Tuberculosis has become one of the world’s most devastating diseases, with more than two million deaths and eight million new cases occurring annually due to the development of drug-resistant strains of Mycobacterium tuberculosis, the breakdown of the immune system of its host by HIV, lapses in public health programmes and the fact that diagnosis of TB is not 100% reliable. Early, affordable, unsophisticated and accurate diagnosis of TB to facilitate timely and proper treatment has become of highest priority to public health. Mycolic acid (MA) is the major lipid cell wall component of Mycobacterium tuberculosis and is unique to mycobacteria and closely aligned genera. Mycolic acids have been shown to be unique antigens for TB diagnosis and have been utilized in standard serodiagnostic techniques, but sensitivity and specificity was found to be unsatisfactory. Two vastly different techniques were investigated in this study – one making use of antibodies and MA, the other, just MA and its unique physical properties of interaction with other MA using fluorescently labelled MA. In the first approach, Sepharose protein-A was employed to trap patient IgG antibodies. The anti-MA antibodies were then quantified by probing with liposomes containing fluorescently labelled MA. Although it generally worked well, a few false –positive and –negative results were obtained. This assay appeared to be more accurate than the standard ELISA immunoassay but it is more labour intensive and not even remotely as amenable to large-scale screening and automation as ELISA. The second approach is based on the release of fluorescent MA from immobilized liposomes on glass by means of the specific attraction that MA in test liposomes or TB patient serum was perceived to have on the immobilized MA. The end-point measured was the remaining fluorescent MA on the surface. Differences were observed between the control and patients’ sera at a very high dilution but not between the HIV negative, TB positive and HIV positive, TB positive patients. This was merely an exploratory investigation and more work still needs to be done before the test is ready for validation with large numbers of serum samples. If subsequent studies confirm these findings, then this concept may be converted into a simple, rapid and affordable TB diagnostic test or be used in combination with the IAsys affinity biosensor to provide a more thorough diagnosis / Dissertation (MSc (Biochemistry))--University of Pretoria, 2009. / Biochemistry / unrestricted

Em todo o mundo, menos de 5% dos doentes com tuberculose (TB), sejam casos novos ou previamente tratados, tem a avaliação dos isolados quanto ao perfil de sensibilidade aos antibioticos. No Rio Grande do Sul, estado localizado no sul do Brasil, cerca de 4700 casos novos de TB são registrados a cada ano, com uma taxa de cura de 68,9%, e uma taxa de abandono de 7,5%. A identificação rápida da resistência às drogas, em isolados clínicos de M. tuberculosis é importante para o estabelecimento de uma quimioterapia eficaz bem como para evitar a propagação de cepas resistentes. Os objetivos deste estudo foram caracterizar os pacientes de TB com maior risco de possuir TB-­‐MDR, analisando o perfil de resistência às drogas dos isolados e o perfil epidemiológico desses pacientes. Além disso utilizou-­‐se as amostras clínicas para avaliar o teste comercial (GenoType® MTBDRplus) e para desenvolver e padronizar um novo teste (Detect-­‐TBMR) para detectar as mutações mais frequentes associadas a resistência à INH e RIF. Uma proporção significativamente maior (75% versus 20%, p = 0,009) de pacientes do gênero masculino foi encontrada entre os casos resistentes às drogas do que entre os casos suscetíveis. 43,8% dos pacientes demoraram mais de 30 dias para procurar assistência médica e no grupo TB MDR, 25% dos casos não tinha sido submetido a qualquer tratamento prévio anti-­‐TB. Em nossas amostras, encontramos uma proporção de 48,3% de TB-­‐ MDR. A família T foi a família de spoligotipo mais frequente. Comparado com o método da proporções, a sensibilidade e especificidade do ensaio MTBDRplus foram 82% e 94% para a resistência à RIF, 60% e 94% para resistência à INH. Comparado com sequenciamento, a sensibilidade e especificidade do ensaio MTBDRplus foi 92% e 97% para a resistência à RIF e 100% e 100% para a resistência à INH, respectivamente. Para detectar resistência à RIF e INH, o ensaio Detect-­‐TBMDR mostrou sensibilidade e especificidade de 79,3% e 77,0% e 100% e 65%, respectivamente, em comparação com o método da proporções. Comparado com o sequenciamento, a sensibilidade e especificidade do ensaio Detect-­‐TBMDR foi de 81,2% e 94,7% e 100% e 96,2%, para detectar e resistência à RIF e INH, respectivamente. Ainda existem discordâncias entre o método das proporções e a abordagem molecular, particularmente em relação a resistência à INH. Contudo, estes métodos são muito importantes para o manejo mais rápido e correto dos pacientes, auxiliando na escolha do melhor esquema terapêutico. / In most parts of the world, less than 5% of new and previously treated tuberculosis (TB) patients are tested for multidrug resistance (MDR) TB. In Rio Grande do Sul state, the southern most Brazilian state; approximately 4700 new cases of TB are recorded each year, with a cure rate of 68.9%, and a noncompliance rate of 7.5%. Rapid identification of drug resistance in clinical isolates of Mycobacterium tuberculosis is important to facilitate rapid and adequate chemotherapy of TB, and to prevent the spread of resistant strains. The aim of this study was to characterize TB patients at higher risk of having MDR-TB, to analyze the drug resistance and epidemiological profile of these patients. Use the clinical samples to assess the commercial test (GenoType® MTBDRplus) and develop and standardize a new test (Detect-MDRTB) for detecting the most frequent mutations associated with resistance to INH and RIF. A significantly higher proportion (75% versus 20%, p = 0.009) of males were found among drug-resistant cases than drug susceptible cases. 43.8% of patients took longer than 30 days to seek medical care and in the MDR group 25% of the cases did not undergo any previous anti-TB treatment. In our samples we found a proportion of 48.3% of MDR-TB. The T family was the most frequent spoligotype family. Compared with the proportion method, the sensitivity and specificity of the MTBDRplus assay were 82% and 94% for RIF-resistance, 60% and 94% for INH resistance. Compared with sequencing, the sensitivity and specificity of the MTBDRplus assay were 92% and 97% for RIF-resistance, 100% and 100% for INHresistance. To detect RIF and INH-resistance, the Detect-TBMDR assay showed a sensitivity and specificity of 79.3% and 77.0%, and 100% and 65%, respectively, compared to proportion method. When compared with sequencing, Detect-TBMDR assay, to detect RIF and INH-resistance, showed a sensitivity and specificity of 81.2% and 94.7% and to 100% and 96.2%, respectively. Discordances still exist between the proportion method and molecular approach, particularly regarding INH-resistance. However, these methods are very important for the management faster and correct patient, helping to choose the best treatment regimen.

La pirazinamida (PZA) es una droga antituberculosa de primera línea, presenta gran actividad in vivo, sin embargo in vitro no es evidente a menos que el pH del medio sea ácido, lo que hace que la susceptibilidad sea difícil de determinar por métodos convencionales. Por lo tanto, el objetivo del presente estudio fue evaluar la prueba molecular Polimorfismo Conformacional de la Hebra simple de ADN (SSCP) en cepas clínicas de Mycobacterium tuberculosis, así como comparar su desempeño con otras pruebas como son el BACTECÔ-460TB, el test de Wayne y el secuenciamiento. Se utilizó la prueba molecular del SSCP para la determinación de la susceptibilidad a PZA trabajando con 157 aislamientos clínicos de M. tuberculosis provenientes del Área de Tuberculosis del Laboratorio de Enfermedades Infecciosas de la Universidad Peruana Cayetano Heredia. / Tesis

Thesis (M. Sc. (Medical Sciences)) -- University of Limpopo, 2015 / Tuberculosis (TB) patients may have mixed infections with both drug-susceptible and drug-resistant Mycobacterium tuberculosis (MTB) strains. This phenomenon termed heteroresistance presents a challenge TB management and is considered a preliminary stage to full resistance. Heteroresistance is more likely to occur in high TB incidence areas and in chronic patients as they have more opportunity to become infected with various strains of TB and has been proven to occur in new cases, treatment failure and relapse. Methods: Sputum samples were collected from new consulting and hospitalised patients who were on treatment for MDR TB. A total of 231 samples were run on MTBDRplus to determine heteroresistance of Mycobacterium tuberculosis to isoniazid and rifampicin. To determine heteroresistance to second-line drugs, 91 samples were run on MTBDRsl. Nineteen (19) samples that were heteroresistant to 2nd line drugs were subjected to spoligotyping to determine the families/lineages they belonged to. Results: A total of 66 were confirmed as Mycobacterium tuberculosis complex by the line probe assays. Out of the 66 MTBC, rifampicin resistance was found in 22 (10%) and 44 (19%) were reported susceptible. Isoniazid resistance was found in 39 (17%) and 27 (12%) were reported susceptible. Of the 66 MTBC positive samples, moxifloxacin resistance was found in 33 (16%) and 14 (7%) were reported susceptible. Kanamycin resistance was found in 17 (8%) and 30 (14%) were reported susceptible. Ethambutol resistance was found in 25 (12%) and 22 (10%) were reported susceptible. Heteroresistance was evident in 22 (10%) samples for the first-line and in 23 (11%) for the second-line drugs. Results of a total of 19 heteroresistant samples subjected to spoligotyping when compared to those in the international spolDB4 database indicated that 4 of them matched existing shared spoligotype international types, 15 were unknown (orphans). Eighteen (18) of 19 heteroresistant samples subjected to spoligotyping were also MDR. Fourteen of the samples that were resistant to both RIF and INH were orphans. Of the 14 MDR, 3 samples belonged to clades T1, T-H37RvV817 and LAM 3 with SITs: 879, 568 and 2301, respectively. One sample with SIT 1196 had an unknown clade was resistant to RIF but susceptible to INH. Conclusion: This study has shown that heteroresistance remains an important phenomenon in clinical tuberculosis, especially in highly endemic areas. According to the current study, heteroresistance was associated more with recurrent cases who are on initiation or continuation phase than new cases and a larger percentage of heteroresistance was reported in second-line drugs than there is in first-line drugs. The T1 genotype was found to be predominant amongst recurrent cases. The LAM3 and T-H37RvV817 lineages were found amongst the new cases. In the present study there was no significant association between heteroresistance and the patient’s treatment history as indicated by a P-value of 0.473 and between heteroresistance and spoligotype families (P-value, 0.991). The predominance of orphan SITs and unknown clades followed by non-Beijing strains in the study may be due to the migration of carriers from the neighboring countries as the Limpopo Province is flanked by Botswana, Zimbabwe and Mozambique. Further studies with larger numbers of patients should focus on the prevalence to associate heteroresistance with patients‟ treatment history and establish the contributing MTBC strain lineages.

Objetivo: Al menos uno de los péptidos sintéticos muestra actividad inhibitoria para PknG Diseño: Esta investigación califica como un estudio in vitro, dado que los experimentos serán realizados bajo condiciones controladas fuera de un organismo vivo.

El texto completo de este trabajo no está disponible en el Repositorio Académico UPC por restricciones de la casa editorial donde ha sido publicado. / Objectives: Diagnosis of tuberculous meningitis (TM) is a challenge in countries with a high burden of the disease and constrained resources and clinical prediction rules (CPRs) could be of assistance. We aimed at developing a CPR for diagnosis of TM in a Latin American setting with high tuberculosis incidence and a concentrated HIV epidemic. Methods: We enrolled adult patients with clinical suspicion of TM attending two hospitals in Lima, Peru. We obtained information on potential anamnestic, clinical and laboratory predictive findings that are easy to collect and promptly available. We independently diagnosed TM according to a composite reference standard that included a series of microbiological tests. We performed bivariate analysis and constructed a logistic regression model to select the predictive findings associated with TM. With the selected predictors included in the model, we developed a score-based CPR. We assessed its internal validity and diagnostic performance. Results: Of 155 analysed patients, 59 (38%) had TM. The CPR we derived includes three predictors: cough for 14 days or more, 10–500 cells in CSF and adenosine deaminase ≥ 6 U/l in CSF. It classifies patients into high-, moderate- or low-score groups and has an overall area under the ROC curve of 0.87. 59% of patients were assigned to either the high- or the low-score group, permitting prompt decision-making. In patients in the high-score group, it attains a positive likelihood ratio for TM of 10.6 and in patients with low scores, a negative likelihood ratio of 0.10. Bootstrap analysis indicated high internal validity. Conclusion: This CPR could support decision-making in patients with clinical suspicion of TM. External validation and further assessment of its clinical impact are necessary before application in other settings. / Revisión por pares

Nonopsonic invasion of mononuclear phagocytes by Mycobacterium tuberculosis (M. tb.) is likely important in the establishment of a primary infection in the lung. M. tb. binds to a variety of phagocyte receptors, of which the mannose receptor and the complement receptor type 3 (CR3) may support nonopsonic binding. CR3, a β₂ integrin, is a target for diverse intracellular pathogens, but its role in nonopsonic binding remains uncertain. We have examined the binding of M. tb. to human CR3 heterologously expressed in Chinese hamster ovary (CHO) cells, thereby circumventing the problems of competing receptors and endogenously synthesised complement, which are inherent in studies with mononuclear phagocytes. The surface expression and functional activity of CR3 were confirmed by rosetting with beads coupled to anti-CR3 monoclonal antibodies (MAbs) and with C3bi-coated microspheres, respectively. We found thatM. tb. binds 4-7-fold more avidly to CR3- expressing CHO cells than to wild-type cells, and importantly, that this binding is very similar in the presence of fresh or heat-inactivated human or bovine sera, or no serum. The binding of M. tb. to the transfected CHO cells is CR3-specific, as it is inhibited by anti-CDllb and anti-CD18 MAbs; interestingly, binding is not inhibited by a MAb (2LPM19c) specific for the C3bi-binding site on CDI lb. Electron micrographs of infected CR3-expressing CHO cells reveal the presence of intracellular bacteria enclosed in well-defined, membrane-bound vacuoles. We conclude that the binding of M. tb. to CR3 is nonopsonic and that the organism likely expresses a ligand that directly binds to CR3.

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