The fight against tuberculosis (TB) continues throughout the world. The causative agents of TB are slow-growing bacteria Mycobacterium tuberculosis complex (MTB), which must be quickly and effectively identified in order to promptly prescribe therapy to the patient. For this purpose, fast and highly sensitive molecular genetic methods are used based on nucleic acid amplification, in particular polymerase chain reaction (PCR). Currently, more rapid methods are used, such as isothermal loop amplification (LAMP). “AmpliTest MTB-LAMP” kit for the detection of MTB DNA in human biological material samples and bacterial cultures has been developed at the Centre for Strategic Planning of FMBA of Russia.
OBJECTIVE
To evaluate effectiveness of new “AmpliTest MTB-LAMP” kit based on isothermal amplification during analysis of biological samples and bacterial cultures in comparison with reference kit during pre-registration clinical trials. The reference kit was identical in intended purpose and registered In Russian Federation as in vitro diagnostic medical device (IVD).
MATERIAL AND METHODS
471 samples of biological material obtained from patients with pulmonary and extrapulmonary tuberculosis localization (sputum (n=110), bronchoalveolar lavage (n=110), urine (n=106), biopsy (surgical material (n=145)) and 100 samples of bacterial cultures were tested. Samples, except for bacterial cultures, were treated with NALC-NaOH [1], and obtained sediment was divided into five equal volumes. Four volumes of the sediment were analyzed using new kit four forms, and a fifth part of the sediment was analyzed using reference “AmpliTest MTB” kit (Centre for Strategic Planning of FMBA of Russia). Amplification was performed using Rotor-Gene 6000, CFX96, DTprime, QuantStudio 5, by hand and special software for automatically results interpretation. Effectiveness indicators (positive and negative agreement of results) of the “AmpliTest MTB-LAMP” kit were calculated using formulas with 95% confidence interval.
RESULTS
The results of sample analysis obtained by new kit and reference kit have completely coincided. There weren’t discordants. Therefore, the effectiveness indicators (positive (PAR) and negative (NAR) agreement of results) of “AmpliTest MTB-LAMP” kit were 100% for all samples.
CONCLUSION
The results obtained indicate the high efficiency of new «AmpliTest MTB-LAMP» kit. It can be used for rapid detection of MTB DNA in order to timely prescribe to a patient antituberculosis regimen and to select samples of MTB DNA which will be appropriate for analysis using kits of AmpliTest brand to identify mutations, associated with MTB resistance to rifampicin and isoniazid.
