Comparison of Culture Methods

Routine methods should have the following characteristics: capacity to support the growth of all strains of MAP; high analytical sensitiv­ity; low contamination rate; short incubation period; ease of identification of MAP; low overall cost; and low occupational health and environ­mental impact.

Not all media support the growth of all strains of MAP. However, suitably modified Middlebrook 7H10 agar, 7H11 agar and 7H9 broth (M7H9C and BACTEC 12B media) appear to do so within reasonable incubation periods (Whittington et al., 2011). Common MAP-S strains grow more slowly, poorly or not at all on HEYM, LJ or in MGIT ParaTB medium (Gumber and Whittington, 2007, 2009; Abendano et al., 2012; Dimareli-Malli et al., 2013; Kawaji et al., 2014). HEYM selects for a narrower range of genetic types of MAP than does liquid culture (Cernicchiaro et al., 2008; Whittington et al., 2011).

The various protocols that are used for cul­ture of MAP on solid media have different sen­sitivities (Jorgensen, 1982). Seemingly small differences between protocols can affect both sensitivity and contamination rate. For example, sensitivity for several methodological variations using HPC and HEYM ranged from 39-68% in one study (Eamens et al., 2000) and varied by 10-fold in another (Stabel, 199 7).

For culture of MAP from faeces, liquid cul­ture methods have greater diagnostic sensitivity than protocols based on solid media, regardless of the strain of MAP (Table 18.6). This table shows data for paired samples cultured in both liquid and solid media. Even though the type of liquid medium that was available changed over time, the outcome was always the same. The lower sensitivity of solid media is disguised by the fact that protocols often specify inoculation of multiple (up to four) solid medium slopes. A reduction from four to three HEYM slopes re­duced sensitivity by 8% (Whitlock et al., 1989).

The sensitivity of culture of MAP from milk in BACTEC 12B and MGIT media was similar (Grant et al., 2003) but analytical sensitivity of HEYM (10⁴CFU∕ml) was two logs less than for BACTEC 12B liquid medium (10² CFU∕ml) (Bradner et al., 2013b).

In addition to the percentage of positive cultures always being higher for liquid media, the growth rate of MAP is faster in liquid media than solid media (Cousins et al., 1995; Pozzato et al., 2011). Thus time to reporting a result is shorter with liquid media than with solid media. For example MAP was evident 5 weeks sooner in BACTEC 12B medium than on HEYM slopes (Damato and Collins, 1990). Comparing the MGIT ParaTB system and 7H10 agar, Kawaji et al. (2014) found the time to obtain a final result in faecal culture was several weeks earlier for the MGIT system than the time to confirm MAP colo­nies on the solid medium.

Growth rates on solid media depend on the type of medium. MAP colonies took 3 weeks longer to appear on HEYM than on 7H10 agar (Damato and Collins, 1990). Some strains grow very slowly on HEYM, a finding that has been confirmed by comparing a wide range of MAP iso­lates on a wide range of media (Whittington et al., 2011). The growth of many different strains of MAP on LJ agar was very slow (>12 weeks to ap­pearance of colonies) compared with 7H10 agar (3-4 weeks) (Whittington et al., 2011).

Table 18.6. Table 18.6.In this Studies in which the sensitivity of culture on solid and liquid media were compared? Various decontamination protocols based on hexadecylpyridinium chloride (HPC) were used.

^aOne study where sample size was not stated was excluded from this table but showed similar trends (Pozzato et al., 2011). ^bSeIected NVSL Interlaboratory check test samples. ^cIdentification by: subculture; molecular detection.

^dna, not available.

^eSensitivity increased with the number of slopes or broths inoculated; HEYM data after 12 weeks of incubation; M7H9G data after 6 weeks of incubation.

Cultivation of Mycobacterium avium subsp. paratuberculosis 289

There are no contemporary cost-benefit analyses for any of the culture methods but in­cubation time and the time taken to clarify the status of contaminated cultures are costly to laboratories. In regulatory applications, costs to the users of test results include business risk associated with the delay between submission of samples and receipt of results and the costs of false-negative test results. There is demand from industry for the development of quicker culture methods.

BACTEC 12B medium is a low-level radio­active source, which creates a waste disposal problem in some jurisdictions, whereas the other liquid systems have fewer environmental and occupational health and safety constraints. All methods create substantial laboratory waste through use of disposable plastic-ware, antibiot­ics and other chemicals.

18.17