Demonstration of a fast and easy sample-to-answer protocol for Mycobacterium tuberculosis diagnosis in point-of-care settings

Abstract

BackgroundTuberculosis (TB) is currently the ninth leading cause of death worldwide and the leading cause from a single infectious agent, ranking above HIV/AIDS. Point of care diagnosis is one of the diagnostic aspects in the health care system that might have the potential to mitigate this worldwide epidemic. Although several qPCR tests are available, most cannot be taken to the field. Therefore, their use in POC settings is limited. Smooth sample preparation and streamlined DNA extraction constitute the biggest challenges for this limitation.\n\nMethodsSeventeen M. tuberculosis samples which were already previously analyzed by GeneXpert or culture technique were subjected to our in-house protocol. Of these samples, ten were positive and seven negatives when tested by GeneXpert, while seven were positive and ten negatives when analyzed by culturing.\n\nResultsHere we present a \"proof of concept\" protocol for sputum liquefaction and disinfection, followed by FTA card DNA extraction. The resulting DNA is rapidly amplified and Mycobacterium tuberculosis (MTB) DNA is detected with the use of a portable qPCR instrument. Our protocol is able to linearly identify down to 2 CFU/mL of MTB, showing great sensitivity on artificial samples. The protocol was challenged with patient samples, and showed excellent agreement with the gold-standard molecular protocol, allowing the detection of 9/10 positive samples (90%, or 10% of false negatives) and 7/7 of the negative (100%, no false positives). When compared to culture, 7/7 culture-positive samples were also found positive (100%, no false negatives), while 2/10 culture-negative were found positive by the present method (20% of false positives).\n\nConclusionThe proposed sample preparation protocol provides a rapid and easy procedure with a small number of reagents and steps, as well as minimal use of equipments, resulting in an easy-to-use tool for M. tuberculosis diagnosis in POC settings.