Rapid and safe diagnosis of Mycobacterium tuberculosis directly from sputum and biological fluids specimens using a low cost molecular biology dedicated system

Author(s): Juan Garberi, Jorge Labrador, Julián Peneipil, Luis Scigliano, Alcides Troncoso

Objective: Early and rapid detection of the causative organism is necessary in tuberculosis. We present here an integrated and dedicated molecular biology system for tuberculosis diagnosis. Methods: One hundred and eighty nine (189) biological specimens from patients strongly suspected by clinical parameters of tuberculosis were studied by Ziehl- Neelsen staining, cultivation on a solid medium and by a balanced heminested fluorometric PCR system (Orange G3TB) that preserves worker safety and produces a rather pure material free of potential inhibitors. DNA amplification was carried out in a low cost, with a tuberculosis termocycler-fluorometer. The double stranded DNA produced is fluorometrically detected. The whole reaction is carried out in one single tube which is never opened after adding the processed sample, thus minimizing the risk of cross contamination with amplicons. The assay is able to detect 30 bacilli per ml of sample having a 99.8 % inter-assay coefficient of variation. Results: PCR were positive in 36 (18,9%) tested samples (33 of them were smear negative). In our study, it yields a preliminary overall sensitivity of 97,4 %. In addition, its overall specificity is 98.7 %. The total run time of the test is four hours with two and a half real working hours. All PCR positive samples also had a positive result by microbiological culture and clinical criteria. Conclusions: The results obtained showed that it could be a very useful tool to increase eficiency in detecting the tuberculosis disease in low bacillus inoculum samples. Furthermore, its low cost and friendly usage make it feasible to be used in regions with poor development.

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