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Early and accurate diagnosis of tuberculosis is extremely important for its control. The classical staining and microbiological methods lack in accuracy and speed respectively. On the other hand, the accurate and faster advanced nucleic acid amplification (NAA) methods have their own limitations and the results need to be interpreted with much caution for therapy. To balance the requirements of accuracy and speed for aiding early and correct drug prescription to the patients, we used a combined classical and molecular biological approach. Using this approach, 244 pulmonary and extra-pulmonary clinical samples from tuberculosis suspects from the Central state of India were tested for the presence of M. tuberculosis. Of these, 84 samples were finally confirmed positive for M. tuberculosis. Most positive samples were from clinical suspects in the age group of 20-24 and 35-39. Analysis of the positive samples showed that the peak at 20-24 was dominated by male while 35-39 was dominated by female gender. We also found gender based difference in the proportion of some of the different type of positive samples. We present here, the status of tuberculosis infection in Central state of India in 2012-2014 tested in our lab.

Aim: Tuberculosis which is an infectious disease caused by M. tuberculosis causing pulmonary and extra pulmonary tuberculosis in clinical suspects in Indore and region around central state of India by IGRA methods. In India, TB which is declared to be ‘notifiable disease of the nation’ by the RNTCP since 2012. We wanted to analyze present state of existence of the same in our city Indore and region around. We wanted to study the present strata of IGRA detected in population of Indore in MP correlating with the Mycobacterium isolates in our laboratory, though the protocol of DOTS have been followed in the country. Experimental design: In present study, we tested clinical suspects using microbiology biology cultivation method and ELISA method for IGRA in Indore for Mycobacterium tuberculosis in the clinical suspects in our Indore lab, MP, India. Place and duration: The study was one in Central lab-Oncquest India Ltd. in Indore between period 2012 to 2014. Methodologies: The present study included 135 patients, including 49 male, and 86 female patients. We used developed TB-TMA method (Oncquest, Ltd.) to detect infection of clinical suspects and utilized culture susceptibility test to detect drug resistance in infecting Mycobacterium causing tuberculosis, tested at Central lab-Oncquest ltd. in India using microbiological methods. The method of drug resistance in Mycobacteria was performed using microbiology methods of drug resistance as described by Songara P, 2015. Results: We found 53% samples to be positive from male group compared to 29% from female group of patients. We could isolate Mycobacterium sp from various clinical suspects using basic microbiology and cultivation methods. Were found 41.6% Mycobacterium to be sensitive to sensitive to INH 36.65 to RIF, 23.3 to PYRA, 305 to ETHM, 25% to STREPTO isolated from various samples from clinical suspects. Conclusion: We were able to detect M. tuberculosis and determine their drug resistance in Mycobacterium method by MDR sure method.

Titelblatt Complete dissertation, Candida albicans is a pathogenic yeast responsible for causing infection in patients under immunosuppressive therapy, and is responsible for recurrent vulvovaginal candidiasis. Although, number of C. albicans derived intrinsic factors and the host factors have been found responsible for virulence and favouring the disease process, there is no antimycotic drug which proves out to be completely fungicidal, suppressing recurrent infections. This work deals with the study of Candida albicans-host cell interaction, to give an insight of the disease process, which could lead to new therapeutic interventions. Earlier studies on D. uninucleata revealed presence of a biologically active 3-HETE derived from exogenously fed Arachidonic acid. It was found, that it is not only important for its own life cycle, but also shows biological activity in human cells. We tried to find if a similar mechanism occurs in pathogenic fungi. It was found that although C. albicans does not contain arachidonic acid, it is able to grow on arachidonic acid as a sole carbon source as efficiently as it can utilise linoleic acid. Metabolism of arachidonic acid by C. albicans occurs both by mitochondrial dependent and independent pathways as shown by inhibitor studies. Arachidonic acid was shown to block the alternative oxidase pathway of energy generation in Candida albicans, and was efficiently metabolised to other fatty acids, carbohydrates, and proteins. Arachidonic acid metabolism by Candida albicans produced a novel compound 3,18-diHETE, which was closely related to previously discovered 3-HETE by Dr. S. Nigam's group. It was also observed that 3,18-diHETE was mainly associated with the cells bearing germ-tube and hyphael forms. This production was found to be aspirin sensitive. We found that C. albicans grown in aspirin showed diminished immunofluorescence with anti-3-OH-oxylipins antibody, indicating decreased amounts of 3,18 diHETE. Apart from this, there was diminished germ- tube formation, which is essential for infection, in C. albicans in presence of aspirin. Aspirin suppressed not only cell growth, but it also reduced the adhesion of C. albicans to the host cells. These observations prompted us to explore a therapeutic role for aspirin. When given along with clotrimazole, aspirin was found to reduce minimum inhibitory concentration (MIC) of clotrimazole against C. albicans. The action of aspirin was directed towards inhibiting 3-hydroxylation mechanism in C. albicans and of clotrimazole towards 18-hydroxylation mechanism and ergosterol synthesis, which occurs via cytochrome p450. This demonstrates a novel approach to potentiate the action of antimycotic drug clotrimazole. Moreover, aspirin is a cyclooxygenase-2 inhibitor, which inhibits formation of PGE2, and thus the infectivity of C. albicans. Our results thus showed that aspirin may be a suitable drug for the therapy of recurrent Candidiasis. It is known that host factors are also responsible for the establishment of infection. HDL, which is lowered in sepsis patients, and therefore substituted, was found to further increase virulence by inducing germtube formation in C. albicans. The first step of infection includes adhesion of pathogen to host cells. C. albicans which adheres to host cells rapidly is found to be more virulent, and is able to proceeds further to establish the infection process. Our results demonstrate that aspirin is able to decrease the adhesion of C. albicans to HeLa cell, thus decreasing its virulence. Upon infection with C. albicans signal transduction pathways are triggered in host cells (HeLa cells). We found upregulation of COX-2 in HeLa cells upon infection with C. albicans as well as increase in PGE2 production. Not only C. albicans but also 3-HETE, upregulated COX-2, showing that 3-HETE was important compound which mediated cell signaling during infection. Using specific chemical inhibitors to various signal transduction pathways, it was established that Protein Kinase C (PKC) and p38 MAP kinase pathway were primarily involved in the COX-2 upregulation. In other systems, involving cytokines and LPS, control of COX-2 transcription involved ERK1/2 and JNK MAP kinase pathways. Further studies were performed to evaluate the exact sequence of activation of kinases involved. GF 203190X (PKC inhibitor) prevented the phosphorylation of p38 MAP kinase, while other inhibitors failed to have any effect. Thus, PKC acts as an upstream factor phosphorylating p38 MAP kinase. C. albicans induced p38 MAP kinase was also observed to be involved in modulating cytoskeletol changes host cell. This effect involved the activation, of phosphorylated HSP27, a factor involved in the modulation of actin, which was abrogated by p38 MAP kinase inhibitor SB 202190.We find that inhibition of free radicals, NAC inhibits COX-2 and PGE2 upregulation suggesting involvement of reactive oxygen species and NFkB during the infection of HeLa cells with C. albicans. The control of transcription occur by the various transcription factors binding to the concerned gene promoter. NFkB is one of the important transcription factors implicated in the COX-2 transcription. The role of NFkB was studied using a NFkB-dependent reporter plasmid and a dominant negative plasmid for IkB (prevents the release of active NFkB). NFkB-dependent transcription was triggered upon infection with C albicans. However, this could be only partially abrogated by the IkB dominant negative plasmid, moreover PI3-kinase pathway is involved in COX-2 upregulation via NFkB pathway. Thus C. albicans brings about redundant signalling for the upregulation and control of COX-2 transcription. Extensive programmed cell death (apoptosis) was observed after 24 h in Hela cells infected with C. albicans as shown by genomic DNA laddering and TUNEL assay. After 6 h postinfection, upregulation of caspase-3 activity was observed. Caspase-3 is an effector caspase which is involved in the cleavage of cellular proteins during apoptosis. Further more, using DNA microarray technology, we showed that C. albicans upregulated PI-3-kinase in HeLa cells. PI-3-kinase is a prominent mediator of anti-apoptotic functions via AKT and p65 NFkB. Its inhibition by wortmannin increased the caspase-3 activity in infected cells. IkB dominant negative transfected cells showed no caspase-3 activity upon infection. This result was surprising as p65 NFkB, whose release is prevented by the dominant negative plasmid, is an anti-apoptotic molecule. This paradoxical reaction was clarified by the observation of truncated forms of p65 NFkB in C. albicans infected HeLa cells. Carboxy terminal truncation of p65 NFkB by caspase-3 renders the molecule inactive thereby preventing its anti-apoptotic function. The pro-apoptotic subunits of NFkB, p50 and c-REL, however, were upregulated in C. albicans infected cells. Thus, the anti- apoptotic pathway, from PI-3-kinase via AKT to p65 NFkB is rendered ineffective by the cleavage of p65 NFkB by caspase-3., 3(R)-Hydroxyoxylipine sind neue Oxygenierungsprodukte von Polyenfettsäuren, die gemeinsam von den Arbeitsgruppen in Berlin (AG Nigam) und Bloemfontein, Südafrika (AG Kock) in der Hefe Dipodascopsis uninucleata entdeckt wurden. Darüber hinaus konnte mittelsImmunfluoreszenzmikroskopie gezeigt werden, daß die entsprechenden Arachidonsäure- und Linolsäuremetaboliten auch in denhumanpathogenen Pilzen, wie Candida albicans vorkommen. In C. albicans wurden 3-Hydroxyoxylipine in den filamentären Strukturformen undKeimschläuchen identifiziert, nicht aber in freien Blastosporen. Vorbehandlung der Pilzkulturen mit Aspirin ließ die 3-Hydroxyoxylipineweitgehend verschwinden. Aspirin bewirkte ebenfalls eine starke Hemmung des Pilzwachstum und zugleich eine Verringerung des Anteilsfilamentärer Strukturelemente. In der vorliegenden Arbeit sind Untersuchungen zur Struktur, Biosynthese und biologischen Funktionen der 3(R)-Hydroxyoxylipine auf 3 verschiedenen Ebenen durchgeführt: auf der Ebene des Pilzstoffwechsels, auf der Ebene der Pathogen- Wirtszell-Interaktion sowie auf der Ebene biologischer Wirkung von C. albicans auf intrazelluläre Signalkette. Desweiteren wurden biologische Wirkungen der bereits von uns isolierten undidentifizierten 3-Hydroxyoxylipinen durch die Transformation von Arachidonsäure auf mammaliäre Zellen und die Funktion dieser Oxylipine für die Morphogenese und Pathogenität von Candida im Hinblick auf ihre mögliche Rolle beim Infektionsprozess untersucht. Da Aspirin sowohl über die Hemmung der Cyclooxygenase in den Wirtszellen als auch der 3-Hydroxyoxylipine beim Pathogen in die Pathogen-Wirtszell-Interaktion eingreift,verspricht die Aspirintherapie auch Erfolg bei der Behandlung resistenter Candida-Stämme.Hinsichtlich der Ergebnisse dieser Untersuchungen sind einige Neuheiten hervorzuheben: (i) C. albicans kann allein auf Arachidonsäure als Substrat gedeihen. Dabei werden primär über "Glyoxalat- Shunt" Kohlenhydrate produziert. Dieser Mechanismus deutet auf die erhöhte Virulenz beteiligter Gene hin. (ii) die Produktion von einem neuartigem, dioxygeniertem Eikosanoid 3,18-DiHETE wurde für die Bildung von "germ-tube" und Hyphen verantwortlich gemacht. (iii) Clomatrizol, das meist- benutzte Mittel gegen vulvovaginale Kandidose, zusammen mit Aspirin wurde als das wirkungsvollste Mittel gegen Kandidose nachgewiesen. (iv) C. albicans hat selektiv COX-2 in HeLa Zellen hochreguliert. Diese Hochregulierung wurde mit der erhöhten Synthese von Prostaglandin E2 begleitet. Prostaglandin E2 wird u.a. für die morphogenetische Veränderung in C. albicans verantwortlich gemacht, und somit für die Infektivität. (v) Die Hochregulierung von COX-2 erfolgt über verschiedene Signaltransduktionswege, wieTyrosinkinase, Proteinkinase C, p38MAP Kinase oder via NFkappaB. NFkappaB und p38MAPK spielen dabei Schlüsselrollen. Dagegen sind andere Stresskinasen, wie ERK1/2, JNK, SAPK offensichtlich nicht beteiligt. (vi) C. albicans triggert auch Apoptose via NFkappaB und/oderPI-3-Kinase in HeLa Zellen. Dabei wechselt NFkappaB ihre anti-apoptotische Wirkung während der Reaktion in eine proapototische Wirkung. Aus den Ergebnissen dieser Arbeit werden neue Aspekte der Regulation des Wachstums und der Morphogenese von 3-hydroxyoxylipin-produzierenden Pilzen sowie neue Ansatzpunkte zur Behandlung von Pilzerkrankungen bei Menschen und Nutzpflanzen erwartet.