AbstractObjectivesDiagnosis of mycobacterial infections poses significant challenges in anatomic pathology. We recently described the use of antimycobacteria immunohistochemistry (IHC) as a sensitive, efficient diagnostic tool and now report the clinical performance of this assay among general, noninfectious disease pathology-trained anatomic pathologists.MethodsOver a 2-year period, all cases were retrospectively identified in which mycobacterial IHC was performed during routine diagnostic workup.ResultsFrom October 2017 to September 2019, mycobacterial IHC was evaluated for 267 cases, resulting in 58 (22%) positive stains. Compared with culture and molecular results, the sensitivity and specificity of IHC were 52% and 80%, respectively. IHC performed significantly better than acid-fast bacilli (AFB) staining (Ziehl-Neelsen) (P < .0001; sensitivity 21%, specificity 92%) but similarly to modified AFB staining (mAFB; Fite-Faraco) (P = .9; sensitivity 61%, specificity 84%). In cases with discordant IHC and mAFB staining, there were no differences in rates of culture or polymerase chain reaction–confirmed positivity.ConclusionsMycobacterial IHC was well adopted with superior clinical performance to AFB and comparable performance to mAFB. These results support the use of IHC as an adjunctive tool in the diagnosis of mycobacterial infections and suggests its potential role as a rapid screening test for molecular testing.