Lic.-avh. (sammanfattning) Stockholm : Karolinska institutet, 2009.

Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 232-250).

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 186-200).

Molecular-epidemiological analysis of Mycobacterium tuberculosis strains isolated in the West-Bohemian Region of the Czech Republic including detailed characterisation of anti-tuberculosis drugs - resistant strains MUDr. Jana Amlerová Abstract Tuberculosis is contagious infectious disease that embodies significant epidemiological and clinical problem worldwide. Tuberculosis incidence differs considerably in various regions of the world but even the countries with low incidence engage strongly in epidemiology of tuberculosis. Tuberculosis belongs to one of the priorities of WHO, cooperation in this matter takes place on a global scale. Tuberculosis is a social illness; accordingly, the countries with high occurrence of tuberculosis are classified as developing countries. Mainly in Africa, there is the situation being complicated by coexistence of HIV. Generally, Europe represents a region with low incidence of tuberculosis. The Czech Republic is a country with the lowest incidence in the world with less than five new cases per 100 000 inhabitants every year. This situation is among others result of high-level tuberculosis surveillance and effective application of epidemiological arrangements based in legislation. This dissertation thesis examines several fields of tuberculosis, mainly focused on modern...

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Despite Africa accounting for ~20% of the global cattle population, prevalence estimates and related risk factors of bovine tuberculosis (bTB), caused by Mycobacterium bovis, are still poorly quantified in many countries across the continent. Control of bTB in Africa is difficult due to poor monitoring of cattle movements and limited abattoir surveillance. Also M. bovis is zoonotic and risk factors for transmission include living in close contact with cattle and consumption of unpasteurised milk. Cattle keeping is integral to some rural populations in Cameroon and understanding the epidemiology of bTB in cattle populations is important both to bovine and public health. Detection of bTB in cattle is difficult due to variability of immune responses to M. bovis infection. The interferon-γ (IFN-γ) assay maybe useful to estimate bTB prevalence and identify bTB risk factors in Cameroon. However its performance can vary at different stages of bTB pathogenesis and in different cattle populations. Recently Fasciola hepatica co-infections have been reported to suppress IFN-γ responses in M. bovis infected cattle but the potential effect with F. gigantica co-infections on bTB prevalence estimates in Cameroon is unknown. An abattoir study was conducted in Cameroon to assess the performance of the IFN-γ assay. In 2012-13; 2064 slaughtered cattle were sampled from Bamenda abattoir (North West Region; NWR) and Ngaoundere abattoir (Vina Division; VD). Individual animal data was collected from routine meat inspection including identification of bTB and Fasciola pathology. Cattle were also tested for bTB using the IFN-γ assay and an M. bovis antibody ELISA. In the absence of a gold-standard diagnostic, the IFN-γ assay was compared to other diagnostic tests to assess agreement and identify factors that affected performance of the assay. Agreement between IFN-γ assay, TB lesion identification and an M. bovis antibody ELISA was poor-moderate, probably partly related to differences in immune response detected. A presence of Fasciola gigantica also increased the odds of false negative IFN-γ assay results. On further investigation co-infected cattle had increased odds of TB lesions and reduced IFN-γ responses that potentially could lead to ~20% reduction in test sensitivity. In an attempt to take into account the potential impact of F. gigantica, when estimating bTB prevalence, an antibody ELISA was developed to detect the exposure in live cattle. To highlight the awareness of disease in cattle-rearing communities, estimate prevalence and identify risk factors of bTB in cattle populations; two cross-sectional studies were conducted in 2013. A stratified clustered cross-sectional study of pastoral cattle herds, in the NWR and the VD, sampled 1448 pastoral cattle reared by 100 pastoralists. A smaller cross-sectional study sampled 60 dairy cattle from 46 small-holder co-operative dairy farmers. Individual animal data and herd-level data were collected and animals were screened by both the single comparative intradermal skin test (SCITT) and IFN-γ assay. Awareness of zoonotic TB was low yet consumption of raw milk was high in cattle-keeping communities highlighting the need for accurate bTB prevalence estimates. Despite the high awareness of the clinical presentation of bTB, clinical signs identified by pastoral herdsmen were not associated with cattle being bTB positive. The SCITT was used to compare two manufacturers cut offs for the IFN-γ assay, ≥0.05 and ≥0.1, and highlighted that these two diagnostics may detect different populations of bTB positive cattle. Using the IFN-γ assay at ≥0.1, bTB prevalence was highest in dairy cattle (21.67%) and was also present in pastoral cattle in the NWR and VD (11.33% and 6.55% respectively). Importantly, as F. gigantica is endemic in Cameroon and its influence could mean the true prevalence of bTB could be higher. Female pastoral cattle were at lower odds of being IFN-γ assay positive potentially due to immunosuppressive factors had lower odds of disease. Husbandry practices also decreased the odds of being IFN-γ assay positive such as drinking from streams, antelope and contact with herds at grazing. Age increased the odds of pastoral cattle being IFN- assay positive potentially being a confounder to chronicity of bTB and other co-infections may influence IFN-γ responses. Dairy cattle herds had different risk factors for being IFN- positive likely due to differences in husbandry practices. Considering the potential risk to public health of M. bovis this thesis highlights the extent of bTB across two major cattle keeping regions in Cameroon and the public health risk in cattle-rearing communities. Furthermore the relationship between Fasciola co-infection and IFN- responses to M. bovis described has potential implications for bTB diagnosis in cattle populations where the parasite is present across the globe.

Mycobacterium tuberculosis (Mtb), a deadly pathogen, has scourged mankind for many centuries and has remained a major threat to global world health. Tuberculosis, the disease caused by this bacterium, is a major cause of death in developing nations and there is potential for its re-emergence in developed countries. An alarming rise in cases of multidrug-resistant and extremely-drug resistant tuberculosis (MDR-TB and XDR-TB) that do not respond to the customary first-line antibiotics necessitates the urgent need for development of new anti-TB drugs. Mtb becomes engulfed in human macrophages post infection of the host, but persists in the harsh environment of the human lungs by utilization of host cholesterol as a carbon source. The P450s CYP125A1, CYP142A1 and CYP124A1 are responsible for catalysing the side-chain degradation of cholesterol, which is critical for cholesterol to be used in the Mtb β-oxidation pathway for energy production. This PhD thesis focuses on understanding the structure/mechanism of the Mtb cholesterol 27-oxidases with the aim of facilitating the development of novel inhibitors of these P450s, which are crucial for Mtb to infect the host and to sustain infection. CYP142A1 and CYP124A1 were purified through three chromatographic steps with contaminating proteins successfully removed to give highly pure forms of these enzymes following the final purification step. Spectrophotometric titrations indicate that CYP142A1 and CYP124A1 bind tightly to cholesterol and cholestenone (and also to branched-chain methyl lipids for CYP124A1), highlighting their physiological roles in sterol and fatty acid metabolism, respectively. Binding analyses with a range of azole antibiotics revealed tight binding to bifonazole, clotrimazole, miconazole and econazole, and weak binding to fluconazole. Studies with compounds from a fragment screening library revealed weak binding to fragment hits for the cholesterol oxidases, but much tighter binding to these enzymes was found for ‘elaborated’ hits from a previous fragment screen on the Mtb cyclodipeptide oxidase CYP121A1, indicative of improved ligand potency achieved via ‘fragment merging’ strategies, and of structural similarities between these diverse Mtb P450s. Light scattering data indicate that CYP142A1 exists in dimeric form in solution, but becomes monomeric when treated with DTT; while CYP124A1 is completely monomeric. Crystal structures of CYP142A1 and CYP124A1 in complex with cholestenone, econazole and fragment library hits were determined. CYP142A1 crystal structures with econazole and fragment hits revealed heme coordination via the heterocyclic nitrogen in an azole group, and provide important data towards design of superior inhibitor drugs. The binding of cholestenone within the active site channels of CYP124A1 and CYP142A1 revealed an alignment favourable for C27 hydroxylation of the cholestenone side chain, which supports the physiological roles of CYP142A1 and CYP124A1 (as well as CYP125A1) in host cholesterol catabolism.

The efficacy of Bacille-Calmette Guerin (BCG) vaccination in protection against pulmonary tuberculosis (TB) is highly variable between populations. One possible explanation is increased exposure of certain populations to non-tuberculous mycobacteria (NTM). Given the variable efficacy of BCG an improved vaccine against TB is required. The novel TB vaccine MVA85A has shown promising results, however, the immunogenicity of the vaccine is reduced when it is administered in the Expanded Programme on Immunisation (EPI) schedule. This thesis aims to explore: (A) the effect of exposure to NTM on the level of protection afforded by BCG vaccination against Mycobacterium tuberculosis (M. tb) and (B) the immunological mechanisms behind EPI interference with MVA85A. The effect of M. avium (MA) exposure via systemic and oral routes on the efficacy of BCG was tested using M. tb aerosol infection in a mouse model. The adaptive immune response was profiled in BCG vaccinated mice with and without exposure to MA pre- and post- M. tb infection. The results showed BCG efficacy could be enhanced by exposure to dead MA by a systemic route; T helper 1 and T helper 17 responses were associated with increased protection. In contrast, BCG efficacy may have been reduced by exposure to live MA by the oral route; T helper 2 and regulatory T cells were associated with reduced protection. To answer the second aim MVA85A was co-administered to mice with aluminium adjuvants or aluminium-containing vaccines to replicate the effect of co-administration in the EPI schedule; the adaptive immune response was profiled. T helper 2 and regulatory T cell responses induced by aluminium-containing vaccines were associated with a reduction in the immunogenicity of MVA85A.

<p>UVOD: Tuberkuloza je odavno poznata bolest koja i danas u 21. veku jo&scaron; uvek predstavlja veliki javnozdravstveni problem, uprkos primeni moćnih antituberkuloznih lekova. Trećina svetske populacije inficirana je bacilom tuberkuloze. Svake godine oboli oko osam miliona, a umre oko dva miliona ljudi, zbog čega je tuberkuloza i dalje infektivno oboljenje sa najvećom stopom smrtnosti. Kasna dijagnoza, multirezistentna tuberkuloza i udruženost sa HIV infekcijom predstavljaju jednu od najvećih prepreka za efikasnu kontrolu ove bolesti u svetu. Rano otkrivanje se oslanja na kvalitetnu bakteriolo&scaron;ku dijagnostiku koja je kamen temeljac svakog nacionalnog programa za kontrolu tuberkuloze. Brza i tačna mikrobiolo&scaron;ka dijagnostika predstavlja osnovu programa kontrole tuberkuloze i zbog toga je uvođenje novih i brzih laboratorijskih testova od veoma velikog značaja. Razvijen je novi komercijalno dostupni imunohromatografski test koji se zasniva na detekciji antigena MPT64 glavnog sekretovanog proteina M. tuberculosis. Test je brz i pouzdan u identifikaciji izolovanih sojeva M. tuberculosis i jeftiniji je od konvencionalnih biohemijskih i molekularnih testova. CILJ: Ciljevi istraživanja su bili da se evaluiraju karakteristike novog brzog imunohromatografskog testa u identifikaciji mikobakterija izolovanih iz respiratornih uzoraka bolesnika sa tuberkulozom pluća i referentnih sojeva klinički značajnih vrsta netuberkuloznih mikobakterija (NTM). MATERIJAL I METODE: Istraživanje je sprovedeno u periodu od 1.1.2010. do 31.12.2013. i obuhvatilo je 43563 respiratornih uzoraka dobijenih od bolesnika hospitalizovanih u Institutu za plućne bolesti Vojvodine. Iz obrađenih respiratornih uzoraka izolovano je 3469 izolata mikobakterija. Identifikacija do nivoa vrste urađena je primenom standardnih biohemijskih testova, molekularnog testa (GenoType&reg; Mycobacterium) i imunohromatografskog testa (BDMGIT Tbc). U istraživanje je uključeno 100 sojeva Gram pozitivnih i Gram negativnih bakterija (n = 19 vrsta) izolovanih iz respiratornih kliničkih uzoraka. Identifikacija do nivoa vrste je potvrđena komercijalnim identifikacionim sistemima. REZULTATI: U toku četvorogodi&scaron;njeg istraživanja izolovano je 3469 izolata mikobakterija iz respiratornih uzoraka. U ispitivanom periodu ne postoji opadajući trend izolacije mikobakterija &scaron;to potvrđuje i koeficijent korelacije (r = 0,31). Svi izolati mikobakterija su identifikovani konvencionalnim biohemijskim ispitivanjima koja pokazuju da je 89% od svih izolata identifikovano kao Mycobacterium tuberculosis (M. tuberculosis), a 11% izolata kao NTM. Mycobacterium xenopi je bila najzastupljenija NTM vrsta identifikovana kod 55,3% izolata. Nakon biohemijske identifikacije kod 300 izolata M. tuberculosis i 100 izolata NTM, identifikacija je potvrđena komercijalno dostupnim molekularnim i imunohromatografskim testom. Na osnovu rezultata testiranja mikobakterija imunohromatografskim testom, senzitivnost, specifičnost, pozitivne i negativne prediktivne vrednosti bile su: 99,7%, 100%, 100% i 99%. U poređenju imunohromatografskog testa sa konvencionalnim biohemijskim ispitivanjima nije nađena statistički značajna razlika (p&gt; 0,5). Kappa vrednost testa je iznosila 0,993, a interval poverenja CI =0,98 &ndash; 1,00. U poređenju imunohromatografskog sa molekularnim testom vrednost kappa je iznosila 0,993, a interval poverenja CI = 0,98 &ndash; 1,00. Slaganje rezultata je potvrđeno i McNemar testom sa vredno&scaron;ću 0,99. Utvrđena je stabilnost sekretovanog antigena MPT64 i posle 5 godina od prvog testiranja. ZAKLJUČAK: Visoka senzitivnost i specifičnost imunohromatografskog testa omogućuju tačnu i preciznu identifikaciju M. tuberculosis kao i pouzdanu diferencijaciju M.tuberculosis od NTM &ndash; a. Imunohromatografski test može da predstavlja zamenu za konvencionalne biohemijske i molekularne testove u identifikaciji M. tuberculosis. Jeftiniji je, jednostavniji za izvođenje i brže se dobijaju rezultati čime seskraćuje vreme za postavljanje dijagnoze.</p> / <p>INTRODUCTION: Tuberculosis (TB) has been known as a disease for a long time, but nevertheless it represents a major public health issue even nowadays in the 21st century, despite potent antituberculous drugs applied. One third of the world population is infected by the TB bacillus. About eight million people get infected and two million die of tuberculosis in a year, so tuberculosis is still an infectious disease with the greatest mortality rate. Late diagnosis, multiresistant tuberculosis and concomitant HIV infection interfere mostly with an efficient control of the disease all over the world. Early TB detection largely depends on the high-quality bacteriological diagnostics, which is the corner stone of each national TB control programme. A fast and accurate microbiological TB diagnosis plays a crucial role in any TB control programme. It is therefore very important to introduce new and fast laboratory tests. A novel commercially available immunochromatographic test has been designed, based on the MPT64 antigen of the major M. tuberculosis &ndash; secreted protein. This is a rapid and reliable test to identify the isolated strains of M. tuberculosis, which is not expensive as conventional biochemical and molecular tests. OBJECTIVE: The objective of the investigation was to evaluate the new immunochromatographic rapid test to identify mycobacteria isolated from respiratory samples from pulmonary TB patients, and referential strains of clinically relevant species of nontuberculous mycobacteria (NTM). MATERIAL AND METHODS: The research was carried out in the period from 1st January, 2010 to 31st December, 2013. It included 43 563 respiratory samples obtained from the patients hospitalized in the Institute for Pulmonary Diseases of Vojvodina, Sremska Kamenica (Serbia). There were 3 469 mycobacterial isolates obtained from the processed respiratory samples. The species &ndash; level identification was performed by standard biochemical tests, the molecular test (GenoType&reg;Mycobacterium), and the immunochromatographic test (BD MGIT Tbc). The study included one hundred (100) of Gram positive and Gram negative bacteria (n = 19 species) isolated from respiratory clinical samples. The species &ndash; level identification was confirmed by commercial identification systems. RESULTS: During the four &ndash; year investigation, 3 469 mycobacterial isolates were obtained from respiratory samples. No declining tendency of mycobacterial isolation was registered in the examined period, as confirmed by the correlation coefficient (r = 0.31). All mycobacterial isolates were identified by conventional biochemical tests showing that 89% of all isolates were identified as M. tuberculosis, and 11% of the isolates as NTM. Mycobacterium xenopi was the most common NTM species identified in 55.3% of the isolates. Following the biochemical identification in 300 M. tuberculosis isolates and 100 NTM isolates, the identification was confirmed by commercially available molecular and immunochromatographic tests. Based on immunochromatographic testing of mycobacteria, the sensitivity, specificity, positive and negative predictive values of the test were 99.7%, 100%, 100% and 99% respectively. There is no statistically significant difference (p&gt; 0.5) when comparing features of immunochromatographic test with conventional biochemical assay. The kappa test value was 0.993, and the confidence interval CI = 0.98 &ndash; 1.00. Comparing the immunochromatographic with the molecular test, the kappa value was 0.993, and the confidence interval CI = 0.98 &ndash; 1.00. The congruence of the tests findings was also confirmed by the McNemar test, estimated to 0.99. The stability of the secreted MPT64 antigen was registered even five years after the first testing episode. CONCLUSION: The high sensitivity and specificity of the imunochromatographic test enable an accurate and precise identification of M. tuberculosis, as well as a reliable differentiation of M. tuberculosis from NTM. The immunochromatographic test may substitute conventional biochemical and molecular tests to identify M. tuberculosis. It is easier to perform and provides faster test results, thus reducing the time of establishing the diagnosis.</p>