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Τα τελευταία χρόνια παρατηρείται παγκοσμίως επιδείνωση των επιδη­μιο­λογικών δεικτών της φυματίωσης με αποτέλεσμα την αναζοπύρωση του διε­θνούς ενδιαφέροντος έρευνας που αφορά τη διακοπή μετάδοσης της αλυσί­δας των μυκοβακτηριδιακών λοιμώξεων. Κύριο στοιχείο ελέγχου και περιστολής της νόσου αποτελεί η αναζήτηση και θεραπεία των πασχόντων με θετικά πτύελα έτσι ώστε να καταστεί δυνατή η διακοπή μετάδο­σης των μυκοβακτη­ριδίων. Τα μικρά ποσοστά της άμεσης μικροβιο­λο­γι­κής επιβεβαίω­σης μιας κλινικής διάγνωσης, η καθυστέρηση των καλλιεργειών λόγω μεγάλου χρόνου επώασης καθώς και το χρονοβόρο και ανεπαρκές των κλασσικών μεθόδων ελέγχου ευαισθησίας στα διάφορα αντιφυματικά φάρμα­κα επιβάλ­λουν συνεχή έρευνα προς ανεύρεση γρήγορων και ευαίσθητων διαγνωστικών μεθόδων. Στην παρούσα μελέτη μελετήθηκαν 3.500 δείγματα προερχόμενα από ασθε­νείς της πενταετίας 1999-2003 που προσήλθαν στο Πανεπιστημιακό Γενικό Νοσοκομείο Πατρών καθώς και στο Ειδικό Νοσοκομείο Νοσημάτων Θώ­­ρακος Δυτικής Ελλάδος με σκοπό την ανεύρεση μυκοβακτηριδίων (Myco­bacterium spp.). Η μελέτη περιελάμ­βα­νε: α) άμεσο έλεγχο σε παρασκεύα­σμα μετά από οξεάντο­χη χρώση Ziehl-Neelsen (ΖΝ) για ανεύρεση οξεά­ντοχων και αλκοολάντοχων βακτηρίων (Acid Fast Bacteria –AFB) σε ποι­κίλα δείγματα του αναπνευ­στικού, αλλά και από άλλα στείρα υγρά σώ­μα­τος, και β) τη μο­ριακή μέθοδο COBAS AMPLICOR αλυσιδωτή αντίδραση πολυμεράσης (CA PCR). Η ευαισθησία, η ειδικότητα, η θετική και αρνητική προγνωστική αξία της CA PCR καθο­ρίστηκε μεταξύ AFB-θετικών και AFB-αρνητικών δειγ­μάτων. Υψηλή ευαισθησία της CA PCR παρατηρήθηκε μεταξύ όλων των AFB-θετικών δειγμάτων, ενώ τα δείγματα πτυέλων που ελήφθησαν μετά από βρογχοσκό­πη­ση (ΜΤΒ-πτύελα) θεωρήθηκαν τα πλέον κατάλληλα δείγματα με ευαισθη­σία 70% και ειδικότητα 98,6% μεταξύ των AFB-αρνητικών δειγμάτων. Η αύξηση τα τελευταία χρόνια της ανθεκτικότητας στελεχών του Μ. tuberculosis καθιστά αναγκαία την καλλιέργεια και την ευαισθησία στα αντιφυ­μα­­τικά φάρ­μακα. Για το σκοπό αυτό, μετά από ειδική επεξεργασία των κλινι­κών δειγ­μά­των (ρευστοποίηση και εμπλουτισμό) σύμφωνα με το πρωτόκολλο της εται­ρείας Becton Dickinson BBL MycoPrep, ακολούθησε καλλιέργεια η οποία έγινε ταυτόχρονα με δυο μεθόδους: α) σε στερεό θρεπτικό υλικό Löwentein-Jensen (L-J BioMerieux, SA Lyon, France), και β) σε φιαλίδια BACTEC MYCO/F Becton-Dickinson για γρήγορη ανί­χνευση με το αυτόματο σύ­στημα BACTEC 9000 ΜΒ. Θετικές καλλιέργειες προερχόμενες και από τις δύο μεθό­δους επιβεβαιώθηκαν με τρεις τρόπους: α) χρώση (Ζ-Ν) του παρασκευά­σματος για οξεάντοχα και αλκοολάντοχα βακτηρίδια (AFB) β) ανακαλλιέργεια σε L-J και γ) CA PCR. Ακολούθησε ταυτο­ποίηση ειδών Mycobacterium spp: α) με βιο­χημικές δοκιμασίες (παραγωγή νιασίνης – παραγωγή θερμοανθεκ­τι­κής καταλάβης), και β) με μεθόδους μοριακής βιολογίας (PCR και υβριδισμό Genotype Mycobacteria). Σε 88 ΜΤΒ απομονωθέντα στελέχη από διαφο­ρε­τι­κούς ασθενείς τα οποία ταυτοποιήθηκαν με PCR και υβρι­δι­σμό, εφαρμό­στηκε δοκιμασία ευαισθησίας στα τέσσερα πρώτης επιλο­γής αντιφυματικά φάρμακα (INH, RIF, STR, EMB) με τρεις διαφορετικές μη αυτόματες μεθόδους: τη μέθοδο αναλογιών σε άγαρ (ΜΟΡ), την MGIT και το E-test. Καλή συμφωνία αποτελεσμάτων σημειώθηκε μεταξύ E-test και ΜΟΡ για την ΙΝΗ και RIF, ενώ μεταξύ των μεθόδων MGIT και ΜΟΡ παρατηρήθηκε καλή συμφωνία για την ΙΝΗ, RIF και STR. Έτσι, ενώ η ΜΟΡ παραμένει μέθοδος εκλογής, αν και χρο­νο­βόρα, εν τούτοις η MGIT παρουσιάζει μεγάλη ευαισθησία και είναι τα­χύ­τερη. Η εφαρμογή νέων αξιόπιστων τεχνικών μειώνει εντυπωσιακά το χρό­­νο έκδοσης αποτελεσμάτων όσον αφορά τις λοιμώξεις από Mycobacterium spp., την ταυτοποίηση και τον έλεγχο αντοχής στα αντιφυ­μα­τικά φάρμακα έτσι ώστε η πρόγνωση της νόσου όσον αφορά την εργα­στη­ριακή διάγνωση της φυμα­τίω­σης και την παρακολούθηση της θεραπείας βελτιώνεται σημαντικά. / Re-emergence of tuberculosis in combination with the appearance of multidrug-resistant strains in recent yearsintensifies the need for application of rapid methods for the identification of mycobacteria. Even though staining for acid-fast bacilli (AFB) to identify Mycobacterium tuberculosis complex (MTB) is usually performed in the first 24 h of specimen receipt, it is considered a method of low sensitivity. Although time consuming, the standard detection method remains the culture of specimens with optimum results obtained after application of both solid and liquid media.The use of nucleic acid amplification techniques provides a sensitive and specific approach for the identification of MTB directly in clinical specimens. The Cobas Amplicor polymerase chain reaction for MTB (CA PCR, Roche Diagnostic Systems, Inc., Branchburg, N.J., USA) is a system that combines specimen processing with automated amplification and detection, easily adopted by a clinical laboratory. In the present study, the sensitivity, specificity, positive and negative predictive values of CA PCR and culture results of AFB-positive and AFB-negative respiratory specimens obtained by different means (expectoration, bronchoscopy, expectoration after broncho­scopy, lavages and aspirations), as well as in samples from normally sterile body fluids, were evaluated. A total of 3414 specimens from 2365 patients (1 to 3 specimens/patient) with clinical suspicion of tuberculosis were processed during the study period (1999-2003) by the Microbiology Laboratory at the University Hospital of Patras. The respiratory specimens consisted of 684 sputa, 1473 bronchoalveolar lavages (BAL), 625 sputa expectorated after bronchoscopy (SAB), 296 tracheal aspirations (TA) and 189 pleural fluids. Furthermore,23 gastric aspirates and 124 samples ofsynovial, pericar­dial, peritoneal and cerebrospinal fluids (CSF) were also examined in the present study. All specimens were processed according to conventional procedures for identification of mycobacteria; cultures were performed by inoculation of sediments directly on Löwenstein-Jensen slants (L-J, bioMerieux, SA Lyon, France) and in BACTEC MYCO/F-Sputa and BACTEC MYCO/F LYTIC culture vials (Becton Dickinson). Statistical analysis was conducted using the SPSS v.12.0 software package for Windows (SPSS Inc.). Comparison of Z-N and PCR results were expressed by means of percent agreement and Kappa statistic. Highest sensitivity of CA PCR was observed among all positive AFB samples, whereas sputa collected after bronchoscopy were the most appropriate specimens, showing 70% sensitivity and 98.6% specificity, among the AFB-negative samples. The increase of M. tuberculosis infections is a global health problem in terms of both the disease and resistance to commonly used drugs. For the reason of continuing develop­ment of resistance and especially multidrug-resistance of M. tuberculosis (MDR), Microbiology Laboratories should provide reliable antibiotic suscep­tibility testing results in the minimum of time. In the present study, antimy­co­bacterial drug susceptibility testing (AST) was performed on 88 non-replicate M. tuberculosis clinical isolates, using the Mycobacterium Growth Indicator Tube (MGIT) System and Etest as compared to the method of proportion (MOP), in order to evaluate the potential application of these manual methods in the routine diagnostic laboratory. Obtained results among four antituberculous agents, isoniazid (INH), rifampin (RIF), ethambutol (EMB) and streptomycin (STR) were compared. Isolates were recovered from different patients and were identified at species level by PCR and hybridization. Resistance to INH was detected in 20.5%, 29.5% and 12.5% of the isolates, followed by STR resistance (19.3% 26.1% and 1.1%), RIF (9.1%, 4.5% and 5.7%) and EMB (2.3%, 11.4% and 2.3%) as showed by the MOP, MGIT and Etest, respe­ctively. Sensitivity of the manual MGIT ranged from 37.5% for RIF resistance to 100% for EMB, while sensitivity of Etest ranged from 5.9% for STR to 62.5% for RIF. In conclusion, whilesputum samples and BAL are the preferable clinical specimens for MTB recovery, sputa expectorated after bronchoscop are those showing the highest sensitivity by PCR, both among Z-N-positive and negative specimens. The combination of Z-N and CA PCR including internal control of collected after bronchoscopy from a patient with clinical signs of infectionundoubtedly contribute to the early diagnosis of tuberculosis. According to our data, the sensitivity of the manual MGIT is higher in testing resistant isolates compared to Etest, with the exception of rifampin. On the other hand, Etest shows higher specificity as well as higher positive predictive values and it may be used for testing rifampin resistance. However, for a routine Clinical Microbiology Laboratory, among the manual methods tested, the method of proportion remains the “gold standard” even though a longer incubation period is needed.

The eis gene of M. tuberculosis is believed to play a role in the intracellular survival of this pathogen. Significantly higher levels of antibodies to Eis were detected by ELISA in the sera of patients with tuberculosis as compared to healthy controls. PBMCs from recovered TB donors were also found to demonstrate significantly higher levels of proliferation in response to stimulation with the Eis protein than PBMCs from either active TB cases or healthy controls. Neither the active TB population nor the healthy controls showed significant levels of IFN- or IL-4 secretion in response to stimulation of PBMC with Eis or ESAT-6. Far Western analysis determined that Eis interacts with a ~65 kDa protein that localizes to the cytoplasmic fraction of M. tuberculosis lysate. Real-time PCR analysis of M. tuberculosis infected U-937 macrophages showed that the eis gene is constitutively expressed during infection. Using immunofluorescence microscopy (IF), the Eis protein was detected within the cytoplasm of M. tuberculosis infected macrophages indicating that the protein was being released/secreted from the mycobacterium containing phagosomes. Western blot analysis of the cytoplasm of macrophages infected with M. tuberculosis expressing green fluorescent protein confirmed these results. Western blot analysis also detected the presence of native Eis both in the culture supernatant of infected macrophages and vesicles released from the macrophages. IF also detected the presence of Eis in uninfected macrophages. The Eis protein in the cytoplasm of M. tuberculosis infected macrophages was also found to colocalize with EEA1, an endosomal marker, indicating a possible association of the protein with early endosomes. Eis was also shown to elicit higher levels of IL-10 secretion than PPD in human monocytes. Infection of monocytes from healthy tuberculin reactors with M. tuberculosis wild type and eis mutant demonstrated that eis plays a role in modulation of IL-10/TNF- secretion in response to infection. Bioinformatic analysis of the amino acid sequence of Eis indicates that Eis is an acetyltransferase of the GCN5 related family of N-acetyltransferases. Further work is required to determine the role Eis plays in the survival of M. tuberculosis within the macrophage.

The microbial degradation of pyrene, a 4-ring polycyclic aromatic hydrocarbon (PAH), has been elucidated with the increasing number of pyrene-degrading bacteria that have been isolated in recent years. A pyrene degrading bacterium identified as Mycobacterium sp. strain S65, was isolated from a jet-fuel contaminated site in Sept-Iles, northern Quebec, Canada. S65 utilized pyrene, phenanthrene, and fluoranthene as sole carbon and energy sources, but did not degrade naphthalene, anthracene, and fluorene. Pyrene mineralization was enhanced by adding benz[a]anthracene, benzy[a]pyrene, or phenanthrene as cosubstrates. When added to PAH contaminated soil as a potential bioaugmentation agent, S65 did not appear to survive well, nor was it effective at degrading PAHs under these conditions. / Pyrene catabolic genes in S65 were partially characterized by Southern hybridization using a probe constructed from the naphthalene inducible pyrene dioxygenase gene, nidA, from the pyrene-degrading bacterium, Mycobacterium sp. strain PYR-1.

Crohn’s disease (CD) is a chronic inflammatory bowel disease, primarily affecting the young, which causes marked morbidity and reduced quality of life. Currently there is no cure for CD, and the causes of this disease are poorly understood. In ruminants, Johne’s disease (JD) is characterised by chronic intestinal inflammation similar to CD and is caused by the pathogen Mycobacterium avium subspecies paratuberculosis (MAP), which invades and replicates within the phagocytes of infected animals, leading to chronic disease. There is increasing molecular and microbiological evidence of Map bacteria in CD patients. However, little is known regarding the role of Map in the aetiology of CD. This thesis demonstrated that a human isolate of Map traffics through THP-1 human monocytes via a similar path to that taken by pathogenic mycobacteria. Flow cytometry demonstrated that Map are phagocytosed via a cholesterol-dependant mechanism, potentially mediated by a cell wall constituent. Once internalised, live Map reside in cholesterol-rich areas of the cell. These compartments exhibit reduced acidity compared to the compartments containing killed-Map, and have atypical retention of markers including the late endosomal marker Rab 7 and cellular TACO protein. Both of these markers were also present on phagosomes of pathogenic mycobacteria, where they interrupt fusion of the compartment with lysosomes. This was confirmed by visualisation of these proteins on phagosomes containing M. bovis,a known mycobacterial pathogen. Cholesterol depletion using simvastatin affected Map persistence in THP-1 cells at 1 and 2 weeks post infection, a finding similar to other studies with M. tuberculosis. Spheroplast-like forms were evident after long term culture of Map with THP-1 monocytes, visualised by light and electron microscopy. These were similar to forms observed in peripheral blood leukocytes from a CD patient. Collectively, these results support the hypothesis that Map may be involved in the aetiology of at least a subset of CD cases.

Tuberculosis (TB) is one of the most prevalent infectious diseases worldwide and causes high morbidity and mortality, despite the widespread availability of effective antibiotics against most strains of Mycobacterium tuberculosis, which is the causative agent of TB. One of the primary reasons that hinder TB control is that many cases of active disease go undetected or are discovered late. This is, in large part, due to the relative insensitivity and limited specificity, amongst other limitations, of the current TB diagnostics tests. Moreover, M. tuberculosis infection can be asymptomatic and latent, or cause active disease. Therefore, an ideal or effective TB diagnostic needs to distinguish between these two states. The aim of this study was to develop novel diagnostic reagents for M. tuberculosis using phage displayed peptides and nucleic acid aptamers with a view to discerning latent from active TB. Using a linear (X12) and constrained (CX7C) phage display libraries, five rounds of selection (biopanning) were performed. Ten phage displayed peptides that bind to the mycobacteria surface were selected. These phage clones were identified using both random clone picking and high throughput (HTP) sequencing. A phage clone displaying the CPLHARLPC peptide was identified by HTP sequencing as the most enriched, representing 82.49% of the selected CX7C phage population. Further characterization showed that it bound better to different mycobacteria species, including M. tuberculosis, than the unselected phage library. Moreover, using surface plasmon resonance (SPR) technology, the chemically synthesised CPLHARLPC peptide was shown to bind M. tuberculosis H37Rv whole cell lysate and not non-mycobacteria lysates. In addition, using the systematic evolution of ligands by exponential enrichment (SELEX) protocol and SPR technology, 2'-Fluoro-pyrimidine-RNA aptamers were selected against the mycobacteria ESX-3 secreted protein, ESX-G. At least five aptamers were identified after five rounds of selection. Two of these aptamers, GH43 and GH78, not only bound EsxG with high affinities, KD 8.04 ± 1.90 nM and KD 78.85 ±9.40 nM respectively, but also preferentially bound EsxG better than the EsxA homologue. Taken together, these findings suggest that a combination of phage display, SELEX and HTP sequencing can be a useful tool for the identification of specific detection reagents that can bind to mycobacteria and its associated targets. These reagents could be exploited to develop alternative molecular probes for TB diagnostics. / Thesis (Ph.D.)-University of KwaZulu-Natal, Durban, 2013.

Graduation date: 2005

Thesis (MScMed)--University of Stellenbosch, 2006. / Bibliography. Also available via the Internet.

Proefschrift Universiteit van Amsterdam. / Met bibliogr., lit. opg. - Met samenvatting in het Nederlands.

Thesis (Ph. D.)--Cornell University, May, 2008. / Vita. Includes bibliographical references (leaves 205-233).