Abstract
Microscopy-based tuberculosis (TB) diagnosis i.e. Ziehl-Neelsen screening still remains the primary diagnostic method in resource poor and high TB burden countries, however this method has poor sensitivity (~60%). Bringing three million TB patients who are left undiagnosed under the treatment has been a major focus as part of END-TB strategy across the world. We have developed a portable set-up called ‘SeeTB’ that converts a bright-field microscope into fluorescence microscope (FM) with minimal interventions. SeeTB, a total internal reflection-based fluorescence excitation system allows visualization of auramine-O stained bacilli efficiently with high signal-to-noise ratio. Along with the device, we have developed a sputum-processing reagent called ‘CLR’ that homogenizes and digests the viscous polymer matrix of sputum. We have compared the performance of SeeTB system in 237 clinical sputum samples along with FM, GeneXpert and liquid culture. In comparison with culture as gold standard, FM has sensitivity of 63.77% and SeeTB has improved sensitivity to 76.06%. In comparison with GeneXpert, FM has sensitivity of 73.91% while SeeTB has improved sensitivity to 85.51%. However, there is no significant change in the specificity between FM and SeeTB system. In short, SeeTB system offers the most realistic option for improved TB case identification in resource-limited settings.
Highlights
Microscopy-based tuberculosis (TB) diagnosis i.e. Ziehl-Neelsen screening still remains the primary diagnostic method in resource poor and high TB burden countries, this method has poor sensitivity (~60%)
Traditional fluorescence microscope (FM) involves Epi-mode of illumination; where the excitation light illuminates www.nature.com/scientificreports the whole cross-section in Z-axis of the sample under analysis. This results in exciting the fluorophores in whole volume of excitation beam and increases the background noise
In TIR, the excitation beam is highly selective to the fluorescent molecules close to the glass surface resulting minimum background from the sample
Summary
Microscopy-based tuberculosis (TB) diagnosis i.e. Ziehl-Neelsen screening still remains the primary diagnostic method in resource poor and high TB burden countries, this method has poor sensitivity (~60%). SeeTB system offers the most realistic option for improved TB case identification in resource-limited settings. In May 2014, World Health Organization (WHO) unanimously passed resolution to support ‘ENDTB strategy[5] This strategy aims to reduce TB deaths by 95%, cut the new cases up to 90% by 2035, and complete eradication of TB by 2050. Fluorescence microscopy (FM) of sputum smear slides for TB detection was proposed in 1930s and shown to improve the sensitivity as compared to the traditional Ziehl Neelsen (ZN) based test[4]. Limited sensitivity cost of device and reproducibility issues are still major barriers in large-scale adaption
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