Treatment alternatives for Mycobacterium kansasii

John R. Graybilla,b,* and Rosie Bocanegraa

a Department of Medicine, University of Texas Health Science Center, San Antonio, TX 78284; b Infectious Diseases Service, Box 111F, Veterans Administration Hospital, 7400 Merton Minter Blvd, San Antonio, TX 78284, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Mycobacterium kansasii was administered intravenously to congenitally athymic (nude) mice. Beginning 1 week later, rifapentine, azithromycin, ethambutol or combined therapy was initiated orally. All three drugs were highly active individually. Although there was no evidence of antagonism, combined therapy was not more effective than either component used alone.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Among non-tuberculous mycobacteria recovered from patients with HIV infection, Mycobacterium avium– intracellulare (MAC) accounts for well over 90%.1 Mycobacterium kansasii in some series is second to MAC as a cause of serious non-tuberculous mycobacterial infection in patients with AIDS.2,3 Optimal treatment of M. kansasii in non-HIV-infected patients includes isoniazid, rifampicin and ethambutol. Patients with AIDS and M. kansasii infection are at a disadvantage with this traditional regimen, because rifampicin greatly accelerates hepatic metabolism of HIV protease inhibitors, rendering them ineffective. Accordingly, Wallace et al.4 and others5 have recommended clarithromycin as a secondary agent for the treatment of rifampicin-resistant M. kansasii.

In the present study we evaluated two other agents, azithromycin and rifapentine, and compared these with ethambutol, a standard treatment drug. Azithromycin was chosen because it is active in vitro against M. kansasii, is not a strong cytochrome enzyme inducer and is active in vivo in mice.58 Rifapentine was chosen because it is a less intense hepatic microsomal enzyme inducer than rifampicin, is cleared more slowly than rifampicin and is active against M. kansasii. We performed dose ranging for survival studies, explored potential combinations using survival studies and finally conducted a study of tissue burden using one regimen.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
M. kansasii

A clinical isolate (Hill) was obtained from a patient with HIV and M. kansasii disease. Before infection, colonies were inoculated on to Lowenstein agar for 14 days. The organisms were scraped from the plate, washed three times in sterile isotonic saline, counted in a haemocytometer and adjusted to 107 organisms per 0.1 mL mouse dose. All inocula were confirmed by serial colony count dilution, and viable counts are reported.

In vitro testing

The MIC after a 2 week incubation in Middlebrook 7H9 broth was <0.5 mg/L for rifapentine and 2 mg/L for azithromycin and clarithromycin.9

Animal model

BALB/c athymic mice were obtained from our breeding colony, which is certified as following the National Institutes of Health Guidelines for Laboratory Animal Medicine. Mice were infected intravenously at approximately 6 weeks of age. In preliminary screening studies, athymic mice lost weight and eventually succumbed between 2 weeks and 2 months after infection. The speed of attrition was dependent on the inoculum dose.

Treatment regimens

One week after infection, treatment was begun with various drug regimens, with the doses based on prior mouse studies with MAC. As the infecting inoculum was generally in the region of 107 cfu, and because control mice succumbed in a similar time frame, some of the results represent pooled studies. Treatment was continued for 21 days, and mice were observed until day 35 for survival studies. Treatment was given as 0.2 mL gavage, and included rifapentine at 0.15, 0.3 or 0.6 mg/kg/day, ethambutol at 10, 25 or 100 mg/kg/day and azithromycin at 10, 15, 30, 50 or 150 mg/kg/day. Mice were killed on day 35. The spleen and liver were removed under aseptic conditions, homogenized in 2 mL of water and serial 10-fold dilutions were made for colony counts. Cultures were incubated for 2–3 weeks, until colonies were readily visible.

Based on the results of monotherapy survival studies, we tested combinations of suboptimal doses of individual drugs. Because survival studies might be less sensitive than tissue burden to effects of combination therapy, we studied tissue burden. Our intravenous model produces a widely disseminated infection targeting the liver and spleen, and these organs were selected.

Statistics

For survival studies, groups were compared by the Wilcoxon test of life tables. For tissue burden studies non-parametric comparison was carried out using Dunnett's two-tailed t-test. P < 0.05 determined significance.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Survival studies were first carried out with individual drugs. Rifapentine was highly active, with a sharp break between effective (0.6 mg/kg, P < 0.001 compared with control) and ineffective doses (0.3 mg/kg, P = 0.11 compared with control) (Figure 1aGo). Azithromycin also significantly prolonged survival, down to 15 mg/kg/dose, with P = 0.02 compared with control (Figure 1bGo). Ethambutol significantly prolonged survival down to 10 mg/kg, the lowest dose tested, with P < 0.001 compared with controls (Figure 1cGo).



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Figure 1. Survival of mice infected with M. kansasii and treated with various regimens. (a) Treatment of 7–14 mice/group with rifapentine given once daily orally, doses in mg/kg: •, untreated control; {blacktriangleup}, 0.6; {diamond}, 0.3; {blacksquare}, 0.15; {circ}, 0.075. (b) Treatment of seven mice/group with azithromycin given once daily orally, doses in mg/kg: {square}, untreated control; {diamond}, 150; {diamondsuit}, 50; •, 15; {circ}, 10. (c) Treatment of seven mice/group with ethambutol given once daily orally, doses in mg/kg: {triangleup}, untreated control; {square}, 100; •, 25; {blacktriangleup}, 10.

 
Doses for combination studies were chosen to reflect the lowest doses showing antimycobacterial activity in the single drug studies. Rifapentine alone was protective (Figure 2aGo), but was not superior to azithromycin (P = 0.9). As shown in Figure 2bGo, azithromycin and ethambutol did not show any increase in protection over individual drugs (P = 0.9 compared with ethambutol alone). A final study of combination therapy was performed with tissue counts of liver and spleen. As shown in Figure 3Go, rifapentine significantly reduced spleen (P = 0.002) and liver (P = 0.026) tissue burden. Azithromycin was ineffective used alone in spleen (P = 0.07) or liver (P = 0.3). The combination, while superior to controls, was not superior to rifampicin alone.



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Figure 2. Survival of seven mice/treatment group after infection with M. kansasii and treatment with (a) rifapentine (Rpt) and/or azithromycin (Az): ——, untreated control; ----, Az 30 mg/kg/ day; --—--, Rpt 0.3 mg/kg/day; —-—, Az 30 mg + Rpt or (b) azithromycin and/or ethambutol (Eth): ——, untreated control; —-—, Az 10 mg/kg/day; ----, Eth 10 mg/kg/day; •, Az + Eth.

 


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Figure 3. Spleen (left columns) and liver (right columns) tissue burden of M. kansasii, expressed as cfu/organ. Treatments were water (control), rifapentine (Rpt), azithromycin (Az) or both drugs. Bars indicate standard deviation.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The use of rifampicin is a significant problem in highly active antiretroviral therapy (HAART) regimens, which depend on HIV protease inhibitors, because rifampicin accelerates degradation of most protease inhibitors. Azithromycin and clarithromycin have both been used for treatment of MAC, both in mice and in patients with AIDS. Azithromycin is concentrated more heavily in macrophages and has a slower clearance than clarithromycin. Clarithromycin and azithromycin have fewer drug interactions than the rifampicin analogues. Rifapentine may cause less induction of hepatic enzymes, and rifabutin even less. Our present studies suggest that azithromycin may be an acceptable substitute for rifampicin in the treatment of disease caused by M. kansasii. Azithromycin presumably does not interfere with protease inhibitor therapy of HIV. Rifapentine was chosen because it is a new analogue of rifampicin, with less tendency to accelerate hepatic cytochrome enzyme activity. Rifapentine was also highly effective in prolonging survival, and also at a very low dose of 0.6 mg/kg/day reduced counts when given as monotherapy. Combination therapy has traditionally been used, both to prevent emergence of resistance to Mycobacterium tuberculosis, and to shorten duration of therapy. Although we have confirmed the activity of azithromycin, we could not show significant benefit of adding azithromycin to either ethambutol or rifapentine. The doses chosen were selected to optimize potential drug interaction. However, there is a broad interface of doses for potential drug interactions, so the strength of this conclusion is limited to the mouse model, and to the few specific regimens we employed.


    Notes
 
* Correspondence address. Infectious Diseases Service, Box 111F, Veterans Administration Hospital, 7400 Merton Minter Blvd, San Antonio, TX 78284, USA. Tel: +1-210-615-111; Fax: +1-210-614-6197; E-mail: graybill{at}uthscsa.edu Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Montessori, V., Phillips, P., Montaner, J., Haley, L., Craib, K., Bessville, E. et al. (1996). Species distribution in human immunodeficiency virus-related mycobacterial infections: implications for selection of initial treatment. Clinical Infectious Diseases 22, 989–92.[ISI][Medline]

2 . Horsburgh, C. R., Jr & Selik, R. M. (1989). The epidemiology of disseminated nontuberculous mycobacterial infection in the acquired immunodeficiency syndrome (AIDS). American Review of Respiratory Disease 139, 4–7.[ISI][Medline]

3 . Parenti, D. M., Symington, J. S., Keiser, J. & Simon, G. L. (1995). Mycobacterium kansasii bacteremia in patients infected with human immunodeficiency virus. Clinical Infectious Diseases 21, 1001–3.[ISI][Medline]

4 . Biehle, J. & Cavalieri, S. J. (1992). In vitro susceptibility of Mycobacterium kansasii to clarithromycin. Antimicrobial Agents and Chemotherapy 36, 2039–41.[Abstract]

5 . Wallace, R. J., Dunbar, D., Brown, B. A., Onyi, G., Dunlop, R., Ahn, C. H. et al. (1994). Rifampin-resistant Mycobacterium kansasii. Clinical Infectious Diseases 18, 736–43.[ISI][Medline]

6 . Perronne, C., Gikas, A., Truffot-Pernot, C., Grosset, J., Vilde, J. L. & Pocidalo, J. J. (1991). Activities of sparfloxacin, azithromycin, temafloxacin, and rifapentine compared with that of clarithromycin against multiplication of Mycobacterium avium complex within human macrophages. Antimicrobial Agents and Chemotherapy 35, 1356–9.[ISI][Medline]

7 . Witzig, R. S. & Franzblau, S. G. (1993). Susceptibility of Mycobacterium kansasii to ofloxacin, sparfloxacin, clarithromycin, azithromycin, and fusidic acid. Antimicrobial Agents and Chemotherapy 37, 1997–9.[Abstract]

8 . Klemens, S. P. & Cynamon, M. H. (1994). Activities of azithromycin and clarithromycin against nontuberculous mycobacteria in beige mice. Antimicrobial Agents and Chemotherapy 38, 1455–9.[Abstract]

9 . Brown, B. A., Wallace, R. J., Jr & Onyi, G. O. (1992). Activities of clarithromycin against eight slowly growing species of nontuberculous mycobacteria, determined by using a broth microdilution MIC system. Antimicrobial Agents and Chemotherapy 36, 1987–90.[Abstract]

Received 5 September 2000; accepted 24 November 2000