No description available.

The aim of this project was to identify growth phase-specific heat shock proteins of Mycobacterium smegmatis LR222. A growth curve was constructed using the ATP assay. This method was shown by B. A. Ntolosi to be the most accurate indicator of when the organism entered the various phases of growth. It was possible to determine that M. smegmatis LR222 entered the exponential phase of growth after a short lag phase of 4 to 8 hours and persisted in this phase for 20 to 22 hours. It then reached the stationary phase, which lasted for 40 to 46 hours. Protein heat shock assays were performed on growth phase-specific samples. This allowed the identification of a 43-46 kDa in molecular weight stationary phase protein on one-dimensional SOS-PAGE. The protein was induced in cells entering the stationary phase of growth and not by heat shock as it was induced under both the control and the heat shock temperatures. The protein was further characterised by two-dimensional gel electrophoresis, which demonstrated resolution into two, strongly age-associated, proteins. Subtractive RNA hybridisation was attempted in order to obtain a subtraction cDNA probe from a stationary phase RNA sample depleted of sequences common in both the exponential and the stationary phase samples. Ribosomal RNA was removed from the total RNA by the process of photobiotinilation. The mRNA was then used as a template to synthesise with reverse transcriptase single stranded cDNA. cDNA/mRNA denatured hybrids were hybridised to the exponential phase RNA sample. The new hybrids were "subtracted" by chemical cross-linking with DZQ and the unique cDNA used to produce by random primers a radioactively labelled probe. A M. smegmatis library was probed but unfortunately no signal was observed. Further adjustments and improvements to this technique are required before it can be used effectively.

Evaluation of resistance to antituberculosis drugs is routinely performed with genotypic or phenotypic methods; however, discordance can be seen between these different methodologies. Our objective was to identify mutations that could explain discordant results in the evaluation of susceptibility to rifampicin and isoniazid between molecular and phenotypic methods, using whole genome sequencing (WGS). Peruvian strains showing sensitive results in the GenoType MTBDRplus v2.0 test and resistant results in the proportions in the agar-plaque test for isoniazid or rifampin were selected. Discordance was confirmed by repeating both tests, and WGS was performed, using the Next Generation Sequencing methodology. Obtained sequences were aligned "through reference" (genomic mapping) using the program BWA with the algorithm "mem", using as a reference the genome of the M. tuberculosis H37Rv strain. Discordance was confirmed in 14 strains for rifampicin and 21 for isoniazid, with 1 strain in common for both antibiotics, for a total of 34 unique strains. The most frequent mutation in the rpoB gene in the discordant strains for rifampicin was V170F. The most frequent mutations in the discordant strains for isoniazid were katG R463L, kasA G269S, and Rv1592c I322V. Several other mutations are reported. This is the first study in Latin America addressing mutations present in strains with discordant results between genotypic and phenotypic methods to rifampicin and isoniazid. These mutations could be considered as future potential targets for genotypic tests for evaluation of susceptibility to these drugs. / Revisión por pares

Se caracterizaron los genotipos de cepas de M. tuberculosis resistente, multidrogorresistente (MDR), MDR asociada a resistencia a drogas de segunda línea (MDR plus) y sensible a las drogas que proceden de los distritos de Lima y Callao. Cuarenta y nueve pacientes con TB fueron incluidos en el estudio. Los genotipos de M. tuberculosis fueron establecidos por PCR usando el primer Mtb2 (5’-CGG-CGG-CAA-CGG-CGG-CA-3’) en combinación con primers situados inversamente en los flancos repetitivos de la IS6110. Se revisaron las historias clínicas de los pacientes para la obtención de información epidemiológica. La susceptibilidad a isoniacida, rifampicina, estreptomicina, etambutol, kanamicina, acido p-amin-salicilico, tioacetazona y pirazinamida fueron estudiados. / --- We characterise the genotypes of Mycobacterium tuberculosis both resistant, multidrug resistant (MDR), multidrug drug resistant plus (MDR plus) and susceptible to drugs strains come from to Lima and Callao. Forty-nine patients with TB were included in the study. The genotypes of the M. tuberculosis isolates were established by PCR using the primer Mtb2 (5’-CGG-CGG-CAA-CGG-CGG-CA-3’) in combination with primers sited at inverted repeats flanking IS6110. Were revised the clinical history of the patients for epidemiological information. The susceptibility to isoniacid, rifampicin, streptomycin, ethambutol, kanamycin, para-amin-salicylic acid, tioacetazon and pyrazinamide was studied. / Tesis

A research report submitted to the Faculty of Health Sciences, University of the Witwatersrand, in partial fulfilment for the Degree of Master of Medicine In the branch of Anatomical Pathology Johannesburg 2015 / Background: Mycobacterium tuberculosis (Mtb) poses a major global health problem. According to the World Health Organization, South Africa is a country with one of the highest reported incidence rates of this disease. Key to overcoming this preventable and treatable disease lies in establishing a reliable and rapid diagnostic approach. Aims and Objectives: This study aims to investigate the validity of the mycobacterium polymerase chain reaction (PCR) assay applied to formalin-fixed, paraffin-embedded tissue in which the histology showed granulomatous inflammation with no demonstrable acid-fast bacilli. Methods: A retrospective, cross sectional and non-interventional study was conducted on 121 histopathology cases showing granulomatous inflammation with a negative Ziehl-Neelsen (ZN) stain. The mycobacterium PCR results obtained in these cases were compared against the results of mycobacterium culture obtained from a specimen derived from the same or related site as the biopsy. Results: The mean age of the study population was 35.3 years and the study cohort included 63 males and 58 females. The sensitivity of nested mycobacterium PCR (detecting the 133 base pair product of the heat shock protein 65 kilo Dalton gene), was 64.1% and the specificity was 68.2%. The positive and negative predictive values were 49% and 80% respectively. Twenty six of the 121 cases studied had a false positive result (21.5%). CONCLUSION: There are many factors that may influence the result of a PCR assay and the interpretation thereof. Some of these factors include the inability of the test to distinguish between live and dead bacilli, the high risk of carry over contamination, and the paucibacillary nature of certain samples with an unequal distribution of the few bacilli that may be present. Although the sensitivity and specificity of mycobacterium PCR on paucibacillary, formalin-fixed, paraffin embedded tissue is suboptimal, the interpretation of these results must be performed in conjunction with the overall clinical presentation of the patient. / MT2016

No description available.

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, one of the top ten causes of death worldwide. One of the genes upregulated in Mtb during macrophage infection is rv2633c, but the structure and function of its gene product remain unknown. Preliminary research has indicated that Rv2633c is a hemerythrin-like protein that exhibits catalase activity and binds two iron atoms using an HHE domain. Additionally, Rv2633c appears to exist as a dimer. The purpose of this project is to identify specific residues outside of the HHE domain that contribute to the protein's iron-binding ability and/or catalase activity, and to determine whether residues on the C terminus are required for dimerization. Conserved residues D37, E42, and E95 were selected due to their proximity in the amino acid sequence to the HHE domain. Each residue was mutated to alanine using site-directed mutagenesis and the mutations were confirmed using Sanger sequencing. The E95A mutant and the C-terminal truncation mutant were expressed in Escherichia coli using the T7 expression system and purified using affinity chromatography. While wild-type Rv2633c eluted as a soluble protein, the C-terminal truncation mutant was not soluble, indicating that the C terminus may be required for Rv2633c folding. The E95A mutant eluted as a soluble protein, but may have lower iron content than wild-type Rv2633c, indicating that this glutamic acid residue could contribute to iron-binding, despite being outside the HHE domain.

No description available.

No description available.

No description available.