Drug-resistant tuberculosis is a major health threat; more than 500,000 cases of multidrug-resistant (MDR) tuberculosis occur each year. Multidrug-resistant tuberculosis is resistant to rifampin and isoniazid, the two drugs most often used as effective treatment against tuberculosis. In addition, strains of extensively drug-resistant tuberculosis (XDR TB) are emerging that are resistant not only to rifampin and isoniazid, but also to quinolones and other drugs, such as aminoglycosides and capreomycin.
Standardized methods for susceptibility testing, including direct and indirect testing and new molecular tools, currently are available for susceptibility testing.
M. TUBERCULOSIS COMPLEX
In vitro drug susceptibility testing should be performed on the first isolate of M. tuberculosis from all patients. Susceptibility testing of M. tuberculosis requires meticulous care in the preparation of the medium, selection of adequate samples of colonies, standardization of the inoculum, use of appropriate controls, and interpretation of results. Laboratories that see very few positive cultures should consider sending isolates to a reference laboratory for testing. Isolates must be saved in sterile 10% skim milk in distilled water at −70° C for possible future additional studies (e.g., susceptibilities if the patient does not respond well to treatment).
Direct Versus Indirect Susceptibility Testing
Susceptibility tests may be performed by either the direct or indirect method. The direct method uses as the inoculum a smear-positive concentrate containing more than 50 acid-fast bacilli per 100 oil immersion fields; the indirect method uses a culture as the inoculum source. Although direct testing provides more rapid results, it is less standardized, and contamination may occur.
Conventional Methods
The development of primary drug resistance in tuberculosis represents an increase in the proportion of resistant organisms. This increase in resistant organisms results from a spontaneous mutation and subsequent selection to predominance of these drug-resistant mutants by the action of a single or ineffective drug therapy. A poor clinical outcome is predicted with an agent when more than 1% of bacilli in the test population are resistant. If an isolate is reported as resistant to a drug, treatment failure is likely if this drug is used for therapy.
Drug resistance is defined for M. tuberculosis complex in terms of the critical concentration of the drug. The critical concentration of a drug is the amount of drug required to prevent growth above the 1% threshold of the test population of tubercle bacilli.
Four general methods are used throughout the world to determine the susceptibility of M. tuberculosis isolates to various antituberculous agents (Table 1). Initial isolates of M. tuberculosis are tested against five antimicrobials, which are referred to as primary drugs (Box 1.) If resistance to any of the primary drugs is detected, a second battery of agents is tested (Box 1).
Table1. Overview of Conventional Methods to Determine Susceptibility of M. tuberculosis Isolates to Antimycobacterial Agents
Box1. Antitubercular Agents Commonly Tested against M. tuberculosis
New Approaches
Several technologies recently introduced show promise of being faster, more reliable, and/or easier to perform than most conventional methods of susceptibility testing. For example, mutations leading to rifampin resistance have been detected using molecular methods. One molecular method, the line probe assay (INNO-LiPA Rif TB; Innogenetics, Ghent, Belgium), is a commercially available, reverse hybridization–based probe assay for rapid detection of rifampin mutations leading to rifampin resistance in M. tuberculosis. Many different genotypic assays are currently available for drug susceptibility testing. Most are based on PCR amplification of a specific region of an M. tuberculosis gene, followed by analysis of the amplicon for specific mutations associated with resistance to a particular drug. The presence or absence of mutations can then be detected by several methods, such as automated sequencing.
As previously mentioned, high-density DNA probe assays have been used to detect rifampin resistance and to identify mycobacterial species identification.
An innovative approach used by Jacobs et al.3 to perform susceptibility testing involved the use of a luciferase-reporter mycobacteriophage (bacterial viruses). The basis for this assay is simple: viable mycobacteria can become infected with and replicate the mycobacteriophage; dead tubercle bacilli cannot. The mycobacteriophage was constructed to have the firefly luciferase gene next to a mycobacterial promoter; therefore, the presence and growth of the mycobacteriophage is detected by chemiluminescence. In brief, the isolate of M. tuberculosis to be tested is grown in the presence and absence of drug, and the specially constructed mycobacteriophage is added. After infection, luciferin, a substrate of luciferase, is added. If organisms are viable (i.e., thereby allowing infection of the bacteriophage and sub sequent transcription and translation of the luciferase gene), the luciferin is broken down and light is emitted that can be measured; the amount of light emitted is directly proportional to the number of viable M. tuberculosis organisms. Therefore, if an organism is resistant to the drug, light is emitted; organisms susceptible to the drug do not emit light. Another commercially available assay that uses mycobacteriophages is the FAST Plaque TB–RIF test (Bio Tec Laboratories, Ipswich, UK).
Susceptibility testing should be repeated if the patient remains culture positive after 3 months following appropriate therapy or fails to respond clinically to therapy.
Therapy
Therapy directed against M. tuberculosis depends on the susceptibility of the isolate to various antimicrobial agents. To prevent the selection of resistant mutants, treatment of tuberculosis requires four drugs: isoniazid, rifampin, ethambutol, and pyrazinamide. Initial therapy includes all four drugs for 8 weeks. However, if drug susceptibility is determined for isoniazid, rifampin, and pyrazinamide, ethambutol may be discontinued. This is the preferred therapy for initial treatment, followed by isoniazid and rifampin for an additional 18 weeks. The most common two-drug regimen is isoniazid (INH, also known as isonicotinylhydrazine) and rifampin. The com bination is administered for 9 months in cases of uncomplicated tuberculosis; if pyrazinamide is added to this regimen during the first 2 months, the total duration of therapy can be shortened to 6 months. Ethambutol may also be added to the regimen. INH prophylaxis is recommended for individuals with a recent skin test conversion who are disease free.
NONTUBERCULOUS MYCOBACTERIA
In general, the treatment of patients infected with NTM requires more individualization of therapy than does the treatment of patients with tuberculosis. This individualization is based on the species of mycobacteria recovered, the site and severity of infection, antimicrobial drug susceptibility results, concurrent diseases, and the patient’s general condition. Currently, sufficient data exist to allow general recommendations for susceptibility testing of MAC, M. kansasii, and M. marinum. Pulmonary infections with M. avium complex are often treated with clarithromycin, rifampin, and ethambutol (or streptomycin or amikacin for severe disease). If the infection is disseminated, clarithromycin, ethambutol, and rifabutin may be prescribed. Pulmonary infections with M. kansasii are treated with isoniazid, rifampin, and ethambutol. M. marinum skin and soft tissue infections may be treated with either clarithromycin and ethambutol, clarithromycin and rifampin, or rifampin and ethambutol.
Susceptibility testing should be performed on clinically significant, rapidly growing mycobacteria (Table 2). Skin and soft tissue infections, if susceptible, are treated with clarithromycin and at least one additional drug based on susceptibility testing. Pulmonary infections with M. abscessus should also be treated with a multidrug regimen that includes clarithromycin, if susceptible, and then additional drugs based on susceptibility testing.
Table2. CLSI Recommendations for Susceptibility Testing of Nontuberculous Mycobacteria
