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Digital Fluorescence Microscopy for Tuberculosis Diagnosis

Abstract

This thesis describes the conceptualization, design, creation, and testing of a portable digital LED fluorescence microscope for use in diagnosing M. tuberculosis in low-resource settings. Over a century after its introduction, sputum microscopy remains an essential technique for tuberculosis diagnosis even in wealthy countries: sputum from a patient is smeared on a slide, stained for visibility, and examined for the presence of microscopic tuberculosis bacteria, requiring both a high resolution microscope and substantial technician skill in interpreting what is observed. Our device is ≤ 3kg and 18 x 18 x 8 cm; battery-powered, charging off 12V solar or auto power; and controlled via USB 2.0 by a low-cost laptop. It is capable of digital fluorescence imaging in direct sunlight of Auramine O-stained sputum smears over a 0.64 x 0.49 mm field of view with a nominal resolution of 0.76 µm resolution and image display at ≳ 2500X magnification. Diagnostic sensitivity is 63% and specificity 85% when used by individuals with ~ 10 hours of training in reading sputum smears. Images are uploaded automatically via the local mobile phone network to district hospitals for quality assurance and record-keeping. We have also developed a diagnostic image-processing algorithm with accuracy equivalent to our human readers when applied to images taken with the device; in the future we will integrate diagnostic image processing at the time of imaging in order to reduce technician training requirements, increase repeatability, and potentially increase sensitivity and specificity. The system is currently deployed in Hanoi, Vietnam as part of an effort to extend tuberculosis diagnosis to peripheral levels of the healthcare system.

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