do sobrenadante, espirometria, pletismografia e medidas da FeNO. Após período de seguimento, os pacientes foram classificados em dois grupos: asma grave controlada e asma grave resistente ao tratamento conforme critérios da ATS/ERS. Resultados: Foram incluídos 40 pacientes (idade média 12,8 anos; 62,5% sexo masculino), sendo 13 (32,5%) classificados como STRA após o período de seguimento. A mediana do número de exacerbações foi maior e do escore de ACT menor nos pacientes STRA e esta diferença foi significativa. Não foram encontradas diferenças significativas: nos dados demográficos, nos parâmetros funcionais espirométricos e de pletismografia (CVF, VEF1, VEF/CV, FEF 25-75%, LTC, RV, RV/LTC, resistência e condutância das vias aéreas) e nos valores de FeNO quando comparado o grupo de pacientes controlados com o de STRA. O padrão inflamatório eosinofílico foi predominante nos dois grupos de pacientes, entretanto, o grupo STRA apresentou porcentagem proporcionalmente maior de neutrófilos no escarro comparados com o grupo de asma grave controlada, na visita 3 e também na visita 1 quando analisados retrospectivamente (p < 0,05). As medianas nos níveis das citocinas IL10, GM-CSF, INFy e TNFalfa no escarro foram significativamente maiores no grupo STRA quando comparado ao grupo controlado (p < 0,05) e o GM-CSF e TNF-alfa apresentaram correlação inversa com escore de ACT. Conclusão: Nesta coorte prospectiva, os parâmetros funcionais e a FeNO não discriminaram crianças com STRA dos que atingiram o controle. A presença de neutrófilos no escarro e das citocinas IL10, INFy e, particularmente, GM-CSF e TNFalfa podem ter para um papel na resistência ao tratamento da asma grave em crianças e adolescentes. Antagonistas específicos dessas citocinas podem no futuro representar uma estratégia na terapêutica / Background: The pathophysiological mechanisms of severe therapyresistant asthma (STRA) in children are not fully elucidated and seem to differ from findings in adults, thus justifying specific research on children. Induced sputum is useful for detecting phenotypes and endotypes of severe asthma via inflammatory markers. The aim of the present study was to investigate the inflammatory patterns of children with STRA by the induced sputum method and to compare them with a group of children who achieved control of severe asthma. Methods: A prospective cohort of children (6-18 years old) diagnosed with severe asthma (Global Initiative for Asthma - GINA criteria) and in treatment for at least 6 months at a reference center was assessed for 3 months (3 consecutive visits). Inhalation technique, adherence to treatment and main comorbidities were assessed. Induced sputum samples were collected for cytology analysis and quantitative assessment of cytokines in the supernatant; the participants were also subjected to spirometry, plethysmography and fractional exhaled nitric oxide (FeNO) measurements. At the end of follow-up, the patients were classified into two groups: controlled severe asthma and STRA according to the European Respiratory Society and American Thoracic Society (ERS/ATS) criteria. Results: Forty patients were included (average age 12.8 years old; 62.5% male); 13 (32.5%) were classified as STRA at the end of follow up. The median number of exacerbations was higher and the Asthma Control Test (ACT) score was lower in the STRA group; these differences were significant. Significant differences were not found relative to demographic data, spirometry and plethysmography function parameters [forced vital capacity (FVC), forced expiratory volume in the first second (FEV1), ratio of FEV1 to slow vital capacity (FEV1/SCV), forced expiratory flow at 25-75% of FVC (FEF 25-75%), total lung capacity (TLC), residual volume (RV), RV/TLC, airway resistance and conductance] and FeNO after comparison of the STRA and controlled asthma groups. The eosinophilic inflammatory pattern predominated in both groups; however, the STRA group showed a proportionally higher percentage of sputum neutrophils compared with the controlled asthma group at visit 3 and visit 1 upon retrospective analysis (p<0.05). The median sputum levels of the cytokines IL-10, GM-CSF, IFN-y and TNF-alpha were significantly higher in the STRA group compared with the controlled asthma group (p < 0.05); GM-CSF and TNF-? showed inverse correlations with ACT scores. Conclusion: In the analyzed prospective cohort, functional parameters and FeNO did not discriminate between children with STRA and children with controlled asthma. The presence of neutrophils and the cytokines IL-10, IFN-y and, more particularly, TNF-alpha and GM-CSF in the sputum might have a role in resistance to treatment for severe asthma among children and adolescents. Antagonists specific for these cytokines might represent a therapeutic strategy in the future

La tuberculose continue d’être une cause majeure de morbidité et de mortalité dans le monde, principalement dans les pays en voie de développement, bien qu’elle soit une maladie curable. Le diagnostic rapide et précis de la TB active est essentiel pour l’initiation rapide du traitement et le contrôle de la maladie. Le développement de nouveaux tests rapides de diagnostic de TB active représente un véritable challenge pour l’optimisation du diagnostic.L’objectif principal de nos travaux de thèse était de développer et d’évaluer des approches de PCR en temps réel ciblant la séquence d’insertion IS6110 pour la détection de l’ADN de MTB dans les expectorations.Nous avons tout d’abord développé une PCR en temps réel ciblant la séquence répétée IS6110 pour la quantification de l’ADN de MTB. L’évaluation des étapes d’optimisation de la sensibilité de la PCR IS6110 a permis de préciser les performances analytiques et le gain de sensibilité comparativement à une PCR ciblant le gène unique senX3. Au terme d’une comparaison de six protocoles de lyse/ extraction la méthode Chelex® s’est avérée être la plus efficace dans la récupération de l’ADN. La performance diagnostique de la PCR optimisée a été évaluée et comparée avec la PCR automatisée Xpert MTB/RIF sur un panel de 62 échantillons respiratoires.Dans un deuxième temps, nous avons comparé la performance diagnostique de la PCR IS6110 optimisée, le test Xpert MTB/RIF et la version ultrasensible récemment commercialisée du test PCR leader Xpert MTB/RIF Ultra pour la détection de l’ADN de MTB dans des expectorations ayant une faible charge bacillaire.Enfin, à partir de 203 LCR collectés dans le cadre du diagnostic de méningites aseptiques au Burkina Faso, nous avons évalué la performance de la PCR en temps réel multiplexe (IS6110, HSV1, HSV2) combinée à l’extraction par la méthode Chelex® pour la détection de l’ADN de MTB et d’Herpès. / TTuberculosis continues to be a major cause of morbidity and mortality worldwide, mainly in developing countries, despite being a curable disease. The rapid and accurate diagnosis of active TB is essential for rapid initiation of treatment and disease control. The development of new rapid diagnostic tests for active TB represents a real challenge for the optimization of the diagnosis.The main objective of our thesis work was to develop and evaluate real-time PCR approaches targeting the IS6110 insertion sequence for the detection of sputum MTB DNA. We first developed a real-time PCR targeting the IS6110 repeat sequence for the quantification of MTB DNA. The evaluation of the sensitivity optimization steps of the IS6110 PCR made it possible to specify the analytical performances and the sensitivity gain compared to a PCR targeting the single gene senX3. After a comparison of six lysis / extraction protocols, the Chelex® method proved to be the most efficient in the recovery of DNA. The diagnostic performance of optimized PCR was evaluated and compared with automated Xpert MTB / RIF PCR on a panel of 62 respiratory specimens.In a second step, we compared the diagnostic performance of the optimized IS6110 PCR, the Xpert MTB / RIF test and the highly marketed ultra-sensitive version of the Xpert MTB / RIF Ultra leader PCR assay for the detection of sputum MTB DNA. having a low bacillary load.Finally, from 203 LCR collected in the context of the diagnosis of aseptic meningitis in Burkina Faso, we evaluated the performance of the multiplexed real-time PCR (IS6110, HSV1, HSV2) combined with extraction by the Chelex® method for the detection of MTB and Herpes DNA.

Orientador: Antonio Fernando Ribeiro / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-11T18:35:30Z (GMT). No. of bitstreams: 1 Amalfi_LiliamMachado_M.pdf: 5120878 bytes, checksum: c21489142714302bf486eaaa938cf04c (MD5) Previous issue date: 2007 / Resumo: Considerando que a fibrose cística é a mais importante doença hereditária, potencialmente letal, incidente na raça branca, que a infecção pulmonar é reconhecida por ter o maior papel na morbimortalidade levando à morte prematura em 90% dos pacientes, e que a principal causa das exacerbações é as infecções recorrentes ou crônicas; torna-se fundamental para um centro de referência, o conhecimento do perfil microbiológico de seus pacientes. A correlação entre a exacerbação dos sintomas pulmonares e a contagem de colônias de bactérias na cultura rotina diagnóstica (CRD) serve para orientar o controle das infecções. Norteados por este fundamento, para o perfil microbiológico utilizaram-se resultados CRD do: Registro eletrônico do Paciente (REP), Arquivo do Laboratório de Microbiologia (ALM) e Prontuário do paciente (PP). As três bases dados pertencem à mesma casuística, sendo que os dois últimos foram utilizados para verificar a coerência entre os perfis e estudo da susceptibilidade. Estatística: x2, Fisher, Pearson, regressão linear, e, nível significância p<0.05 e IC 95%. Foram resgatados 38.480 registros do REP, referentes a 975 CRD's, 402 nos ALM's e 371 dos PP's. Foram isoladas: Pseudomonas aeruginosa em 80,9% REP (43% pertenciam ao morfotipo não mucóide), nos ALM em 70,8% (43% não mucóide) e em 100% dos PP (60,6% não mucóide) e; Staphylococcus aureus em (50,1% REP, 48% ALM, e em 55% PP). Microrganismos emergentes como Burkholderia cepacia em (3,69% REP, 1% ALM, e em 3,5% dos PP's), Strenotrophomonas maltophilia em (3% REP, 2,8% ALM e 1,6% PP). Observamos uma elevada prevalência dos P. aeruginosa, durante o 1° ano de vida (57%), diferença significante se comparadas aos resultados da Cystic Fibrosis Fundation e Consenso Europeu que relatam uma prevalência de 20% entre 0-5 anos. Em relação à prevalência da B. cepacia, foi utilizado o meio seletivo, elevando a prevalência para 13% no ano de 2003, e, se observados ao longo dos anos, o valor se assemelha aos demais estudos (3,6%). Em relação ao estudo de susceptibilidade antimicrobiana, foram encontradas 13 cepas de P.aeruginosa multidroga-resistente aos 5 antibióticos incluídos no estudo (3,23% CRD's em 3 pacientes). Das P.aeruginosa 13,4% foram sensíveis a todos os antimicrobianos (ALM). Foi possível observar resistência das cepas P. aeruginosa aos antibióticos freqüentemente utilizados como a Gentamicina (50%) e Amicacina (31%). As mesmas cepas foram sensíveis à Ceftazidima (45%), Ciprofloxacina (48,7%). Foram isoladas em 389 dos 402 CRD's (ALM), ambos morfotipos (mucóide e não mucóide) apresentando elevada sensibilidade a Ceftazidima, Imipenem e Amicacina (66,9, 56,3% e 60% respectivamente) e, dentre eles, o que apresentou maior eficácia e menor resistência foi a Ceftazidima (5,4%). As cepas de S. aureus apresentaram elevada sensibilidade (72,7% ALM e 100% PP) sendo que dos 183 antibiogramas somente 11 cepas (6% em 5 pacientes ALM) apresentaram-se Oxa-Resistente, apesar de uma prevalência elevada e persistente. O perfil microbiológico, utilizando REP, foi coerente com ALM e PP, sugerindo que dados eletrônicos podem se amoldar às perspectivas de futuras pesquisas, levando-se em consideração a necessidade de novos estudos e maior interação entre as equipes, ajustadas as correções de possíveis vieses / Abstract: Considering that the Cystic Fibrosis is the most important hereditary illness potentially lethal incident in Caucasoid, and the pulmonary infections has been recognized as having the greatest role in the morbid mortality, being cause of death in 90% of the patients, and the main exacerbations cause are the recurrent or chronic infection/ the knowledge in the microbiological profile from patients becomes basic for all reference centers. When infected, the treatment will depends of microorganisms characteristics (antimicrobial resistance and ambient conditions) and, the prognostic depends of the nutritionals and immunological conditions; The correlation between the exacerbation from pulmonary symptoms and the counting of bacteria colonies by the culture routine diagnosis (CRD), serves to guide and control the infections. Guided by this bedding, our objective was to delineate this profile, using the CRD results from: Electronic Patient Register (REP), Archives from Microbiology Laboratory (ALM) and Handbooks of Patient (PP). All the databases belong to the same casuistry, being ALM data and PP collected to verify if the electronic registers correspond to the findings and notations in these archives. For the statistical calculations: descriptive analysis, x2, fisher, correlation Pearson and linear regression and the significance were p <0, 05 and IC 95%. Were analyzed 975 CRD's results between 38.480 registers from REP (100 patients), 402 from ALM (100 patients) and 371(9 patients) from PP's. Were identified: Pseudomonas aeruginosa in 80,9% from REP (43.1% belonged to morphotype mucoid), from ALM in 70,8% (27.8% mucoid) and PP (51.4% mucoid); Staphylococcus aureus in (50.1% REP, 48% ALM, and in 55% PP). Emergent microorganisms as Burkholderia cepacia in (3.69% REP, 1% ALM, and in 3,5% of the PP's), Strenotrophomonas maltophilia in (3% REP, 2.8% ALM and 1.6% PP). In relation of microorganisms prevalence, we observed a high significant prevalence of the Pseudomonas aeruginosa during the first year life (57%), greater than the results from Cystic Fibrosis Foundation and European Consensus (20% of 0-5 yrs). In relation of Burkholderia cepacia prevalence, the selective media was used (year 2003), increasing the prevalence to 13%, if observed during a long period, it would be equivalent to found (3,6%). About antimicrobial susceptibility, 13 of P. aeruginosa multi-drugs resistant were found to all usual antibiotics (3,3% CRD of 3 patients). The P. aeruginosa, 13,4% were sensitive to all antimycrobiane. We observed a great resistance between P. aeruginosa against to usual antibiotics as Gentamicine (50%) and Amicacine (31%). The same cepas were sensitive to Ceftazidima (45%), Ciprofloxacin (48,7%). Of 389 CRD's (ALM), were simultaneously found the both morphotypes (mucoid and nonmucoid) that showed a high sensitive to Ceftazidima, Imipenem e Amicacin (66,9, 56,3% e 60%) and the more effective and less resistant was Ceftazidima (5,4%). The Staphylococcus aureus were high sensitive (72,7% ALM and 100% PP) and just 11 cepas in 183 (5,9%) were Oxa-Resistant in 5 patients, even thought the high and persistent prevalence. The microbiological profile of cystic fibrosis patient from REP corresponds to the results from ALM and PP, suggesting that the electronic register can be molder to the perspectives of future researches, with news studies on this subject, and to plan more interaction between teams to correct any possible vises to appropriate research / Mestrado / Saude da Criança e do Adolescente / Mestre em Saude da Criança e do Adolescente

No description available.

Thesis (MScMedSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Setting This study was conducted in the Tygerberg district, Cape Town, in the Western Cape, South Africa Background The evaluation of early tuberculosis (TB) treatment response is based on month 2 sputum culture status. This method of evaluation has a number of limitations: the test requires relatively advanced laboratory infrastructure and procedures, it takes several weeks to obtain results and is a relatively a poor marker at predicting treatment response. The discovery of potential host markers which reflect the efficacy of early treatment would be of great importance for clinical management of individual patients. The treatment failure would be detectable earlier than at week 8 of treatment. The duration of clinical trials of new anti-tuberculosis drugs may also be substantially reduced by such markers if these would be measurable earlier than at week 8 of therapy. Objectives 1) To evaluate diluted, 7-day whole blood cultures stimulated with live Mycobacterium tuberculosis (M.tb) for the presence of host markers of early TB treatment response 2) To evaluate an overnight, undiluted, M.tb antigen stimulated whole blood culture Quantiferon Gold In Tube (QFT-GIT) supernatants for host markers of early TB treatment response The study designs were as follows: In study one, baseline samples and samples from week 1, week 2 and week 4 of treatment from 30 cured TB patients were selected from a larger biomarker study, in which whole blood was stimulated with live M.tb or left unstimulated. Fifty seven host markers were measured in supernatants by multiplex cytokine arrays. In study two, baseline samples and samples from week 2 and week 8 of treatment from 19 cured TB patients were randomly selected from the placebo group in a micronutrient supplement study. QFT-GIT supernatants from these participants were assessed through multiplex cytokine arrays for levels of fifty seven host markers. All of the participants in both studies were Human Immunodeficiency Virus (HIV) negative. Changes in marker expression over time and between fast and slow responders to treatment were evaluated. Comparability between the two culture methods was assessed for markers that were evaluated in both studies. Results In study one, the majority of host markers showed significant changes over time in the unstimulated supernatants. Only GRO and IL-1beta changed significantly in an antigen-specific manner (background levels subtracted). No significant changes were observed between fast and slow responders. In study two, the majority of host markers showed significant changes over time in the unstimulated supernatants whereas only MDC and IL-4 changed during the observation period in antigen stimulated levels. Significant differences were observed between fast and slow responders at pre-treatment for IL-13 Ag-Nil and IL-1betaAg-Nil . Conclusion This study revealed, antigen-specific responses showed only limited potential for early TB treatment response monitoring, but may have potential in differentiating between treatment outcomes. Future investigations may have to include later time points during treatment as these were not included in the present assessment. The QFT-GIT samples do not appear to be equivalent to live M.tb stimulated 7-day whole blood assays. / AFRIKAANSE OPSOMMING: Instelling Die studie is uitgevoer in die Tygerbergdistrik, Kaapstad, Wes-Kaap, Suid-Afrika. Agtergrond Die evaluering van die respons op vroeë tuberkulose (TB) behandeling word gebaseer op die status van maand 2 sputum kulture. Hierdie evalueringsmetode het ‘n paar beperkinge: die toets benodig relatief gevorderde laboratorium infrastruktuur en prosedures, die toetsuitslae is eers na ‘n paar weke beskikbaar en dit is n relatiewe swak merker om repons op behandeling te voorspel. Die ontdekking van potensiële selfmerkers wat die doeltreffendheid van vroeë behandeling weerspieël sal van groot belang wees vir die kliniese bestuur van individuele pasiënte. Mislukking van die behandeling sal sodoende voor week 8 van behandeling waargeneem kan word. Die tydsduur van kliniese proewe van nuwe anti-tuberkulose medikasie mag ook baie verkort word met sulke merkers as dit voor week 8 van behandeling gemeet kan word. Doelwitte 1) Om verdunde, 7-dae oue volbloedkulture, met lewende Mikobakterium tuberkulosis (M.tb) gestimuleer, te evalueer vir die teenwoordigheid van vroeë TB behandeling respons selfmerkers. 2) Om die supernatant van oornag, onverdunde, M.tb antigeen gestimuleerde volbloedkulture Quantiferon Gold In Tube (QFT-GIT) vir vroeë behandeling respons selfmerkers te evalueer. Die studie-ontwerpe was soos volg: Met studie een is basislynmonsters en monsters verkry na week 1, week 2 en week 4 van behandeling van 30 geneesde TB-pasiënte geselekteer uit ‘n groter biomerkerstudie waarin die volbloed met lewende M.tb gestimuleer is of ongestimuleer gelaat is. Sewe-en-vyftig selfmerkers is in die supernatante gemeet deur middel van multipleks sitokine arrays. Met studie twee is basislynmonsters en monsters verkry na week 2 en week 8 van behandeling van 19 geneesde TB-pasiënte lukraak uit die plasebo-groep in ‘n mikrovoedingstowwe-aanvullingstudie geselekteer. Vlakke van 57 selfmerkers is in die QFT-GIT supernatante van hierdie deelnemers, deur middel van die multipleks sitokine arrays, bepaal. Al die deelnemers van beide studies was HIV negatief. Veranderinge in merker-uitdrukking oor tyd, asook tussen vinnige en stadige respons tot behandeling, is ge-evalueer. Die vergelykbaarheid van die twee kultuurmetodes is geassesseer ten opsigte van die ge-evalueerde merkers in albei studies. Resultate Met studie een het die meerderheid van die selfmerkers in die ongestimuleerde supernatante kenmerkende verandering oor tyd gewys. Slegs GRO en IL-1beta het aansienlik verander in die antigeenspesifieke wyse (agtergrond vlakke afgetrek). Geen kenmerkende veranderinge was waargeneem tussen die vinnige en stadige respons pasiënte nie. Met studie twee het die meerderheid van die selfmerkers aansienlike veranderinge oor tyd in die ongestimuleerde supernatante gewys, in vergelyking waar net die MDC en IL-4 veranderinge gedurende die observasie periode in antigeen gestimuleerde vlakke getoon het. Kenmerkende verskille is tussen die vinnige en stadige respons pasiënte in voorbehandeling vir IL-13 Ag-Nil en IL-1betaAg-Nil waargeneem. Gevolgtrekking Die studie bewys dat antigeenspesifieke response slegs beperkte potensiaal vir vroeë TB behandeling respons monitering het, maar mag die potensiaall vir onderskeidende behandeling uitkomste hê. Toekomstige ondersoeke sal dalk latere tydpunte gedurende die behandeling moet insluit aangesien dit nie in hierdie evaluasie ingesluit is nie. Die QFT-IT monsters verskyn nie as gelykwaardig met die lewendig M.tb gestimuleerde 7-dae volbloed toetse nie.