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Chapter 73 : Susceptibility Test Methods: Mycobacteria, , and Other Actinomycetes

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This chapter includes a description of nonradioactive broth culture systems and rapid molecular systems for detection of drug resistance, as well as the standard agar proportion method. The antimicrobial agents that are used in the treatment of mycobacterial infections are discussed in the chapter for the most commonly encountered species. The Clinical and Laboratory Standards Institute (CLSI) document on drug susceptibility testing of mycobacteria currently recommends that first-line testing include ethambutol (EMB), RMP, INH, and pyrazinamide (PZA). The chapter describes drug susceptibility testing of complex. Broth microdilution is the method recommended by the CLSI for susceptibility testing of nontuberculous mycobacteria (NTM). The CLSI provides guidelines for testing complex (MAC), , , and the rapidly growing mycobacteria. General recommendations regarding the broth microdilution method and QC that apply to all NTM are discussed in the chapter. The chapter describes specific details related to MAC, , , and the rapidly growing mycobacteria. It also talks about incubation temperature and time for each species or group. The chapter explains that spp. and other aerobic actinomycetes (, , , , and rarely spp.) can cause serious disease in immunocompromised and occasionally even healthy hosts. The recommended method for testing and other aerobic actinomycetes is broth microdilution.

Citation: Woods G, Lin S, Desmond E. 2011. Susceptibility Test Methods: Mycobacteria, , and Other Actinomycetes , p 1215-1238. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch73

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Citation: Woods G, Lin S, Desmond E. 2011. Susceptibility Test Methods: Mycobacteria, , and Other Actinomycetes , p 1215-1238. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch73
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Tables

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TABLE 1

Antimicrobial agents recommended for primary treatment of common mycobacterial infections

Recommendations for NTM are from reference .

Almost all (>95%) are resistant to pyrazinamide.

Azithromycin is an acceptable alternative agent.

Surgical debridement may be important for successful therapy.

Most common species are group, , and .

Currently there are no drug regimens of proven efficacy. Antimicrobial therapy may provide symptomatic improvement and disease regression. Surgical resection of limited disease (if possible) and multidrug therapy are optimal.

Citation: Woods G, Lin S, Desmond E. 2011. Susceptibility Test Methods: Mycobacteria, , and Other Actinomycetes , p 1215-1238. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch73
Generic image for table
TABLE 2

Genes associated with drug resistance in

Adapted from reference .

Citation: Woods G, Lin S, Desmond E. 2011. Susceptibility Test Methods: Mycobacteria, , and Other Actinomycetes , p 1215-1238. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch73
Generic image for table
TABLE 3

Test concentrations of antimycobacterial agents against

Where more than one concentration for an agent is listed, the lower concentration is the “critical concentration.”

The concentrations shown are from reference .

About 30% of RMP-resistant isolates are rifabutin susceptible.

INH and EMB may first be tested at the critical concentration. When INH or EMB has tested resistant at the critical concentration, the higher concentration of the drugs may be tested with other second-line agents.

NR, not recommended.

Laboratories may choose to test only the lower concentration.

Citation: Woods G, Lin S, Desmond E. 2011. Susceptibility Test Methods: Mycobacteria, , and Other Actinomycetes , p 1215-1238. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch73
Generic image for table
TABLE 4

Guidelines for selection of the dilution of a specimen concentrate prior to inoculation of 7H10 medium for susceptibility testing using the direct method

Dilutions of a concentrated specimen are prepared based on the number of bacilli observed in the initial acid-fast smear. Sterile distilled water is used to prepare the dilutions; the carbol fuchsin stain is examined with the oil immersion objective (1,000×), and the fluorochrome stain is examined with the high-dry objective (450×). If the patient is receiving therapy, not all bacilli observed in the smear may be viable; therefore, the undiluted specimen should be tested as well as the appropriate dilution based on the microscopic criteria given in this table.

rom reference .

Citation: Woods G, Lin S, Desmond E. 2011. Susceptibility Test Methods: Mycobacteria, , and Other Actinomycetes , p 1215-1238. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch73
Generic image for table
TABLE 5

Antimycobacterial agents and interpretative criteria for complex

Table and footnotes are adapted from reference . S, susceptible; I, intermediate; R, resistant.

pH is 7.3 to 7.4 for broth microdilution and 6.8 for BACTEC 460TB

Clarithromycin is the class drug for macrolides and is the only drug that need be tested.

If BACTEC 460TB pH 7.3 to 7.4 is used, breakpoints are ≤4 mg/ml (susceptible), 8 to 16 μg/ml (intermediate), and ≥32 μg/ml (resistant).

Citation: Woods G, Lin S, Desmond E. 2011. Susceptibility Test Methods: Mycobacteria, , and Other Actinomycetes , p 1215-1238. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch73
Generic image for table
TABLE 6

Secondary antimycobacterial agents and MIC values indicating resistance for testing

Table and footnotes are adapted from reference .

Ciprofloxacin and levofloxacin are interchangeable, but both are less active in vitro than moxifloxacin.

Clarithromycin is considered a primary drug in patients receiving the shortcourse and/or intermittent therapeutic regimens consisting of rifampin, ethambutol, and clarithromycin. For patients receiving the classic regimen of rifampin, ethambutol, and isoniazid, clarithromycin is a secondary agent. Clarithromycin is the class representative for the “newer” macrolides (clarithromycin, azithromycin, roxithromycin).

Breakpoints to establish susceptibility and resistance for NTM have not been established. Report the MIC value only, with no interpretation, for these drugs.

Citation: Woods G, Lin S, Desmond E. 2011. Susceptibility Test Methods: Mycobacteria, , and Other Actinomycetes , p 1215-1238. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch73
Generic image for table
TABLE 7

Broth microdilution interpretive criteria for rapidly growing mycobacteria

Table and footnotes are adapted from reference .

Isolates of with an MIC of ≥64 mg/ml should be retested. If the repeat result is ≥64 mg/ml, the MIC should be reported with the following comment: The MIC is greater than expected for this species; if the drug is being considered for therapy, the laboratory should be notified so the isolate can be sent to a reference laboratory for confirmation of resistance.

Ciprofloxacin and levofloxacin are interchangeable. Both are less active than the newer 8-methoxyfluoroquinolones.

The final reading for nonpigmented rapidly growing mycobacteria should be at 14 days to ensure detection of inducible macrolide resistance, unless the isolate is resistant at an earlier reading. Clarithromycin is the class representative for newer macrolides (i.e., azithromycin and roxithromycin).

If the MIC is .> μg/ml for group, group, and group, the test should be repeated with an incubation period of no more than 3 days. If the repeat result is .8 mg/ml, the MIC should be reported with the following comment: The MIC is greater than expected for this species; if the drug is being considered for therapy, the laboratory should be notified so the isolates can be sent to a reference laboratory for confirmation of resistance. Imipenem results do not predict results for meropenem or ertapenem. Activity against rapidly growing mycobacteria is greater for imipenem than for meropenem or ertapenem.

Tobramycin is used predominantly for treatment of infections. If the MIC to tobramycin is >4 μg/ml for an isolate of , the test should be repeated. If the repeat result is >4 mg/ml, the MIC should be reported with the following comment: The MIC is greater than expected for this species; if the drug is being considered for therapy, the laboratory should be notified so the isolate can be sent to a reference laboratory for confirmation of resistance.

Citation: Woods G, Lin S, Desmond E. 2011. Susceptibility Test Methods: Mycobacteria, , and Other Actinomycetes , p 1215-1238. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch73
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TABLE 8

Broth microdilution breakpoints for

Table and footnotes are adapted from reference .

Ciprofloxacin and levofloxacin are interchangeable. Both are less active than the newer 8-methoxyfluoroquinolones.

Class representative for newer macrolides.

Citation: Woods G, Lin S, Desmond E. 2011. Susceptibility Test Methods: Mycobacteria, , and Other Actinomycetes , p 1215-1238. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch73

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