Jessica Gentry Indiana State Department of Health Background  Approx. 100 cases of TB annually (2014=108)  Largest Burmese population outside of Burma  State PHL tests approximately 2000 specimens/year  Majority of samples submitted by county health departments 2014 ISDH TB Annual Report Background, (con’t.)  TB Control regulated by the Indiana Communicable Disease Rule (CDR)  Investigation must be performed immediately by local health officer  Infectiousness determined by set of 3 sputa 3 smear negative sputa=release from isolation   As a direct result of the CDR, timeliness of smear results is critical AFB Testing at ISDH  AFB and culture performed for every specimen  Heat fixed on slide warmer for 2 hours  Stained with Auramine o-phenol  AFB rated with CAP scale >50/field, >10/field, 1-10/field, <1/field, <1/smear (very few)   PCR performed for smear positives and high risk smear negatives April 2012—Diagnostic Mycobacteriology Course  False-Positive and false-negative smear and culture results--Ken Jost Acceptable methods  2 hours at 65-75 °C  20 minutes at 80 °C  5% phenol in 70% ethanol for 5 minutes  80 °C Validation  Attempted in August 2012  Panel of 20 smear positive sputa specimens  Tested in duplicate  14/20 specimens exhibited lower AFB counts at 80 °C AFB not adhering  sufficiently to slide?  Additionally, problems observed with hot and cold spots on slide warmer Chedore et. al. Phenol/Alcohol Validation  August 2012  Panel of 40 samples tested in duplicate  30 previously tested sputa 20 smear positives with a  range of AFB counts 10 smear negatives   10 AFB cultures 5 MGITs  5 7H11 plates  Smear Fixing Procedure  Step 1: Pipette a few drops of processed sputa onto the slide  Step 2: Place the slide on the slide warmer for 10 minutes to dry  Step 3: Place the slides in a staining rack over absorbent paper  Step 4: Flood the slide with 5% phenol in 70% ethanol  Step 5: Let the slide soak for 5 minutes  Step 6: Immediately stain the Step 4: Flooding the slide with phenol/alcohol slide Validation Results  100% correlation  Slightly improved smear counts for 13% of smear positive samples Sample Sample Type 65-75 °C Phenol/alcohol 7 Sputa <1/field 1-2/field 14 Sputa <1/field 1-2/field 18 Sputa 8-9/field >10/field 26 7H11 plate Pos (very few) Positive  Could potentially result in the detection of AFB in samples with very low bacillary loads—the difference between a positive and a negative result