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Mycolic acid as antigen or analyte in tuberculosisGomes, Monica Nunes 16 July 2008 (has links)
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
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Validação e performance de novos métodos moleculares no diagnóstico da tuberculose resistente / Validation and performance of new molecular methods for the diagnosis of resistant tuberculosisMaschmann, Raquel de Abreu January 2013 (has links)
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.
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Genotipificación de Mycobacterium tuberculosis complex mediante herramientas molecularesJiménez Arias, Ana Patricia 18 April 2016 (has links)
[EN] In the last years, various genotyping techniques were developed for isolation of Mycobacterium tuberculosis complex (MTBC) that has demonstrated a high discriminatory power. In this study, after the identification of selected strains at level of species by Genotype MTBC technique, we evaluated the profit of the simplified amplified-fragment Length Polymorphism technique (AFLPs) and the Mycobacterial Interspersed Repetitive Units technique (MIRU-15). A total of 131 mycobacterium tuberculosis isolates were analyzed, 68 isolates were collected in Ecuador, from the Clinical Laboratory of Hospital Alli Causai located in city of Ambato, and the Laboratory of Bacteriology in Carlos Andrade Marín Hospital located in the capital city Quito. The remaining 63 isolates were harvested in Spain and belong to microorganism's collection of Microbiology Services of Consorcio Hospital General Universitario and Hospital Clínico Universitario of the city of Valencia. Among these isolates, 126 were identified by conventional methods such as molecular MTBC, corresponding to 106 patients. The Mycobacterium tuberculosis control strain ATCC 25177 was also identified as such by this method.
The AFLPs technique allowed as to group the strains in twelve patterns (P1 to P8, P10, P12, P13, P14), of which the most prevalent were patterns P1 with 77 (61.1%) and P2 with 27 (21, 4%) isolates, representing 82.5% of the same. These were followed by the pattern P5 with 5 (3.9%) isolates, the patterns P3, P4 and P6 grouped 3 isolates each one (2.4%), the patterns P8 and P12 with 2 isolates (1.6% ) and finally the patterns P7, P10, P13 and P14 with 1 isolated each one (0.8%). The control strain M. tuberculosis ATCC 25177, showed a restriction profile that prevented their inclusion in any of the patterns described. The discriminatory power of the Hunter-Gaston discriminatory index (HGDI) method was 0.5812 against to 0.9843 of the MIRU-15 technique, which grouped 69 strains (54.8%) in 20 clonal complex and 57 unique patterns (45.2%). In the case of Spain, the strains were related mostly to the lineage 4 or Euro-American including: Cammeroon (1.59%), Haarlem (36.51%), S (31.75%), and LAM (19.05%); the lineage 6 or West Africa I (9.53%), the lineage 1 or EIA (1.59%) In the case of Ecuador the strains were related to the lineage 4: Haarlem (42.86%), S (33.33%) and LAM (22.22%) and Beijing lineage 2 (1.59%) from Asia. The MIRU-VNTR Variable-Number Tandem Repeats (15 loci) technique proved to be a stable system, reproducible and high discriminatory power in comparison with AFLPs system, allowing the use of it to conduct prospective population studies with the aim of contributing to the public health programs to control Tuberculosis (TB). / [ES] En los últimos años se han desarrollado diversas técnicas de genotipificación para aislados de Mycobacterium tuberculosis complex (MTBC) que han demostrado tener un alto poder discriminatorio. En este estudio, tras identificación de las cepas seleccionadas al nivel de especie mediante la técnica comercial GenoType MTBC, se ha evaluado la utilidad de la técnica simplificada del Polimorfismo de Longitud de Fragmentos Amplificados (AFLPs) y la técnica de Unidades Repetitivas Intercaladas Micobacterianas (MIRU-15). Se analizaron un total de 131 aislados clínicos de los cuales 68 aislados fueron recolectados en Ecuador, provenientes tanto del Laboratorio Clínico del Hospital Alli Causai ubicado en la ciudad de Ambato, provincia de Tungurahua como del Laboratorio de Bacteriología del Hospital Carlos Andrade Marín ubicado en la ciudad capital Quito, provincia de Pichincha. Los 63 aislados restantes fueron recolectados en España y pertenecían colección de microorganismos de los Servicios de Microbiología del Consorcio Hospital General Universitario y Hospital Clínico Universitario de la ciudad de Valencia, provincia de Valencia. De éstos aislados, 126 fueron identificados por métodos convencionales y moleculares como MTBC, correspondientes a 106 pacientes. La cepa control Mycobacterium tuberculosis ATCC 25177 también fue identificada como tal mediante este método.
La técnica AFLPs permitió agrupar a las cepas en doce patrones (P1 a P8, P10, P12, P13, P14), de los cuales los más prevalentes fueron los patrones P1 y P2 con 77 (61,1%) y 27 (21,4%) aislados respectivamente, lo que supone el 82,5% del total de los mismos. Le siguieron en frecuencia el patrón P5 con 5 (3,9%) aislados, los patrones P3, P4 y P6 agruparon a 3 aislados cada uno (2,4%), los patrones P8 y P12 con 2 aislados (1,6%) y finalmente los patrones P7, P10, P13 y P14 con 1 aislado cada uno (0,8%). La cepa control M. tuberculosis ATCC 25177, mostró un perfil de restricción que no permitió su inclusión en ninguno de los patrones descritos. El poder discriminatorio del método (HGDI) fue de 0,5812 frente a 0.9843 de la técnica MIRU-15, que agrupó a 69 cepas (54,8%) en 20 complejos clonales y 57 patrones únicos (45,2%). Para el caso de España, las cepas estuvieron relacionadas en su mayoría con el linaje 4 o Euro-Americano que incluye: Cammeroon (1,59%), Haarlem (36,51%), S (31,75%), y LAM (19,05%); el linaje 6 o West Africa I (9,53%), el linaje 1 o EIA (1,59%), Para el caso de Ecuador las cepas estaban relacionadas con el linaje 4: Haarlem (42,86%), S (33,33%), y LAM (22,22%) y el linaje 2 Beijing (1,59%) originario de Asia. La técnica MIRU-VNTR (15 loci) demostró ser un sistema estable, reproducible y con un poder discriminatorio alto en comparación con AFLPs lo que permitiría emplearlo para realizar estudios poblacionales prospectivos con la finalidad de contribuir a los programas de Salud Pública para el control de la Tuberculosis (TB). / [CAT] En els últims anys s'han desenvolupat diverses tècniques de genotipificació per aïllats de Mycobacterium tuberculosis complex (MTBC) que han demostrat tenir un alt poder discriminatori. En aquest estudi, després de la identificació de les soques seleccionades al nivell d'espècie mitjançant la tècnica comercial GenoType MTBC, s'ha avaluat la utilitat de la tècnica simplificada del Polimorfisme de longitud de fragments amplificats (AFLPs) i la tècnica d'Unitats repetitives Intercalades micobacterianes (Miru-15). Es van analitzar un total de 131 aïllats clínics dels quals 68 aïllats van ser recollectats a Equador, provinents tant del Laboratori Clínic de l'Hospital Alli Causai situat a la ciutat d'Ambato, província de Tungurahua com del Laboratori de Bacteriologia de l'Hospital Carlos Andrade Marín ubicat a la ciutat cabdal Quito, província de Pichincha. Els 63 aïllats restants van ser recollectats a Espanya i pertanyien a la collecció de microorganismes dels Serveis de Microbiologia del Consorci Hospital General Universitari i Hospital Clínic Universitari de la ciutat de València, província de València. D'aquests aïllats, 126 van ser identificats per mètodes convencionals i moleculars com MTBC, corresponents a 106 pacients. La soca control Mycobacterium tuberculosi ATCC 25177 també va ser identificada com a tal mitjançant aquest mètode.
La tècnica AFLPs va permetre agrupar les soques en dotze patrons (P1 a P8, P10, P12, P13, P14), dels quals els més prevalents van ser els patrons P1 i P2 amb 77 (61,1%) i 27 (21, 4%) aïllats respectivament, fet que suposa el 82,5% del total dels mateixos. El van seguir en freqüència el patró P5 amb 5 (3,9%) aïllats, els patrons P3, P4 i P6 van agrupar a 3 aïllats cadascun (2,4%), els patrons P8 i P12 amb 2 aïllats (1,6%) i finalment els patrons P7, P10, P13 i P14 amb 1 aïllat cadascun (0,8%). La soca control M. tuberculosis ATCC 25177, va mostrar un perfil de restricció que no va permetre la seva inclusió en cap dels patrons descrits. El poder discriminatori del mètode (HGDI) va ser de 0,5812 enfront de 0,9843 de la tècnica MIRU-15, que va agrupar a 69 soques (54,8%) en 20 complexos clonals i 57 patrons únics (45,2%). Per al cas d'Espanya, les soques van estar relacionades majoritàriament amb el llinatge 4 o Euro-Americà que inclou: Cammeroon (1,59%), Haarlem (36,51%), S (31,75%), i LAM (19,05%); el llinatge 6 o West Africa I (9,53%), el llinatge 1 o EIA (1,59%), Pel cas de l'Equador les soques estaven relacionades amb el llinatge 4: Haarlem (42,86%), S ( 33,33%), i LAM (22,22%) i el llinatge 2 Beijing (1,59%) originari d'Àsia. La tècnica Miru-VNTR (15 loci) va demostrar ser un sistema estable, reproduïble i amb un poder discriminatori alt en comparació amb AFLPs, el que permetria emprar-lo per realitzar estudis poblacionals prospectius amb la finalitat de contribuir als programes de salut pública per al control de la Tuberculosi (TB). / Jiménez Arias, AP. (2016). Genotipificación de Mycobacterium tuberculosis complex mediante herramientas moleculares [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/62681 / TESIS
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Aplicación de la prueba polimorfismo conformacional de la hebra simple de ADN (SSCP) en la determinación de la susceptibilidad a pirazinamida en Mycobacterium tuberculosisMéndez Aranda, Melissa Marlene January 2008 (has links)
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
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Determination of heteroresistant mychobacterim tubeculosis strains and their association with patients tuberculosis treatment history in Limpopo ProvinceMohatli, Matema Constance January 2015 (has links)
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.
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Evaluación de la actividad inhibitoria de péptidos sintéticos seleccionados sobre la proteína quinasa G (PknG) de Mycobacterium tuberculosisBustillos Higuchi, Hideki, Vega, Estefany 07 November 2019 (has links)
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.
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Development of a clinical prediction rule for tuberculous meningitis in adults in Lima, PeruSolari, L, Van der Stuyft, P, Soto, Alonso 04 1900 (has links)
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
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Binding of Mycobacterium tuberculosis to complement receptor type 3 expressed in mammalian cells : dependence on serum opsoninsCywes, Colette 20 July 2017 (has links)
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.
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Mutations in Mycobacterium tuberculosis Isolates with Discordant Results for Drug-Susceptibility Testing in PeruSolari, L., Santos-Lazaro, D., Puyen, Z. M. 01 January 2020 (has links)
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
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Caracterización molecular de cepas de mycobacterium tuberculosis aislados de pacientes con fracaso terapéutico mediante la técnica genotipaje basado en PCRTello Ayllón, Carlos Alberto January 2008 (has links)
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
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