RESUMEN
BACKGROUND: Tuberculosis (TB), especially drug-resistant TB, is a global public health problem. This study aimed to validate a new molecular diagnostic test, the FluoroType® MTBDR. METHOD: Samples underwent routine diagnostic procedures (fluorescence microscopy, culture, species differentiation and phenotypic drug susceptibility testing). Left over samples stored at -20° underwent DNA extraction using the Fluorolyse® kit, followed by FluoroType® MTBDR and Genotype MTBDRplus testing. RESULTS: A total of 350 respiratory and 59 lymph node samples were included in the study; 71 respiratory and 16 lymph node samples were culture positive for M. tuberculosis complex (MTBC). The sensitivity of the FluoroType® MTBDR to detect MTBC DNA was 91.4% (95%CI 82.3-96.8%), 68.4% (95%CI 43.4-87.4%) and 62.5%, (95%CI 35.4-84.8%) for respiratory, smear negative respiratory and lymph node samples respectively. The correlating sensitivities of the GenoType MTBDRplus were 85.9% (95%CI 75.6-93.0%), 52.6% (95%CI 28.9-75.6%) and 56.3% (29.9-80.2). Sensitivity of the FluoroType® MTBDR to detect RMP and INH resistance for respiratory samples was 96.5% (95%CI 82.2-99.9) and 70% (95%CI 45.7-88.1), respectively. The GenoType MTBDRplus revealed sensitivities of 97.1% (95% 85.1-99.9) 70.6% (95%CI 52.5-84.9) for detection of RMP and INH resistance. Indeterminate results were 13/64 (20.3%), 23/64 (35.9%) and 16/64 (25.0%) for rpoB, katG and inhA using the FluoroType® MTBDR. CONCLUSION: The FluoroType® MTBDR has a high sensitivity to detect MTBC DNA. However, the high proportion of indeterminate results across all three genes needs to be addressed.
Asunto(s)
Técnicas Bacteriológicas , ADN Bacteriano/genética , Ganglios Linfáticos/microbiología , Mycobacterium tuberculosis/genética , Esputo/microbiología , Tuberculosis Ganglionar/diagnóstico , Tuberculosis Pulmonar/diagnóstico , Antituberculosos/farmacología , Proteínas Bacterianas/genética , Catalasa/genética , Análisis Mutacional de ADN , ADN Bacteriano/aislamiento & purificación , ARN Polimerasas Dirigidas por ADN/genética , Farmacorresistencia Bacteriana/genética , Genotipo , Humanos , Isoniazida/farmacología , Mutación , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/aislamiento & purificación , Oxidorreductasas/genética , Fenotipo , Valor Predictivo de las Pruebas , Reproducibilidad de los Resultados , Rifampin/farmacología , Espectrometría de Fluorescencia , Tuberculosis Ganglionar/microbiología , Tuberculosis Pulmonar/microbiologíaRESUMEN
In beta-thalassemia, the mechanism driving ineffective erythropoiesis (IE) is insufficiently understood. We analyzed mice affected by beta-thalassemia and observed, unexpectedly, a relatively small increase in apoptosis of their erythroid cells compared with healthy mice. Therefore, we sought to determine whether IE could also be characterized by limited erythroid cell differentiation. In thalassemic mice, we observed that a greater than normal percentage of erythroid cells was in S-phase, exhibiting an erythroblast-like morphology. Thalassemic cells were associated with expression of cell cycle-promoting genes such as EpoR, Jak2, Cyclin-A, Cdk2, and Ki-67 and the antiapoptotic protein Bcl-X(L). The cells also differentiated less than normal erythroid ones in vitro. To investigate whether Jak2 could be responsible for the limited cell differentiation, we administered a Jak2 inhibitor, TG101209, to healthy and thalassemic mice. Exposure to TG101209 dramatically decreased the spleen size but also affected anemia. Although our data do not exclude a role for apoptosis in IE, we propose that expansion of the erythroid pool followed by limited cell differentiation exacerbates IE in thalassemia. In addition, these results suggest that use of Jak2 inhibitors has the potential to profoundly change the management of this disorder.