Molekularbiologische und biochemische Nachweismethoden des Tuberkulose-Komplexes aus Blut und klinischem Material

Abstract

Tuberculosis (TB) remains the leading life-threatening infectious disease worldwide. In particular the systemic co-infection of HIV and Mycobacterium tuberculosis (HIV-TB) has a high mortality rate. The immunosuppression of the HIV infected patients leads to Mycobacterium tuberculosis bacteremia and delayed cultural diagnosis. Molecular PCR tests are rapid, but have yet not been shown sensitive enough for the direct diagnosis of TB in blood. In this approach molecular and biochemical tests were used for the detection of TB bacteria from blood. 1ml and 10ml of negative EDTA blood were spiked with a dilution series of a M. bovis BCG culture. The pre-analytical tool MolYsisTM (MOLZYM, Germany) was used for the isolation of mycobacterial DNA. Three molecular assays, SepsiTestTM 16S PCR, FluoroType MTB and GeneXpert MTB/RIF, were tested for sensitivity effects. In addition clinical samples from patients suspected of TB were analyzed. Furthermore, a Western blot analysis of the heat shock protein Hspx in M. bovis BCG was performed. A high analytical sensitivity (2.5 BCG cfu/ml) was obtained with FluoroType MTB and GeneXpert MTB/RIF when 10ml blood was processed. This sensitivity matches the low load of TB bacteria in the blood of HIV infected patients. The molecular assays in combination with a higher sample volume are a promising basis for the sensitive and rapid detection of M. tuberculosis in blood.