A research report submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in partial fulfilment of the requirements for the degree of Master of Medicine in the branch of Haematology. Johannesburg, 2015. / Introduction: The Gene Xpert MTB/Rif assay (Xpert) is a nucleic acid amplification technique that has been studied in the diagnosis of both pulmonary and, to a lesser extent, extrapulmonary tuberculosis (TB). This study was performed in the National Health Laboratory Services (NHLS) laboratory at Charlotte Maxeke Hospital which services a population with a high prevalence of Mycobacterium Tuberculosis Complex (MTBC) infection. The study aimed to develop a protocol for the processing of pleural and ascitic fluid samples to be run on Xpert for MTBC diagnosis, to evaluate the sensitivity and specificity of the Xpert assay as compared to the gold standard MTBC culture assays and to assess the utility of the Xpert assay as part of the diagnostic algorithm for fluid samples received in high prevalence MTBC laboratories. Materials and methods: A total of 392 pleural and ascitic fluid specimens were received for routine immunophenotypic analysis between August 2012 and February 2013 at the NHLS flow cytometry laboratory in Charlotte Maxeke hospital. Of these specimens, 229 had sufficient residual volume (>0.5ml) after routine immunophenotypic analysis to be tested on Xpert. Specimens were processed as per the manufacturer’s guidelines for pulmonary specimens and results were compared to the gold standard culture for Mycobacterium tuberculosis. Results: Xpert positivity was detected in 8.7% (20/229) of the total specimens. Only 43% (99/229) of these specimens were submitted for concurrent MTBC liquid culture (Mycobacterium Growth Indicator tube, MGIT) testing based on the laboratory information system history. Positivity on Xpert was shown in 9% (9/99) of specimens compared to 17% (17/99) on MGIT. One false positive was detected on Xpert. More than half of the specimens, 57% (130/229) were not referred for concurrent MTBC culture. The Xpert detected MTBC in 8.5% (11/130) of these specimens, with 1 Rifampicin resistant case identified. Xpert sensitivity and specificity in this study were 50% (CI:26-75%) and 99% (CI:91-100%) respectively Conclusion: The sensitivity and specificity of Xpert in this study was comparable to that found in other studies performed on fluid samples. Importantly, this study demonstrates that in a high burden HIV/TB setting like South Africa, more than 50% of fluid specimens referred for immunophenotypic analysis to exclude lymphoma are not referred for concurrent MTBC culture testing. Incorporation of Xpert into the laboratory diagnostic algorithm (LDA) in the immunophenotypic laboratory would, therefore, have a number of benefits, improving overall patient work-up and care. Implementation and policy uptake, however, would require a full costing analysis as Xpert testing would be performed in addition to, and not instead of, routine testing.

Thesis (M. Med. Sc.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 50-57).

Thesis (M.Med.Sc.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 50-57). Also available in print.

A dissertation submitted to the Faculty of Health Sciences, university of the witwatersrand, in fulfilment ofthe requirements for the degree of Master of Medicine (paediatrics) Johannesburg, 20 I 6 / Background: Though features of infants with congenital tubercuiosis (TB) are known including being low-birth-weight (LBw), features of in-utero TB-exposed infants including non-infected are not well reported. Infants bom to TB-infected women are at risk of contracting TB post-delivery, therefore chemoprophylaxis is recommended, and this includes use of isoniazid and rifampicin combination, but littie is known about its effectiveness. Objective: To determine features of in-utero TB-exposed infants and proportion with TB after chemoprophylaxis with isoniazid and rifampicin. Methods: Retrospective review of records of TB-infected women and their infants, from ZA07-20rc. Clinical features of mothers and infants at time of delivery; and follow-up of infants after completion of isoniazid and rifampicin are described. Results: Eighty-eight infants bom to 86 women with a diagnosis of TB were studied. TB diagnosis was made peripartum in24.4Yoof women, 23.3%had exka-pulmonary TB. Among those diagnosed antepa$um 46.2o/owere on treatment for >2 months. Human immunodeficiency virus (HIV) was positive in 97.7Yo;wi& CD4 count <200 cells/mm3 in 74'6yo' Eight mothers (9.3%) died before discharge. There were 56 {63.6%)LBW and 45 (51'2W preterun infants. Culture for acid-fast-bacilli was positive in 4 (4.5%)infants. At 3- months follow-up, 17 (20.2%)defaulted, and among 67 who returned, 7 OAoA)did not return for Mantoux test reading, 1160 (1.7%)had positive Manroux. Conclusion: Majority of TB-exposed infants are born to mothers with TB/ HIV co-infection. A high proportion of TB-exposed infants are born preterm and LBW. The high attrition rate made it difficult to assess effectiveness of chemoprophylaxis with isoniazid and rifampicin / MT2016

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Tuberculosis (TB) is a highly contagious disease caused by the bacterium known as Mycobacterium tuberculosis which is widely spread in South Africa, especially in the rural areas of the Western Province. Rifampicin, isoniazid and pyrazinamide are the three most effective drugs against this organism. However, most of the current commercial anti-TB formulations are inconvenient to administrate. This results in patient non-compliance which has increased with incomplete tuberculosis treatment and further has intensified the mortality rate. The matter is especially severe amongst the paediatric and geriatric patients. Therefore, creating a "user-friendly" but non-alcoholic liquid formulation should improve the whole situation. The key to a successful formulation relies on sufficient concentrations of the drugs within the formulation together with acceptable stability of these drugs. Therefore, during the pre-formulation stage, the solubility and stability studies of rifampicin, isoniazid and pyrazinamide are to be conducted. Rifampicin, isoniazid and pyrazinamide were fully characterized and identified by means of spectroscopic and thermal techniques. A HPLC method for simultaneous analysis of the three drugs was developed and validated. This HPLC method was employed for all the solubility and stability assessments. Unbuffered HPLC water of pH value 7.01 was chosen as the aqueous solvent. This was decided after the stability of rifampicin, isoniazid and pyrazinamide was studied at a pH range of 2 to 10. The solubility and the stability studies of rifampicin, isoniazid, pyrazinamide, rifampicin with isoniazid, rifampicin with pyrazinamide, isoniazid with pyrazinamide and rifampicin combined with both isoniazid and pyrazinamide were performed in the presence of various agents. These agents can be categorized into three groups: the surfactants (poloxamer 188, poloxamer 407 and sorbitol) which could increase the intrinsic solubility or the drugs by altering the surface tensions of the aqueous solution medium, the suspending agents (carbopol 934 and carbopol 974P) which could enable the amount of dosage required to be homogeneously suspended in the formulation without considering the low intrinsic solubility factor of the drugs, and the complexing agents (ß-cyclodextrin, hydroxypropyl-ß-cyclodextrin and -cyclodextrin) which could initiated the inclusion complex between the host cyclodextrin and the drugs, thus further enhance the solubility of the drugs . The stability assessments were performed after 7-days stability trail with the HPLC method developed. Each drug/combination of drugs were stored in closed ampoules and subjected to 25, 40 and 60° C with or without nitrogen flushing while in the presence of the above mentioned agents. While assessing the solubility/stability of the drugs in the presence of the above mentioned surfactants, the phase-solubility curves indicate that both rifampicin and pyrazinamide fail to achieve the desired concentration. Moreover, the stability-time plots clearly indicate that these surfactants fail to enhance the general stabilities of the drugs. When the stabilizing effects of the above mentioned suspending agents were investigated, it was found that although the desired concentration could be easily accomplished by suspending the drug in the aqueous carbopol solutions, the stabilities of the different drug combinations were still below the regulatory level. Cyclodextrins are well known to form inclusion complexes with less polar drug molecules. The inclusion complexation could enhance both the solubility and the stability of the included drug molecules. The computer force field generated models of the cyclodextrin-drug were used to predict the complexation sites. The results indicated the all the inclusion complexation between the drugs and the cyclodextrins were favourable, but do not necessary protect the potential degradation sites of the drugs. The stability results confirmed the above findings as the cyclodextrins did not enhance the stability of the drugs. Various drug-drug interaction pathways were also predicted from the experimental observations which clearly indicated the stability reductions of these drugs in combination. This leads to the conclusion that a liquid formulation combining rifampicin, isoniazid and pyrazinamide should not initiate the use of aqueous solutions as the protic ions of the solution are able to initiate the degradation of these drugs.

Study objective: The standard daily dose of rifampin in directly observed treatment of Mycobacterium tuberculosis (TB) is 600 mg, taken orally. The purpose of this study was to assess the efficacy of standard dose rifampin therapy in patients who were slow to respond to routine directly observed therapy (DOT). Methods: Patients with non-drug-resistant pulmonary TB who were receiving 600 mg of oral rifampin by DOT were eligible for inclusion. Patients were deemed slow to respond if their sputum smears and cultures remained positive for M tuberculosis and if the patient’s condition did not improve clinically or radiographically after 3 months of treatment. Serum rifampin levels were ascertained to determine the adequacy of the standard rifampin dosing. Patients with subtherapeutic blood levels had their rifampin dose increased to 900 mg, and rifampin levels were repeated. Rifampin dosage was increased again if blood levels were still subtherapeutic. No antitubercular medications were added to the treatment regimen. The total weekly dose of the other standard treatment drugs was not increased. Results: Of 124 new patients with active pulmonary TB, 6 patients were identified as slow to respond to the standard antitubercular DOT. All six patients had subtherapeutic serum rifampin levels. All six patients responded clinically, radiographically, and mycobacteriologically after an increase in rifampin dosage to reach target drug blood level. Conclusions: Standard dosing with rifampin resulted in a poor clinical response and subtherapeutic serum levels in six patients. Increasing the dosage of rifampin improved the outcome without additional side effects. In TB patients who are slow to respond to standard treatment, an inadequate dose of rifampin should be suspected. Current antituberculer drug administration does not include adjusted dosage for rifampin.

<p> The ansamycin antibiotics are unique antibacterial agents that inhibit bacterial DNAdependent RNA polymerase II. Clinical use of this class of antibiotics has primarily been focused on the treatment oftuberculosis using the semi-synthetic rifamycin derivative, rifampin. As drug resistance among different classes of antibiotics continues to rise, there is increased interest in new applications ofrifamycins for diseases other than tuberculosis. Clinical resistance to rifampin has largely been the result of point mutations in the target, RpoB, however chromosomal and transposon mediated enzyme-associated resistance is well documented. As rifamycin antibiotic use becomes more widespread, enzymatic resistance will inevitably become more prevalent. Here we describe the characterization of one of the principle enzymes associated with rifampin inactivation, the rifampin ADP-ribosyl transferase enzyme (ARR). Two chromosomally encoded ARR enzymes from MYcobacterium smegmatis, and Streptomyces coelicolor, and the Tn-encoded ARR-2, widely distributed in Gram negative pathogens, were overexpressed and characterized. These enzymes exhibit comparable, substrate specific steady state kinetic features, and substrate-induced conformational changes that suggest ARR enzymes may demonstrate a preferred order of substrate binding. To gain further insight into the interaction between ARR enzymes and rifampin and NAD+, the three-dimensional crystal structure of ARR from M smegmatis was solved in complex with rifampin. Based on the threedimensional structure of ARRm, an SNl type reaction has been predicted for rifampin ADPribosyl transferase enzymes. This is the first detailed examination of these novel antibioticmodifying enzymes, relevant to their increased use in the clinic. </p> / Thesis / Master of Science (MSc)

Rifampicin is one of the major antibiotics used in the treatment of Mycobacterium tuberculosis. This organism causes tuberculosis. Other related nocardioform bacteria which include the Rhodococci are opportunistic pathogens in AIDS patients. These organisms cause tuberculosis-like disease and are currently treated with rifampicin and other drugs. The presence of a low level rifampicin resistance mechanism was identified in seven rhodococcal strains and five other related and unrelated bacteria. Abbreviated Abstract. Open document to view full version] / GR2017

Thesis (Ph. D.)--University of Washington, 1999. / Vita. Includes bibliographical references (leaves 298-336).