Fluorescence Microscopy for TB Testing Dr. Lipton Consultant/Inventor Mycobacteria tuberculosis Epidemiology & Life Cycle – Many millions of Active TB infections worldwide – Spread by droplet infection – Affects: • Stressed, prisoners • Malnourished- the world’s poor • Non-healthy, drug abusers, HIV • Institutionalized patients 2 World TB Incidence World TB incidence. Cases per 100,000 red => 300 orange = 200–300 yellow = 100–200 Delegates green = 50–100 at this blue =< 50 meeting grey =n/a Mycobacteria tuberculosis • Acute Disease • Pulmonary Disease • Extra-pulmonary Disease – Spinal Fluid – Brain → Meningitis – Lymph Nodes – GI and GU – Skeletal, Potts disease 4 Mycobacteria tuberculosis Pathology Most patients have: • Hemoptysis • Productive cough • Fever • Weight loss 5 Mycobacteria tuberculosis Pathology Quiescent Disease Histiocytic phagocytosis 6 Mycobacteria groups Runyon Groups – M. tuberculosis – M. bovis I Slow-growing M. kansasii photochromogens M. marinum II Slow-growing M. scrofulaceum scotochromogens III Slow-growing non- M. avium-intracellulare photochromogens M. xenopi IV Rapid-growing M. fortuitum (<1 week) 7 Processing Specimens Mycobacteria Reagents - TB Digestion Fluid SPUTUM DIGESTION SMEAR CULTURE Methods for TB diagnosis 1. Antibody 2. Antigen 3. PCR 4. Skin testing 5. Culture 6. Microscopy 9 TB Antibody detection Advantages • Rapid assays • Easy to use Disadvantages • Antigens may vary from different suppliers • Lot to lot variability • Specificity poor - <40% of TB detected • May be expensive 10 TB Antigen detection Advantages • Simpler to detect Disadvantages • Machinery may be needed • May be costly • Unknown data on specificity and sensitivity 11 TB PCR detection Advantages – Highly specific- good for epidemiology – Rapid turnaround time (~2.5-7 hours) Disadvantages – Expensive – Requires highly trained personnel – Requires special space for processing 12 TB skin testing Advantages • 65% sensitivity with a 96% specificity • Easy to read • Inexpensive Disadvantages • May not be sufficiently sensitive • Needs trained personnel • Cannot be used as epidemiological tool • Takes up to a week to read. • Hard to read on dark skin 13 TB culture detection Advantages • Accurate • Less expensive than newer techniques Disadvantages • slow to medium slow (7-15 days) • Education needed • Cumbersome • Further tests needed for confirmation Identification - Mycobacteria Biochemical Tests Examples • Catalase Test • Tellurite Reduction Test • Tween 80 Hydrolysis • Urease Test … etc. 15 Types of microscopy • Light Microscopy • Electron Microscopy • Fluorescent Microscopy Mycobacteria Stains– LIGHT MICROSCOPY Carbol Fuchsin • Ziehl Neelson Acid Fast Stain • Kinyoun Stain 17 Mycobacteria Stains LIGHT MICROSCOPY Advantages • Easy to perform • Easy to read • Expensive equipment not needed Disadvantages – Not specific – Not sensitive – Messy – Hard to see organisms in weak infection – Some expertise to perform 18 Mycobacteria Stains- Fluorescent Stains Fluorochromes • AcriFluor- Acridine orange • Auramine Rhodamine - Auramine O & Rhodamine • Modified Auramine O – Auramine O Principle of fluorescence step 1 • Energy is absorbed by the atom which becomes excited Principle of fluorescence step 2 • The electron jumps to a higher energy level Principle of fluorescence step 3 • Soon the electron drops back to ground state • Emitting a photon or packet of light Auramine O Advantages • Fast • Easy to perform 1 minute • Specific for Mycobacteria • Easy to read • Inexpensive Disadvantages • UV Microscope necessary • unfamiliarity 1 minute 23 QBC ParaLens 24 Principle of Diode F.A.S.T. Auramine O TB Stain 26 Study #1 Study: Comparison of 3 commercially Auramine O stains Results: - 185 patients – 13(7%) were positive - F.A.S.T. Auramine O was more sensitive. (Weak positives seen) - F.A.S.T. Auramine O was more cost effective. - Brighter fluorescence observed with F.A.S.T. Auramine O. - It took less time to perform F.A.S.T. Auramine O. - presentation at ASM nat. meeting C-324, 2006 Study #1 Visual Comparison MCC QBC BD REMEL Study #2 Study: Comparison between two Auramine O Stains Results: Both had similar sensitivity and specificity. F.A.S.T. Auramine O gave less background fluorescence, less expensive, and faster to perform J.Clin.Micro.:47:4,2009. EZ Smear Advantages Specimen • Ease of use STAIN STAIN • Portability • Cost • Ease of reading Add Water • Little experience necessary Mix Disadvantages • Slides capable of breaking Wash • Fluorescent microscope necessary Add 2 drops EZ Smear TB Control Slide Advantages • Standardizes staining procedure • Standardizes operator parameters • Meets governmental regulations Disadvantages • Adds to the cost of each assay • Additional slide to read 32 Patient Slide The end … but really the beginning! 34 .