CLINICAL LABORATORY IMMUNOLOGY

Rapid Mycobacterial Liquid Culture-Screening Method for Mycobacterium avium Complex Based on Secreted Antigen-Capture Enzyme-Linked Immunosorbent Assay

Sung Jae Shin, Kelly Anklam, Elizabeth J. B. Manning, Michael T. Collins
DOI: 10.1128/CVI.00461-08

Article Figures & Data

Figures

  • FIG. 1.
    FIG. 1.

    Comparison of single antibody cross-reactivity pre- and postabsorption. Chicken anti-MAC IgY (A) and rabbit anti-MAC IgG (B). Lanes for both panels: 1, Mycobacterium avium subsp. paratuberculosis ATCC 19968; 2, Mycobacterium avium subsp. avium ATCC 35712; 3, Mycobacterium intracellulare ATCC 25122; 4, Mycobacterium phlei ATCC 11758; 5, Mycobacterium terrae ATCC 15755; 6, Mycobacterium scrofulaceum ATCC 19981; 7, Corynebacterium pseudotuberculosis clinical isolate; 8, Escherichia coli ATCC 25922; 9, a mixture of environmental bacteria, including Aeromonas hydrophila, Enterobacter aerogenes, Enterococcus faecalis, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Proteus vulgaris.

  • FIG. 2.
    FIG. 2.

    Schematic diagram of the MAC-ELISA procedure. Key reagents for each step of the MAC-ELISA are indicated in the figure key. MAP, Mycobacterium avium subsp. paratuberculosis; O.D., optical density.

  • FIG. 3.
    FIG. 3.

    Analytical sensitivity of the MAC-ELISA. (A) Enhanced specificity and sensitivity of the MAC-ELISA by absorption of chicken anti-M. avium subsp. paratuberculosis IgY capture antibody and rabbit anti-MAC IgG detector antibody. Lanes: 1, Mycobacterium avium subsp. paratuberculosis ATCC 19968; 2, Mycobacterium avium subsp. avium ATCC 35712; 3, Mycobacterium intracellulare ATCC 25122; 4, Mycobacterium phlei ATCC 11758; 5, Mycobacterium terrae ATCC 15755; 6, Mycobacterium scrofulaceum ATCC 19981; 7, Corynebacterium pseudotuberculosis clinical isolate; 8, Escherichia coli ATCC 25922; 9, a mixture of environmental bacteria, including Aeromonas hydrophila, Enterobacter aerogenes, Enterococcus faecalis, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Proteus vulgaris. (B) Analytical detection limit of the MAC-ELISA using purified M. avium subsp. paratuberculosis (MAP) CFA. (C) Analytical detection limit of the MAC-ELISA using purified M. avium subsp. avium (MAA) CFA.

  • FIG. 4.
    FIG. 4.

    Preliminary assessment of the MAC-ELISA using 1,275 well-defined clinical cultures. (A) Scatter plot (left) of MAC-ELISA OD values for MAC and mycobacteria other than MAC. Each spot represents the MAC-ELISA OD value for a single culture, and the horizontal bar represents the mean OD of the group. Bar and whisker plot (right) of MAC-ELISA OD values. The boxes represent ±standard errors of the means, and the error bars represent 95% CIs. (B) ROC analysis of the scatter plot data. AUC, area under the curve.

  • FIG. 5.
    FIG. 5.

    Clinical application of the MAC-ELISA in conjunction with the MGIT ParaTB medium and MGIT 960 instrument. Neg, negative; Pos, positive; AF, acid-fast; MAP, Mycobacterium avium subsp. paratuberculosis; 2x signal-pos, second time tube signaled positive (see Materials and Methods).

Tables

  • TABLE 1.

    Bacterial strains used to assess specificity of MAC-ELISAc

    Species or organismTotal no. of strainsReference strains included in total no. testedSources other than ATCCaDetection time with MAC-ELISA (wk)b
    Mycobacterium spp.
        M. avium subsp. paratuberculosis13ATCC 19698, K-10JTC3
        M. avium subsp. avium4ATCC 35712, ATCC 25291JTC1 or 2
        M. avium subsp. hominissuis6104JTC, EPA, WSLH1 or 2
        M. intracellulare9ATCC 13950, ATCC 25122JTC, EPA, WSLH1
        M. silvaticum1ATCC 498843
        M. abscessus1ATCC 19977N
        M. asiaticum4ATCC 25276JTCN
        M. bovis3ATCC 19210JTCN
        M. celatum4ATCC 51130JTCN
        M. flavescens2ATCC 14474JTCN
        M. fortuitum2ATCC 49404WSLHN
        M. gordonae2ATCC 14470JTCN
        M. kansasii3ATCC 12478JTCN
        M. lentiflavum2ATCC 51985WSLHN
        M. malmoense1ATCC 29571N
        M. marinum2ATCC 927WSLHN
        M. nonchromogenicum1ATCC 19530
        M. phlei1ATCC 11758N
        M. scrofulaceum7ATCC 19981JTCN
        M. simiae2ATCC 25275WSLHN
        M. smegmatis2ATCC 14468, mc2155N
        M. terrae3ATCC 15755JTCN
    Nonmycobacterial species
        Aeromonas hydrophila1WSLHN
        Corynebacterium pseudotuberculosis1JTCN
        Enterococcus faecalis1ATCC 29212WSLHN
        Enterobacter aerogenes1WSLHN
        Escherichia coli4ATCC 25922WSLHN
        Klebsiella pneumoniae1WSLHN
        Proteus vulgaris1WSLHN
        Pseudomonas aeruginosa1WSLHN
    Unidentified fungi6JTCN
    Total92

    • ↵ a Isolates were identified using mutiplex PCR and HPLC. JTC, Johne's Testing Center, Madison, WI; EPA, Environmental Protection Agency, Cincinnati, OH; WSLH, Wisconsin State Laboratory of Hygiene, Madison, WI.

    • ↵ b Detection time indicates the amount of time for CFA to be completely detected in the assay, when a starting inoculum of 102 CFU/ml of all strains was cultivated in 7H9 broth, up to 8 weeks. N, not detected.

    • ↵ c ATCC, American Type Culture Collection, Manassas, VA.

  • TABLE 2.

    Comparison of time to positive culture between MAC-ELISA and MGIT cultures

    Inoculum CFU/mlTime to positive culture (days)c
    M. avium subsp. paratuberculosis JTC303M. avium subsp. avium ATCC 35712M. phlei ATCC 11758
    MGITaMAC-ELISAbMGITMAC-ELISAMGITMAC-ELISA
    106-1074.873.570.7ND
    105-1067.275.372.6ND
    104-10510.1146.974.3ND
    103-10412.8218.7146.0ND
    102-10317.12110.61410.4ND
    101-10221.52812.72114.8ND
    100-10139.13515.821NDND
    10−1-100NDNDNDNDNDND

    • ↵ a The MGIT 960 instrument measures fluorescence as an indication of microbial growth hourly.

    • ↵ b Culture fluid was tested by the MAC-ELISA weekly.

    • ↵ c ND, not detectable by the MAC-ELISA up to 56 days of incubation.

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KEYWORDS

Antigens, Bacterial
Mass Screening
Mycobacterium avium Complex
Mycobacterium avium-intracellulare Infection
Paratuberculosis

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