title: Rapid mycobacterial liquid culture-screening method for Mycobacterium avium complex based on secreted antigen-capture enzyme-linked immunosorbent assay.
abstract: Sensors in automated liquid culture systems for mycobacteria, such as MGIT, BacT/Alert 3D, and Trek ESP II, flag growth of any type of bacteria; a positive signal does not mean that the target mycobacteria are present. All signal-positive cultures thus require additional and often laborious testing. An immunoassay was developed to screen liquid mycobacterial cultures for evidence of Mycobacterium avium complex (MAC). The method, called the MAC-enzyme-linked immunosorbent assay (ELISA), relies on detection of MAC-specific secreted antigens in liquid culture. Secreted MAC antigens were captured by the MAC-ELISA with polyclonal anti- Mycobacterium avium subsp. paratuberculosis chicken immunoglobulin Y (IgY), detected using rabbit anti-MAC IgG, and then revealed using horseradish peroxidase-conjugated goat anti-rabbit IgG. When the MAC-ELISA was evaluated using pure cultures of known mycobacterial (n = 75) and nonmycobacterial (n = 17) organisms, no false-positive or false-negative MAC-ELISA results were found. By receiver operator characteristic (ROC) analysis of 1,275 previously identified clinical isolates, at the assay optimal cutoff the diagnostic sensitivity and specificity of the MAC-ELISA were 92.6% (95% confidence interval [95% CI], 90.3 to 94.5) and 99.9% (95% CI, 99.2 to 100), respectively, with an area under the ROC curve of 0.992. Prospective evaluation of the MAC-ELISA with an additional 652 clinical samples inoculated into MGIT ParaTB medium and signaling positive per the manufacturer's instructions found that the MAC-ELISA was effective in determining those cultures that actually contained MAC species and warranting the resources required to identify the organism by PCR. Of these 652 MGIT-positive cultures, the MAC-ELISA correctly identified 96.8% (of 219 MAC-ELISA-positive cultures) as truly containing MAC mycobacteria, based on PCR or high-performance liquid chromatography (HPLC) as reference tests. Only 6 of 433 MGIT signal-positive cultures (1.4%) were MAC-ELISA false negative, and only 7 of 219 MGIT signal-negative cultures (3.2%) were false positive. The MAC-ELISA is a low-cost, rapid, sensitive, and specific test for MAC in liquid cultures. It could be used in conjunction with or independent of automated culture reading instrumentation. For maximal accuracy and subspecies-specific identification, use of a confirmatory multiplex MAC PCR is recommended.
authors.affiliation: Department of Microbiology and Infection Signaling Network Research Center, College of Medicine, Chungnam National University, Daejeon, South Korea.
authors.forename: Sung Jae
authors.initials: SJ
authors.lastname: Shin
authors.forename: Kelly
authors.initials: K
authors.lastname: Anklam
authors.forename: Elizabeth J B
authors.initials: EJ
authors.lastname: Manning
authors.forename: Michael T
authors.initials: MT
authors.lastname: Collins
mesh_terms: D000942:Antigens, Bacterial / Q000276:immunology / Q000302:isolation & purification*; D003951:Diagnostic Errors; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D008403:Mass Screening / Q000379:methods*; D015269:Mycobacterium avium Complex / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D015270:Mycobacterium avium-intracellulare Infection / Q000175:diagnosis / Q000382:microbiology / Q000662:veterinary*; D010283:Paratuberculosis / Q000175:diagnosis*; D012680:Sensitivity and Specificity
chemical_list: D000942:Antigens, Bacterial