Activity of Clarithromycin against Mycobacterium avium Infection in Patients with the Acquired Immune Deficiency Syndrome A Controlled Clinical Trial1 - 3
BERTRAND DAUTZENBERG, CHANTAL TRUFFOT, SOPHIE LEGRIS, MARIE-CAROLINE MEYOHAS, H. CHRISTINE BERLIE, ALAIN MERCAT, SYLVIE CHEVRET, and JACQUES GROSSET
Introduction common complication in patients with late-stage acquired immune deficiency syndrome (AIDS) is disseminated infection with Mycobacterium avium complex. In specimens taken at autopsy from nonselected patients with AIDS, investigators found that 23 to 50070 of cultures show positive results for M. avium (1-3). Studies suggest that several single (4-10) or combined drugs (11-14) may have activity against M avium, but their activity has been implicated only in vitro, in macrophage culture (15-19), and in animal experiments using beige mice infected with M avium (20-25). Antituberculous and antimicrobial agents given in combination have been reported to lower fever and offer transient improvement in some M. avium infections in humans (26-29). No single agent has shown clear antimicrobial effects against the causative organisms (12, 20, 26, 30, 31). Recently, several groups have demonstrated that clarithromycin, a new macrolide, is active against M avium both in vitro and in vivo; its reported minimum inhibitory concentration for 90070 of M. avium strains (MIC 9 o ) measured on 7HlO or 7Hll agar medium has ranged from 4 (20) to 16 (32) ug/ml. It has been found to inhibit the multiplication of M. avium in macrophage culture (15) and in the spleens and lungs of beige mice infected with M. avium (20). Patients receiving clarithromycin in Phases 2 and 3 trials for clinical conditions other than AIDS have tolerated the drug well. Because of these promising findings, we designed a multicenter trial to test the short-term activity of clarithromycin against disseminated M avium infection in humans with AIDS. A randomized, double-blind, placebo-con-
A
564
SUMMARY Disseminated Mycobacterium av;um Infection is common in patients with acquired Immune deficiency syndrome (AIDS), but no drug studies have been reported establishing antlmicrobial activity against this organism in a controlled, randomized trial. Clarithromycln, a new macrolide, has activity against M. avium In vitro and In animals, but It has not been studied In humans. In this randomized, double-blind, placebo-controlled trial, we measured the ability of clarithromycin to reduce M. Bvium bacteremia In patients with AIDS and disseminated Infection. Of 23 patients initially enrolled, 15 men with late-stage AIDS qualified for the stUdy. One group received clarlthromycln alone for 6 wk, then placebo plus rifampln, Isoniazid, ethambutol, and clofazlmine for 6 wk. The other group received placebo alone, then clarlthromycln plus the other four drugs. Colony-forming units (CFU) of M. avlum per milliliter of blood were determined by quantitative cultures taken at baseline and every 2 wk thereafter. Minimum Inhibitory concentration of clarlthromycln for 90% of the strains Isolated from patients at baseline, as measured on 7H11 agar at pH 6.6,was 81!g/ml. Eight eligible patients with Initial positive cultures who were initially receiving clarlthromycln alone had marked declines In blood M. Ivium CFU; In six cases, CFU decreased to zero. When seven patients were switched to placebo plus the other four drugs, CFU rose In four patients and remained undetectable In three. The five eligible patients Initially treated with placebo had progressive CFU Increases; when three were switched to clarlthromycin plus the four drugs, their CFU declined. Weconclude that clarlthromycln has consistent activity against M. avium and may benefit patients with AIDS and disseminated M. evtum infection. AM REV RESPIR DIS 1991; 144:564-569
trolled, modified crossover design was used. Methods
Patients Twenty-three patients were initially enrolled in the study. Inclusion criteria were: positive serologic test results for human immunodeficiency virus, clinical signs and symptoms consistent with M avium infection (i.e., periodic fever, night sweats, malaise, and/or cough), no signs or symptoms suggestive of other infections, pretreatment blood or bone marrow cultures positive for M avium, and age 18 yr or older. Patients were excluded if they had a history of allergy to macrolides, if they were currentlytaking any antimycobacterial or investigational drugs, or if they were judged unlikely to complete the study (i.e., a Karnofsky score < 30 or other signs of imminent decline). Fifteen patients fulfilled the criteria and qualified for evaluation. The pretreatment characteristics of these patients are listed in table 1.
The 15 qualified patients received care in five university-affiliated hospitals. The study design was approved by the Institutional Ethical Review Board of the Groupe Hospitalier Pitie-Salpetriere, Paris; each patient gave signed informed consent in conformity with the Helsinki Declaration and French law. Pa-
(Received in original form December 13, 1990and in revised jorm April 10, 1991) 1 From the Pulmonary Department and the Bacteriology/Virology Laboratories, Groupe Hospitalier Pitie-Salpetriere, Paris; Abbott France, Rungis; Bacteriology Laboratory and Department of Infectious Diseases, Hopital Saint-Antoine, Paris; and the Department of Biostatistics, Hopital St. Louis, Paris, France. 2 Supported by a grant from Abbott Laboratories, Abbott Park, Illinois. J Correspondence and requests for reprints should be addressed to Professor Bertrand Dautzenberg, Service de Pneumologie Groupe Hospitalier Pitie-Salpetriere, 47 Boulevard de l'hopital, 75651, Paris, Cedex 13, France.
565
ACTIVITY OF CLARITHROMYCIN AGAINST M. AVIUM INFECTION IN AIDS
TABLE 1 CHARACTERISTICS OF ELIGIBLE PATIENTS AT START OF STUDY
Race: white/black/Asian Mean age, yr Body weight, kg Risk factors: homosexual/hemophilia Months since HIV diagnosis Opportunistic infections in last 12 months, n Diagnoses in last 12 months, n of patients Pneumocystosis Cytomegalovirus infection Cryptococcosis Kaposi sarcoma Positive M. avium cultures Blood < 5 CFU/ml ~ 5 CFU/ml Urine Sputum Patients receiving other medications, n Karnofsky index Dyspnea score Temperature, °C White blood cell count, /mm 3 Lymphocytes, /mm 3 CD 4 count, /mm 3 Platelets, /mm 3 Hemoglobin, mg/100 ml Alkaline phosphatase, UlL Serum glutamic-oxaloacetic transaminase, U/L
Group 1 (n = 8)
Group 2 (n = 7)
6/1/1 37 ± 11 57 ± 9 6/126 ± 16 3.4 ± 0.7
7/0/0 34 ± 8 52 ± 8 6/1 32 ± 18 3.7 ± 2.8
4
3
2 2
1 1
1
2
5
4
3 1 3
3 2
2
8t 63 0.2 38.0 4,380 453 19.4 258,000 9.4 317 22.3
± ± ± ± ± ± ± ± ± ±
7:1: 21 0.5 0.7 2,390 164 11.4 196,600 1.5 479 13.7
56 ± 0.7 ± 38.1 ± 2,110 ± 193 ± 12.1 ± 157,1OO± 3.1 ± 170 ± 54.4 ±
15 1.0 0.5 700§ 91§ 20.9 83,200§ 1.6 109 33.2
- One patient not included had no discernible risk factors but was from Zaire. Drugs used by patients in this group included zidovudine (AZT) (three), pentamidine (three), and fluconazole (six). :j: Drugs used by patients in this group included zidovudine (six), pentamidine (four), and fluconazole (five). § Significantly less than values for Group 1 patients.
t
tients wereentered between October 1988and November 1989.
to be the main parameters to assess the activity of clarithromycin.
Study Design The patients were randomized into two groups. During Phase 1 of the study (6 wk), patients in Group 1 received 1,000 mg clarithromycin orally twice daily for 6 wk, and those in Group 2 received placebo (figure 1). At the end of the 6 wk, patients were crossed over to Phase 2 (also 6 wk). Those who received clarithromycin in Phase 1 were given placebo plus a four-drug combination: rifampin (10 mg/kg daily), isoniazid (5 mg/kg daily), ethambutol (20 mg/kg daily), and clofazimine (100 mg daily). Patients receiving placebo during Phase 1 received clarithromycin in the dosage cited above plus the same four-drug combination during Phase 2. The protocol made provision for termination after 3 wk in Phase 1 if the patient's clinical condition deteriorated; the patient could then switch to Phase 2. At no point was the doubleblind broken. The main parameter for efficacy of treatment was difference in numbers of colonyforming units (CFU) of M. avium in the two study groups as determined by quantitative blood cultures. Blood was cultured within 48 h before therapy was begun and every2 wk thereafter to determine patients' responses to clarithromycin or placebo in Phase 1 and the combination of drugs in Phase 2. Clinical outcomes were recorded but were not considered
Culture Methods On the days when blood was sampled, three lO-ml aliquots were drawn at 30-min intervals.Samples werecollectedinto isolator tubes (DuPont Instruments, Newtown, CT) and immediately transferred to the laboratory. Each sample was centrifuged at 3,000 x g for 30 min, and the pellet was divided into two parts: a OJ-ml pellet was mixed with 0.9 ml sterile water, and 0.25 ml of this was inoculated onto each of four tubes of Loewenstein-Jensen medium. The remaining undiluted pellet was divided into seven equal parts of about 0.25 ml each; these wereinoculated onto five tubes of plain Loewenstein-Jensen medium and onto two tubes of Loewenstein-Jensen medium containing 0.5% sodium pyruvate. All inoculations were completed within 3 h after venipuncture. All tubes except one were incubated at 37° C; the remaining tube, inoculated with undiluted pellet, was incubated at 42° C. The tubes were examined weekly for as long as 3 months for bacterial growth and enumeration of colonies. By this method, the lowest detectable CFU value was 0.03 per ml of blood, i.e., 1 CFU/30 ml of blood. Isolated strains of M avium were sent to a central bacteriology laboratory for identification, including serotyping, and drug susceptibility testing; identification was done according to standard procedures. At the end
of the study, pretreatment strains and those isolated from the last cultures were tested simultaneously for susceptibility to the drugs used.
Drug Susceptibility Testing and Serotyping Minimum inhibitory concentrations (drug concentration required to inhibit ~ 99% of CFU) of clarithromycin, rifampin, and clofazimine were determined on 7Hll agar at pH 6.6 (Difco Laboratories, Detroit, MI) containing twofold concentrations of drugs ranging from 0.5 to 16,0.5 to 512,and 0.06to 2Ilg/ml, respectively. Inocula consisted of 0.05 ml of two dilutions (10-3 and 10-5 mg/ml wet weight) of 7-day-old cultures of M. avium in Tweenalbumin broth (Dubos medium). Cultures were incubated at 37° C with 5OJo carbon dioxide for 21 days before determinations of MIC. Susceptibility to isoniazid (0.1,0.2, 1.0, and 10ug/ml) and ethambutol (2Ilg/ml) was determined on Loewenstein-Jensen medium, using the proportion method routinely employed in the laboratory (33). Clinical Status and Safety Patients had their general condition monitored weekly as part of their overall medical care. General status was scored using the Karnofsky performance scale.The followingwere rated on a scale of zero to 3: sputum appearance (0 = absent, 3 = green); cough (0 = absent, 3 = severe enough to cause chest pain and prohibit normal activities); dyspnea (0 = absent, 3 = severe enough to prohibit normal activities); night sweats (0 = absent, 3 = five or more times a week); malaise (0 = none present, 3 = severe).Rales, rhonchi, and cyanosis were scored as 0 (absent) or 1 (present) and patients' subjective assessment of their quality of life as 1 (better), 2 (no change since last visit), or 3 (worse). All spontaneous complaints wererecorded. Safety of treatment was also monitored by the followinglaboratory studies: blood count with complete differential and platelet count, lymphocyte count, coagulation tests, blood urea nitrogen, serum glutamic-oxaloacetic transaminase (SGOT), serum glutamic-pyruvic transaminase (SGPT), alkaline phosphatase, bilirubin, protein/albumin, sodium, potassium, and uric acid. Urinalysis was also done once a week. After half of the patients had entered the study, clinical hypoacusis was noted in one patient; thereafter, audiograms were performed at the beginning and end of each phase. Statistical Analysis The numbers of patients with no change, increase, or decrease in CFU in the two groups were compared over time using Fisher's test. A model was also constructed to analyze changes in CFU in each group (log scale)using a linear model. Therefore, only patients with at least two measures (one within the 48 h before treatment and one after treatment) wereanalyzed. The slopes of the linear models
566
DAUTZENBERG, TRUFFOT, LEGRIS, MEYOHAS, BERLlE, MERCAT, CHEVRET, AND GROSSET
for the two groups were compared using the Mann-Whitney nonparametrical test. A similar approach was used for three selected clinical parameters: fever, Karnofsky index, and dyspnea. All patients with available data were analyzed. Values are expressed as mean ± standard error of the mean (SEM). Because of some treatment differences in Phase 1, valid comparisons between the two groups could not be made for Phase 2 results.
cal status was deteriorating (Patients 5 and 6). Twowere dropped from the study near the end of Phase 1because their condition worsened (Patients 10 and 13). During Phase 2, two patients receiving placebo plus the drug combination were removed from the study because of clinical deterioration, one after 2 wk (Patient 2) and the other before the end of the sixth week (Patient 7).
Results
Drug Susceptibility Jesting
Comparability of Groups and Attrition Of the 23 patients originally enrolled in the study, eight were excluded, seven because of negative pretreatment blood cultures for M avium and one because of poor compliance. Thus, 15patients qualified for analysis: eight in Group 1 and seven in Group 2 (table 1). Because of two early deaths, however, the activity of clarithromycin could be analyzed in only 13 patients, eight in Group 1 and five in Group 2. Characteristics of the patients in the two groups were comparable at the time of entry into the study except for somewhat lower counts of white blood cells, lymphocytes, and platelet counts in Group 2 (table 1). Details of attrition and early crossover are as follows: One patient from Group 1 discontinued participation after 2 wk during Phase 1 because of acute hepatitis (Patient 8). Twoothers were switched to Phase 2 at the end of 4 and 5 wk because the physicians felt that their clini-
Minimum inhibitory concentrations of clarithromycin as measured on 7Hll agar at pH 6.6 (Difco) for 50 and 90070 of pretreatment strains were 8 and 8 ug/ml, respectively. The corresponding values for clofazimine were0.25 and 0.5 ug/ml, respectively; for rifampin, the values were 16 and 32 ug/ml, respectively. All strains were resistant to isoniazid and ethambutol (MIC: 2 to 10and 2 ug/ml, respectively). The last positive culture for each patient was studied for susceptibility to clarithromycin and the other four drugs, and no significant change in the MIC was found. Among the eight strains isolated from Group 1 patients, one belonged to Serotype 6, two to Serotype 8, two to Serotype 9, and one to Serotype 23; the serotype could not be determined for the remaining two. Among the seven strains isolated from Group 2 patients, two belonged to Serotype 8 and one to Serotype 9; serotype could not be determined for the remaining four.
M. avium in Blood Cultures The initial concentrations of M. avium in blood specimens differed widely among patients in the study. In Group 1 patients, initially treated with clarithromycin, values ranged from 2 to 7,670 CFU/30 ml (0.07 to 253 CFU/m1); in Group 2 patients, initially treated with placebo, values ranged from 3 to 18,240 CFU/30 ml (0.3 to 640 CFU/ml). However, the numbers of CFU obtained from three blood specimens taken from patients at each sampling time were always very consistent; the differences from one sample to another were less than 1 log. As shown in table 2, there were no significant inconsistences between M avium CFU recovered from undiluted blood samples and those recovered from samples diluted 1:10. During Phase 1, M avium disappeared from blood cultures of five clarithromycin-treated patients by 2 wk oftreatment; in two others M avium disappeared by 4 wk. One of the latter, Patient 1, had 1 CFU isolated at 6 wk. One patient in the clarithromycin-treated group (Patient 3) had cultures that remained weakly positive, probably because of his high CFU count before treatment (table 2 and figure 1). The mean decrease in M avium in blood during the 6 wk of clarithromycin treatment was 2.65 log. Unlike clarithromycin-treated patients, all five placebo-treated patients had increasesin M avium CFU (mean increase, 1.55 log over 6 wk).
TABLE 2 COLONY-FORMING UNITS OF M. AVIUM ISOLATED FROM UNDILUTED AND 10:1 DILUTED LYSATES OF 30-ML BLOOD SAMPLES FROM PATIENTS WITH AIDS BEFORE AND DURING TREATMENT Phase 2
Phase 1
Patient No.
2 Weeks
Pretreatment Undil
Dil
Undil
4 Weeks
Dil
Dil
Undil
Crossover Pretreatment
6 Weeks Undil
Dil
Undil
808 161 7,100 20 27 2 6 5
0 32 570 0 7 0 0 0
3 1 25 0 0 0 0 0
9 10 11 12 13 14 15
14 20 530 3 18,240 62 2,162
0 79 78
322 830 1,300 4 23,200 Died Died
0 2 3
O· 0 0 0 0
0 0 1 ND 0 0 0 DT
0 0 2 NO 0 0 0 DT
1
d 2 0 EC EC 0 DT
0 0 0 0 EC EC 0 DT
1 0 2 0 0 0 0 DT
EC DT 137
31 DT 1,400 34 DT
O· 960 3 0
O· 800
126 930 1,580 29 22,800
523 430 265
o·
1,200
Definition of abbreviations: EC = early crossover; OT = discontinued therapy; NO
• Only 10 ml of blood cultured.
4 Weeks
Dil
Undil
6 Weeks
Dil
Undil
Dil
0 0 0 0 0 0 0 DT
17 3 2 0 1 0 0 DT
0 0 0 0 0 0 0 DT
12 DT 3 0 0 ND 0 DT
0 DT 1 0 0 ND 0 DT
41 DT 8 0 0 0 DT DT
0 DT 0
O· 0 0 DT DT
Group 2: Clarithromycin + four-drug combination
Group 2: Placebo 39 122 46
2 Weeks Undil
Group 1: Placebo + four-druq combination
Group 1: Clarithromycin 1 2 3 4 5 6 7 8
Dil
Ed DT 1,400 34 DT
=
O· DT
48 DT 137
O· DT
3 DT 54 0 DT
2 DT 17
O· DT
not done; Undil = undiluted culture; Oil = culture diluted 10:1.
6 DT 3 0 DT
0 DT 1
O· DT
6 DT 0 0 DT
0 DT 0
o·
DT
567
ACTIVITY OF CLARITHROMYCIN AGAINST M. AVIUM INFECTION IN AIDS
Phase 1: Clarithromycin
Phase 2: Placebo
1000 100 Fig. 1. Changes in CFU of M. avium in blood cultures of Group 1 patients receiving clarithromycin during Phase 1 of the trial and placebo plus a fourdrug combination (rifampin, isoniazid, ethambutol, and clofazimine) during Phase 2. Dashed line indicates early crossover. Open triangles = Patient 1; closed squares = Patient 2; X = Patient 3; closed triangles = Patient 4; open circles = Patient 5; crosses = Patient 6; open squares = Patient7; closed circles = Patient 8.
E
:5 LL o
e
::J oS; CQ
4
During Phase 2, among patients who initially received clarithromycin and were crossed over to placebo plus a four-drug combination, the numbers of CFU increased in those with positive blood cultures at the end of Phase 1. M avium reappeared in the blood cultures of one patient who previously had negative cultures after 4 wk of clarithromycin treatment (Patient 2) and in another who was an early crossover to Phase 2 (Patient 5). Both of these patients had more than 1 CFU/ml before the trial (figure 1). However, this rebound in the number of bacteria in the blood did not reach statistical significance. During Phase 2, among patients initially given placebo and crossed over to clarithromycin plus the four-drug combination, the CFU
Clinical Outcomes and Safety During Phase 1, the patients treated with clarithromycin had a mean decrease in fever ( - 0.2 ± 0.4) and improvement in Karnofsky score (1.4 ± 12.1); conversely, the patients treated with placebo had an increase in fever (0.2 ± 0.6) and worsening of Karnofsky score (-12.0 ± 13.0). However, the differences observed
Phase 1: Placebo
1000 100 Fig. 2. Changes in CFU of M. avium in blood cultures of Group 2 patients receiving placebo during Phase 1 of the trial and clarithromycin plus a four-drug combination (rifampin, isoniazid, ethambutol, and clofazimine) during Phase 2. Dashed line indicates early crossover. Open squares = Patient 9; open circles = Patient 10; closed squares = Patient 11;closed circles = Patient 12;open triangles = Patient 13;closed triangles = Patient 14; crosses = Patient 15.
counts decreased. Of the three. patients switched to Phase 2, two had no detectable M. avium in blood cultures after 6 wk of treatment with clarithromycin (figure 2). The third had 6 CFU/30 ml as compared with 646 CFU/30 ml at the beginning of Phase 2. The mean decrease in CFU during the 6 wk of treatment was 1.34 log.
E
:5 LL c
o§ ~
CQ
0.0
o
Phase 2: Clarithromycin
in the two groups during Phase 1 did not reach statistical significance. During Phase 2, the patients treated with placebo and four drugs had an increase in fever (0.4 ± 1.1)and worsening of Karnofsky score (-12.9 ± 21.4). Conversely, the patients treated with clarithromycin and four drugs had a decrease in fever ( - 0.7 ± 0.3) and a small change in Karnofsky score (- 3.5 ± 5.8). Statistical evaluation was not done during Phase 2 because the groups were no longer comparable. Of the 11 patients who underwent at least one audiogram, one had hypoacusis before treatment was started, one developed it during Phase 1 placebo treatment, and one developed it after clarithromycin treatment. In all cases, decrease in hearing was mild. One patient had hypothermia (34 0 C) while receiving placebo during Phase 1. In one patient receiving clarithromycin during Phase 1, treatment was stopped because of an increase in SOOT (25 to 55 U/L) and alkaline phosphatase (65 to 266 U/L). While receiving placebo during Phase 1, another patient had increases in SOOT (51 to 645 U/L); the level rose further during administration of clarithromycin and the four-drug combination (as much as 1,130 U/L), but because of clinical improvement the patient was continued in the trial. No difference in gastrointestinal tolerance was observed between the groups. Only two patients spontaneously complained of nausea during Phase 1, one receiving clarithromycin and the other receiving placebo. Discussion The main finding of our trial, based on blood cultures, was that clarithromycin has definite activity against M avium in patients with AIDS. All eight patients who were treated with clarithromycin during Phase 1 had steep decreases in their M avium CFU counts in blood. In contrast, all seven patients treated with placebo had persistent positive cultures and gradually increasing CFU counts. The effectiveness of clarithromycin was also strongly suggested by the results of blood cultures during Phase 2 of the trial. Patients who initially received placebo and were crossed over to clarithromycin plus the four-drug combination (rifampin, isoniazid, ethambutal, and clofazimine) had marked declines in their CFU levels. We could not attribute this effectiveness to the four-drug combination during Phase 2 because four
568
of the patients initially treated with clarithromycin and then crossed over to placebo plus the four-drug combination either had cultures that converted from negative to positive during Phase 2 or had increases in their CFU counts. Therefore, our results suggest that the four-drug combination was not effective in affectmg the M avium load in blood of patients with AIDS. Although our data indicate that clarithromycin shows definite activity against M avium within 6 wk of treatment, this interval is definitely not enough to cure all patients with AIDS of the infection. Blood cultures reflect only the concentration of organisms released from the tissue and circulating in the blood. A much longer period of treatment is probably required to eradicate M avium from both tissues and blood. Our clinical observations are consistent with encouraging results from studies of disseminated M avium infection in beige mice treated with clarithromycin (50 or 100 mg/kg daily) (20) and from experiments using human macrophages infected with M avium and cultured in a medium containing clarithromycin (4 ug/ml) (15). However, we found a discrepancy between clinical activity and our in vitro data: in our study, the MIC 9 0 of clarithromycin against M. avium was 8 ug/rnl, whereas the peak serum level reported in pharmacokinetic studies in humans was only about 4 J,1g/ ml after administration of 1,000mg twice daily. Taking into account the fact that M. avium is located primarily inside the cells of patients with AIDS, it may be that the discrepancies between MIC 9 0 values in vitro and plasma levelsachieved clinically could be due to concentration of clarithromycin by the cells. Indeed, in vitro studies have demonstrated an liE ratio (intracellular to extracellular concentration) of 9.2 (34). Alternatively, clarithromycin may be inactivated by the medium used to determine MIC, i.e., 7Hll at pH 6.6 on agar medium (Difco). In agar most drugs are absorbed, and macrolides are partially inactivated at low pH (35). It is likely that the MICs of clarithromycin against M avium would be lower if determined at pH equivalent to that of blood. Wewere unable to demonstrate more clinical improvement because the sample sizesof the two groups were small and the overallsymptoms of our patients were moderate (no high fever, etc.). Side effects after clarithromycin ad-
DAUTZENBERG. TRUFFOT, LEGRIS, MEYOHAS, BERLlE, MERCAT, CHEVRET, AND GROSSET
ministration weremild when they did occur. Transient hypoacusis, although observed after clarithromycin treatment, was also found before treatment or during placebo treatment. Elevation of SGPT and/or alkaline phosphatase occurred in two patients, one receiving clarithromycin and the other receiving placebo. In summary, our data suggest that clarithromycin has clear activity against M avium and may benefit patients with AIDS and disseminated M. avium infection. However, we must emphasize that our findings are based on a short-term trial involving only small numbers of patients. Moreover, the assessment of antimicrobial activity was based mainly on blood cultures. Future studies must be designed to obtain information about optimal dosage, length of treatment required to achieve the best therapeutic results, and the potential risk of acquired resistance during longer periods of clarithromycin monotherapy. Therefore, the search for active companion drugs that will lessen or prevent the risk of acquired resistance is of the utmost importance. Acknowledgment The writers thank the following physicians who participated in the protocol and entered patients into the study: V. Averous, D. Terminassian, G. Arlet, J. Modai', and J. M. Decazes, Hopital Saint Louis, Paris; J. Merrer and M. Le Neveu, Hopital de Poissy; D. Quinsat and T. Fosse, Hopital d'Antibes, Nice; P. Boudes and L. Desforges, Hopital Henri Mondor, Creteil; M. Siffert and C. Perez, Centre Medico Chirurgical Gui de Chauliac, Montpellier; G. Guermonprez, D. Tosoni, A. Coutelier, and F. Antoun, Groupe Hospitalier Pitie-Salpetriere, Paris; P. Brion and S. Duborgel, Hopital de la Tronche, Grenoble; J. Lacronique and G. Paul, Hopital Cochin, Paris; M. Eliazewitch and C. Kristetter, Hopital Saint-Antoine, Paris; Y. Laurian, Hopital Bicetre, Kremlin-Bicetre; J. P.Chauvin, Abbott France, Rungis; Cl. Chastang, Biostatistician, DBIM Hopital St. Louis, Paris; and P. Prokocimer, Abbott Laboratories, USA. The writers also thank Barbara Cox, Biomedical Editor, for her assistance in preparing the manuscript for publication. References 1. Truffot-Pernot C, Lecoeur H, Maury L, Dautzenberg B, Grosset J. Results of blood cultures for detection of mycobacteria in AIDS patients. Tubercle 1989; 70:187-91~ 2. Wilkes MS, Fortin AH, Felix JC, Godwin TA, Thompson WG. Valueof necropsy in acquired immunodeficiency syndrome. Lancet 1988; 2:85-8. 3. Wallace JM, Hannah JB. Mycobacterium avium complex infection in patients with the acquired immunodeficiency syndrome. A clinicopathologic study. Chest 1988; 93:926-32.
4. Yajko DM, Nassos PS, Hadley WK. Broth microdilution testing of susceptibilities to 30 antimicrobial agents of Mycobacteriumavium strains from patients with acquired immune deficiency syndrome. Antimicrob Agents Chemother 1987; 31: 1579-84. 5. Gangadharam PRJ, Kesavalu L, Rao PNR, Perumal VK, Iseman MD. Activity of amikacin against Mycobacterium avium complexunder simulated in vivo conditions. Antimicrob Agents Chemother 1988; 32:886-9. 6. Cynamon MH, KlemensSP. Newantimycobacterial agents. Clin Chest Med 1989; 10:355-64. 7. Heifets LB, Lindholm-LevyPJ, Flory MA. Bactericidal activity in vitro of various rifamycins against Mycobacterium avium and Mycobacterium tuberculosis. Am Rev Respir Dis 1990; 141: 626-30. 8. Fenlon CH, Cynamon MH. Comparative in vitro activities of ciprofloxacin and other 4-quinolones against Mycobacterium tuberculosis and Mycobacterium intracellulare. Antimicrob Agents Chemother 1986; 29:386-8. 9. Inderlied CB, Young LS, Yamada JK. Determination of in vitro susceptibility of Mycobacterium avium complex isolates to antimycobacterial agentsby various methods. Antimicrob Agents Chemother 1987; 31:1697-702. 10. Khardori N, Rolston K, Rosenbaum B, Hayat S, BodeyGP. Comparative in vitro activity of twenty antimicrobial agents against clinical isolates of Mycobacterium avium complex. J Antimicrob Chemother 1989; 24:667-73. 11. Banks J, Jenkins PA. Combined versus single antituberculosis drugs on the in vitro sensitivity patterns of non-tuberculous mycobacteria. Thorax 1987; 42:838-42. 12. Heifets LB, Iseman MD, Lindholm-Levy PJ. Combination of rifampin or rifabutine plus ethambutol against Mycobacterium avium complex: bactericidal synergistic, and bacteriostatic additive or synergistic effects. Am Rev Respir Dis 1988; 137: 711-5. 13. Yajko DM, Kirihara J, Sanders C, Nassos P, Hadley WK. Antimicrobial synergism against Mycobacterium avium complex strains isolated from patients with acquired immune deficiencysyndrome. Antimicrob Agents Chemother 1988; 32: 1392-5. 14. Heifets LB. Synergistic effect of rifampin, streptomycin, ethionamide, and ethambutol on Mycobacterium intracellulare. Am Rev Respir Dis 1982; 125:43-8. 15. Perrone C, Gikas A, Truffot-Pernot C, Grosset J, Pocidalo J J, Vilde JL. Clarithromycin, sulisoxazoleand rifabutin activity against Mycobacterium avium complex multiplication within human macrophages. Antimicrob Agents Chemother 1990; 34:1508-11. 16. Yajko DM, Nassos PS, Sanders CA, Hadley WK. Killing by antimycobacterial agents of AIDSderived strains of Mycobacterium avium complex inside cells of the mouse macrophage cell line J 774. Am Rev Respir Dis 1989; 140:1198-203. 17. Crowle AJ, Elkins N, May MH. Effectiveness of ofloxacin against Mycobacterium tuberculosis and Mycobacterium avium, and rifampin against M tuberculosis in cultured human macrophages. Am Rev Respir Dis 1988; 137:1141-6. 18. Bermudez LEM, Young LS. Activities of amikacin, roxithromycin, and azithromycin alone or in combination with tumor necrosis factor against Mycobacterium avium complex. Antimicrob Agents Chemother 1988; 32:1149-53. 19. Perumal VK, Gangadharam PRJ, Iseman MD. Effect of rifabutin on the phagocytosis and intracellular growth of Mycobacterium intracellulare in
ACTIVITY OF CLARITHROMYCIN AGAINST M. AV/UM INFECTION IN AIDS
mouse resident and activated peritoneal alveolar macrophages, Am Rev Respir Dis 1987; 136:334-7. 20. Fernandes PB, Hardy DJ, McDaniel D, Hanson CW, Swanson RN. In vitro and in vivo activities of ciarithromycin against Mycobacterium avium. Antimicrob Agents Chemother 1989; 33: 1531-4. 21. Gangadharam PRJ, Peru mal VK, Jairam BT, et al. Activity of rifabutin alone or in combination with ciofazimine or ethambutol or both against acute and chronic experimental Mycobacterium intracellulare infections. Am Rev Respir Dis 1987; 136:329-33. 22. Saito H, Saito K. Activity of rifabutin alone and in combination with ciofazimine, kanamycin and ethambutol against Mycobacterium intracellulare infections in mice. Tubercle 1989; 70:201-5. 23. Gangadharam PRJ, Perumal VK, Podapati NR, Kesavalu L, Iseman MD. In vivo activity of amikacin alone or in combination with ciofazimine or rifabutin or both against acute experimental Mycobacterium avium complex infection in beige mice. Antimicrob Agents Chemother 1988; 32: 1400-3. 24. Inderlied CB, Kolonoski PT, Wu M, Young LS. Amikacin, ciprofloxacin, and imipenem treat-
ment for disseminated Mycobacterium avium complex infection in beige mice. Antimicrob Agents Chemother 1989; 33:176-80. 25. Cynamon MH, Swenson CE, Palmer GS, Ginsberg RS. Liposome-encapsulated amikacin therapy of M avium complex infection in beige mice. Antimicrob Agents Chemother 1989; 33: 1179-83. 26. Agins BD, Berman DS, Spicehandler D, El Sadr W, Simberkoff MS, Rahal J J. Effect of combined therapy with ansamycin, clofazimine, ethambutol and isoniazid for Mycobacterium avium infection in patients with AIDS. J Infect Dis 1989; 159:784-7. 27. American Thoracic Society and Centers for Disease Control. Mycobacterioses and the acquired immunodeficiency syndrome. Joint Position Paper. Am Rev Respir Dis 1987; 136:492-6. 28. Hoy J, Mijch A, Sandland M, Grayson L, Lucas R, Dwyer B. Quadruple-drug therapy for Mycobacterium avium-intracellulare bacteremia in AIDS patients. J Infect Dis 1990; 161:801-5. 29. Chieu J, Nussbaum J, Bozzerre S, et al. Treatment of disseminated Mycobacterium avium complex infection in AIDS with amikacin, ethambutol, rifampin and ciprofloxacin. Ann Intern Med 1990;
569 113:358-61. 30. Hawkins CC, Gold JWM, Whimbey E, et al. Mycobacterium avium complex infections in patients with the acquired immunodeficiency syndrome. Ann Intern Med 1986; 105:184-8. 31. Young LS. Mycobacterium avium complex infection. J Infect Dis 1988; 1988; 157:863-7. 32. Berlin OGW, Young LS, Floyd-Reising SA, Bruckner DA. Comparative in vitro activity of the new macrolide A-56268 against mycobacteria. Eur J Clin Microbiol 1987; 6:486-7. 33. Canetti G, Rist N, Grosset J. Mesurede la sensibilite du bacille tuberculeaux aux drogues antibacillaires par la methode des proportions. Methodologie, criteres de resistance, resultats, interpretation. Rev Tuberc Pneumol1963; 27:217-72. 34. Anderson R, Joone G, Van Rensburg CEJ. An in vitro evaluation of the cellular uptake and intraphagocytic bioactivity of clarithromycin (A = 56268, TE-301), a new macrolide antimicrobial agent. J Antimicrob Chemother 1988; 22:923-33. 35. Barry AL, Jones RN, Thornsberry C. Disk diffusion and disk elution test with A 56268 and erythromycin. Eur J Clin Microbiol1987; 6:109-11.
BERTRAND DAUTZENBERG, CHANTAL TRUFFOT, SOPHIE LEGRIS, MARIE-CAROLINE MEYOHAS, H. CHRISTINE BERLIE, ALAIN MERCAT, SYLVIE CHEVRET, and JACQUES GROSSET
Introduction common complication in patients with late-stage acquired immune deficiency syndrome (AIDS) is disseminated infection with Mycobacterium avium complex. In specimens taken at autopsy from nonselected patients with AIDS, investigators found that 23 to 50070 of cultures show positive results for M. avium (1-3). Studies suggest that several single (4-10) or combined drugs (11-14) may have activity against M avium, but their activity has been implicated only in vitro, in macrophage culture (15-19), and in animal experiments using beige mice infected with M avium (20-25). Antituberculous and antimicrobial agents given in combination have been reported to lower fever and offer transient improvement in some M. avium infections in humans (26-29). No single agent has shown clear antimicrobial effects against the causative organisms (12, 20, 26, 30, 31). Recently, several groups have demonstrated that clarithromycin, a new macrolide, is active against M avium both in vitro and in vivo; its reported minimum inhibitory concentration for 90070 of M. avium strains (MIC 9 o ) measured on 7HlO or 7Hll agar medium has ranged from 4 (20) to 16 (32) ug/ml. It has been found to inhibit the multiplication of M. avium in macrophage culture (15) and in the spleens and lungs of beige mice infected with M. avium (20). Patients receiving clarithromycin in Phases 2 and 3 trials for clinical conditions other than AIDS have tolerated the drug well. Because of these promising findings, we designed a multicenter trial to test the short-term activity of clarithromycin against disseminated M avium infection in humans with AIDS. A randomized, double-blind, placebo-con-
A
564
SUMMARY Disseminated Mycobacterium av;um Infection is common in patients with acquired Immune deficiency syndrome (AIDS), but no drug studies have been reported establishing antlmicrobial activity against this organism in a controlled, randomized trial. Clarithromycln, a new macrolide, has activity against M. avium In vitro and In animals, but It has not been studied In humans. In this randomized, double-blind, placebo-controlled trial, we measured the ability of clarithromycin to reduce M. Bvium bacteremia In patients with AIDS and disseminated Infection. Of 23 patients initially enrolled, 15 men with late-stage AIDS qualified for the stUdy. One group received clarlthromycln alone for 6 wk, then placebo plus rifampln, Isoniazid, ethambutol, and clofazlmine for 6 wk. The other group received placebo alone, then clarlthromycln plus the other four drugs. Colony-forming units (CFU) of M. avlum per milliliter of blood were determined by quantitative cultures taken at baseline and every 2 wk thereafter. Minimum Inhibitory concentration of clarlthromycln for 90% of the strains Isolated from patients at baseline, as measured on 7H11 agar at pH 6.6,was 81!g/ml. Eight eligible patients with Initial positive cultures who were initially receiving clarlthromycln alone had marked declines In blood M. Ivium CFU; In six cases, CFU decreased to zero. When seven patients were switched to placebo plus the other four drugs, CFU rose In four patients and remained undetectable In three. The five eligible patients Initially treated with placebo had progressive CFU Increases; when three were switched to clarlthromycin plus the four drugs, their CFU declined. Weconclude that clarlthromycln has consistent activity against M. avium and may benefit patients with AIDS and disseminated M. evtum infection. AM REV RESPIR DIS 1991; 144:564-569
trolled, modified crossover design was used. Methods
Patients Twenty-three patients were initially enrolled in the study. Inclusion criteria were: positive serologic test results for human immunodeficiency virus, clinical signs and symptoms consistent with M avium infection (i.e., periodic fever, night sweats, malaise, and/or cough), no signs or symptoms suggestive of other infections, pretreatment blood or bone marrow cultures positive for M avium, and age 18 yr or older. Patients were excluded if they had a history of allergy to macrolides, if they were currentlytaking any antimycobacterial or investigational drugs, or if they were judged unlikely to complete the study (i.e., a Karnofsky score < 30 or other signs of imminent decline). Fifteen patients fulfilled the criteria and qualified for evaluation. The pretreatment characteristics of these patients are listed in table 1.
The 15 qualified patients received care in five university-affiliated hospitals. The study design was approved by the Institutional Ethical Review Board of the Groupe Hospitalier Pitie-Salpetriere, Paris; each patient gave signed informed consent in conformity with the Helsinki Declaration and French law. Pa-
(Received in original form December 13, 1990and in revised jorm April 10, 1991) 1 From the Pulmonary Department and the Bacteriology/Virology Laboratories, Groupe Hospitalier Pitie-Salpetriere, Paris; Abbott France, Rungis; Bacteriology Laboratory and Department of Infectious Diseases, Hopital Saint-Antoine, Paris; and the Department of Biostatistics, Hopital St. Louis, Paris, France. 2 Supported by a grant from Abbott Laboratories, Abbott Park, Illinois. J Correspondence and requests for reprints should be addressed to Professor Bertrand Dautzenberg, Service de Pneumologie Groupe Hospitalier Pitie-Salpetriere, 47 Boulevard de l'hopital, 75651, Paris, Cedex 13, France.
565
ACTIVITY OF CLARITHROMYCIN AGAINST M. AVIUM INFECTION IN AIDS
TABLE 1 CHARACTERISTICS OF ELIGIBLE PATIENTS AT START OF STUDY
Race: white/black/Asian Mean age, yr Body weight, kg Risk factors: homosexual/hemophilia Months since HIV diagnosis Opportunistic infections in last 12 months, n Diagnoses in last 12 months, n of patients Pneumocystosis Cytomegalovirus infection Cryptococcosis Kaposi sarcoma Positive M. avium cultures Blood < 5 CFU/ml ~ 5 CFU/ml Urine Sputum Patients receiving other medications, n Karnofsky index Dyspnea score Temperature, °C White blood cell count, /mm 3 Lymphocytes, /mm 3 CD 4 count, /mm 3 Platelets, /mm 3 Hemoglobin, mg/100 ml Alkaline phosphatase, UlL Serum glutamic-oxaloacetic transaminase, U/L
Group 1 (n = 8)
Group 2 (n = 7)
6/1/1 37 ± 11 57 ± 9 6/126 ± 16 3.4 ± 0.7
7/0/0 34 ± 8 52 ± 8 6/1 32 ± 18 3.7 ± 2.8
4
3
2 2
1 1
1
2
5
4
3 1 3
3 2
2
8t 63 0.2 38.0 4,380 453 19.4 258,000 9.4 317 22.3
± ± ± ± ± ± ± ± ± ±
7:1: 21 0.5 0.7 2,390 164 11.4 196,600 1.5 479 13.7
56 ± 0.7 ± 38.1 ± 2,110 ± 193 ± 12.1 ± 157,1OO± 3.1 ± 170 ± 54.4 ±
15 1.0 0.5 700§ 91§ 20.9 83,200§ 1.6 109 33.2
- One patient not included had no discernible risk factors but was from Zaire. Drugs used by patients in this group included zidovudine (AZT) (three), pentamidine (three), and fluconazole (six). :j: Drugs used by patients in this group included zidovudine (six), pentamidine (four), and fluconazole (five). § Significantly less than values for Group 1 patients.
t
tients wereentered between October 1988and November 1989.
to be the main parameters to assess the activity of clarithromycin.
Study Design The patients were randomized into two groups. During Phase 1 of the study (6 wk), patients in Group 1 received 1,000 mg clarithromycin orally twice daily for 6 wk, and those in Group 2 received placebo (figure 1). At the end of the 6 wk, patients were crossed over to Phase 2 (also 6 wk). Those who received clarithromycin in Phase 1 were given placebo plus a four-drug combination: rifampin (10 mg/kg daily), isoniazid (5 mg/kg daily), ethambutol (20 mg/kg daily), and clofazimine (100 mg daily). Patients receiving placebo during Phase 1 received clarithromycin in the dosage cited above plus the same four-drug combination during Phase 2. The protocol made provision for termination after 3 wk in Phase 1 if the patient's clinical condition deteriorated; the patient could then switch to Phase 2. At no point was the doubleblind broken. The main parameter for efficacy of treatment was difference in numbers of colonyforming units (CFU) of M. avium in the two study groups as determined by quantitative blood cultures. Blood was cultured within 48 h before therapy was begun and every2 wk thereafter to determine patients' responses to clarithromycin or placebo in Phase 1 and the combination of drugs in Phase 2. Clinical outcomes were recorded but were not considered
Culture Methods On the days when blood was sampled, three lO-ml aliquots were drawn at 30-min intervals.Samples werecollectedinto isolator tubes (DuPont Instruments, Newtown, CT) and immediately transferred to the laboratory. Each sample was centrifuged at 3,000 x g for 30 min, and the pellet was divided into two parts: a OJ-ml pellet was mixed with 0.9 ml sterile water, and 0.25 ml of this was inoculated onto each of four tubes of Loewenstein-Jensen medium. The remaining undiluted pellet was divided into seven equal parts of about 0.25 ml each; these wereinoculated onto five tubes of plain Loewenstein-Jensen medium and onto two tubes of Loewenstein-Jensen medium containing 0.5% sodium pyruvate. All inoculations were completed within 3 h after venipuncture. All tubes except one were incubated at 37° C; the remaining tube, inoculated with undiluted pellet, was incubated at 42° C. The tubes were examined weekly for as long as 3 months for bacterial growth and enumeration of colonies. By this method, the lowest detectable CFU value was 0.03 per ml of blood, i.e., 1 CFU/30 ml of blood. Isolated strains of M avium were sent to a central bacteriology laboratory for identification, including serotyping, and drug susceptibility testing; identification was done according to standard procedures. At the end
of the study, pretreatment strains and those isolated from the last cultures were tested simultaneously for susceptibility to the drugs used.
Drug Susceptibility Testing and Serotyping Minimum inhibitory concentrations (drug concentration required to inhibit ~ 99% of CFU) of clarithromycin, rifampin, and clofazimine were determined on 7Hll agar at pH 6.6 (Difco Laboratories, Detroit, MI) containing twofold concentrations of drugs ranging from 0.5 to 16,0.5 to 512,and 0.06to 2Ilg/ml, respectively. Inocula consisted of 0.05 ml of two dilutions (10-3 and 10-5 mg/ml wet weight) of 7-day-old cultures of M. avium in Tweenalbumin broth (Dubos medium). Cultures were incubated at 37° C with 5OJo carbon dioxide for 21 days before determinations of MIC. Susceptibility to isoniazid (0.1,0.2, 1.0, and 10ug/ml) and ethambutol (2Ilg/ml) was determined on Loewenstein-Jensen medium, using the proportion method routinely employed in the laboratory (33). Clinical Status and Safety Patients had their general condition monitored weekly as part of their overall medical care. General status was scored using the Karnofsky performance scale.The followingwere rated on a scale of zero to 3: sputum appearance (0 = absent, 3 = green); cough (0 = absent, 3 = severe enough to cause chest pain and prohibit normal activities); dyspnea (0 = absent, 3 = severe enough to prohibit normal activities); night sweats (0 = absent, 3 = five or more times a week); malaise (0 = none present, 3 = severe).Rales, rhonchi, and cyanosis were scored as 0 (absent) or 1 (present) and patients' subjective assessment of their quality of life as 1 (better), 2 (no change since last visit), or 3 (worse). All spontaneous complaints wererecorded. Safety of treatment was also monitored by the followinglaboratory studies: blood count with complete differential and platelet count, lymphocyte count, coagulation tests, blood urea nitrogen, serum glutamic-oxaloacetic transaminase (SGOT), serum glutamic-pyruvic transaminase (SGPT), alkaline phosphatase, bilirubin, protein/albumin, sodium, potassium, and uric acid. Urinalysis was also done once a week. After half of the patients had entered the study, clinical hypoacusis was noted in one patient; thereafter, audiograms were performed at the beginning and end of each phase. Statistical Analysis The numbers of patients with no change, increase, or decrease in CFU in the two groups were compared over time using Fisher's test. A model was also constructed to analyze changes in CFU in each group (log scale)using a linear model. Therefore, only patients with at least two measures (one within the 48 h before treatment and one after treatment) wereanalyzed. The slopes of the linear models
566
DAUTZENBERG, TRUFFOT, LEGRIS, MEYOHAS, BERLlE, MERCAT, CHEVRET, AND GROSSET
for the two groups were compared using the Mann-Whitney nonparametrical test. A similar approach was used for three selected clinical parameters: fever, Karnofsky index, and dyspnea. All patients with available data were analyzed. Values are expressed as mean ± standard error of the mean (SEM). Because of some treatment differences in Phase 1, valid comparisons between the two groups could not be made for Phase 2 results.
cal status was deteriorating (Patients 5 and 6). Twowere dropped from the study near the end of Phase 1because their condition worsened (Patients 10 and 13). During Phase 2, two patients receiving placebo plus the drug combination were removed from the study because of clinical deterioration, one after 2 wk (Patient 2) and the other before the end of the sixth week (Patient 7).
Results
Drug Susceptibility Jesting
Comparability of Groups and Attrition Of the 23 patients originally enrolled in the study, eight were excluded, seven because of negative pretreatment blood cultures for M avium and one because of poor compliance. Thus, 15patients qualified for analysis: eight in Group 1 and seven in Group 2 (table 1). Because of two early deaths, however, the activity of clarithromycin could be analyzed in only 13 patients, eight in Group 1 and five in Group 2. Characteristics of the patients in the two groups were comparable at the time of entry into the study except for somewhat lower counts of white blood cells, lymphocytes, and platelet counts in Group 2 (table 1). Details of attrition and early crossover are as follows: One patient from Group 1 discontinued participation after 2 wk during Phase 1 because of acute hepatitis (Patient 8). Twoothers were switched to Phase 2 at the end of 4 and 5 wk because the physicians felt that their clini-
Minimum inhibitory concentrations of clarithromycin as measured on 7Hll agar at pH 6.6 (Difco) for 50 and 90070 of pretreatment strains were 8 and 8 ug/ml, respectively. The corresponding values for clofazimine were0.25 and 0.5 ug/ml, respectively; for rifampin, the values were 16 and 32 ug/ml, respectively. All strains were resistant to isoniazid and ethambutol (MIC: 2 to 10and 2 ug/ml, respectively). The last positive culture for each patient was studied for susceptibility to clarithromycin and the other four drugs, and no significant change in the MIC was found. Among the eight strains isolated from Group 1 patients, one belonged to Serotype 6, two to Serotype 8, two to Serotype 9, and one to Serotype 23; the serotype could not be determined for the remaining two. Among the seven strains isolated from Group 2 patients, two belonged to Serotype 8 and one to Serotype 9; serotype could not be determined for the remaining four.
M. avium in Blood Cultures The initial concentrations of M. avium in blood specimens differed widely among patients in the study. In Group 1 patients, initially treated with clarithromycin, values ranged from 2 to 7,670 CFU/30 ml (0.07 to 253 CFU/m1); in Group 2 patients, initially treated with placebo, values ranged from 3 to 18,240 CFU/30 ml (0.3 to 640 CFU/ml). However, the numbers of CFU obtained from three blood specimens taken from patients at each sampling time were always very consistent; the differences from one sample to another were less than 1 log. As shown in table 2, there were no significant inconsistences between M avium CFU recovered from undiluted blood samples and those recovered from samples diluted 1:10. During Phase 1, M avium disappeared from blood cultures of five clarithromycin-treated patients by 2 wk oftreatment; in two others M avium disappeared by 4 wk. One of the latter, Patient 1, had 1 CFU isolated at 6 wk. One patient in the clarithromycin-treated group (Patient 3) had cultures that remained weakly positive, probably because of his high CFU count before treatment (table 2 and figure 1). The mean decrease in M avium in blood during the 6 wk of clarithromycin treatment was 2.65 log. Unlike clarithromycin-treated patients, all five placebo-treated patients had increasesin M avium CFU (mean increase, 1.55 log over 6 wk).
TABLE 2 COLONY-FORMING UNITS OF M. AVIUM ISOLATED FROM UNDILUTED AND 10:1 DILUTED LYSATES OF 30-ML BLOOD SAMPLES FROM PATIENTS WITH AIDS BEFORE AND DURING TREATMENT Phase 2
Phase 1
Patient No.
2 Weeks
Pretreatment Undil
Dil
Undil
4 Weeks
Dil
Dil
Undil
Crossover Pretreatment
6 Weeks Undil
Dil
Undil
808 161 7,100 20 27 2 6 5
0 32 570 0 7 0 0 0
3 1 25 0 0 0 0 0
9 10 11 12 13 14 15
14 20 530 3 18,240 62 2,162
0 79 78
322 830 1,300 4 23,200 Died Died
0 2 3
O· 0 0 0 0
0 0 1 ND 0 0 0 DT
0 0 2 NO 0 0 0 DT
1
d 2 0 EC EC 0 DT
0 0 0 0 EC EC 0 DT
1 0 2 0 0 0 0 DT
EC DT 137
31 DT 1,400 34 DT
O· 960 3 0
O· 800
126 930 1,580 29 22,800
523 430 265
o·
1,200
Definition of abbreviations: EC = early crossover; OT = discontinued therapy; NO
• Only 10 ml of blood cultured.
4 Weeks
Dil
Undil
6 Weeks
Dil
Undil
Dil
0 0 0 0 0 0 0 DT
17 3 2 0 1 0 0 DT
0 0 0 0 0 0 0 DT
12 DT 3 0 0 ND 0 DT
0 DT 1 0 0 ND 0 DT
41 DT 8 0 0 0 DT DT
0 DT 0
O· 0 0 DT DT
Group 2: Clarithromycin + four-drug combination
Group 2: Placebo 39 122 46
2 Weeks Undil
Group 1: Placebo + four-druq combination
Group 1: Clarithromycin 1 2 3 4 5 6 7 8
Dil
Ed DT 1,400 34 DT
=
O· DT
48 DT 137
O· DT
3 DT 54 0 DT
2 DT 17
O· DT
not done; Undil = undiluted culture; Oil = culture diluted 10:1.
6 DT 3 0 DT
0 DT 1
O· DT
6 DT 0 0 DT
0 DT 0
o·
DT
567
ACTIVITY OF CLARITHROMYCIN AGAINST M. AVIUM INFECTION IN AIDS
Phase 1: Clarithromycin
Phase 2: Placebo
1000 100 Fig. 1. Changes in CFU of M. avium in blood cultures of Group 1 patients receiving clarithromycin during Phase 1 of the trial and placebo plus a fourdrug combination (rifampin, isoniazid, ethambutol, and clofazimine) during Phase 2. Dashed line indicates early crossover. Open triangles = Patient 1; closed squares = Patient 2; X = Patient 3; closed triangles = Patient 4; open circles = Patient 5; crosses = Patient 6; open squares = Patient7; closed circles = Patient 8.
E
:5 LL o
e
::J oS; CQ
4
During Phase 2, among patients who initially received clarithromycin and were crossed over to placebo plus a four-drug combination, the numbers of CFU increased in those with positive blood cultures at the end of Phase 1. M avium reappeared in the blood cultures of one patient who previously had negative cultures after 4 wk of clarithromycin treatment (Patient 2) and in another who was an early crossover to Phase 2 (Patient 5). Both of these patients had more than 1 CFU/ml before the trial (figure 1). However, this rebound in the number of bacteria in the blood did not reach statistical significance. During Phase 2, among patients initially given placebo and crossed over to clarithromycin plus the four-drug combination, the CFU
Clinical Outcomes and Safety During Phase 1, the patients treated with clarithromycin had a mean decrease in fever ( - 0.2 ± 0.4) and improvement in Karnofsky score (1.4 ± 12.1); conversely, the patients treated with placebo had an increase in fever (0.2 ± 0.6) and worsening of Karnofsky score (-12.0 ± 13.0). However, the differences observed
Phase 1: Placebo
1000 100 Fig. 2. Changes in CFU of M. avium in blood cultures of Group 2 patients receiving placebo during Phase 1 of the trial and clarithromycin plus a four-drug combination (rifampin, isoniazid, ethambutol, and clofazimine) during Phase 2. Dashed line indicates early crossover. Open squares = Patient 9; open circles = Patient 10; closed squares = Patient 11;closed circles = Patient 12;open triangles = Patient 13;closed triangles = Patient 14; crosses = Patient 15.
counts decreased. Of the three. patients switched to Phase 2, two had no detectable M. avium in blood cultures after 6 wk of treatment with clarithromycin (figure 2). The third had 6 CFU/30 ml as compared with 646 CFU/30 ml at the beginning of Phase 2. The mean decrease in CFU during the 6 wk of treatment was 1.34 log.
E
:5 LL c
o§ ~
CQ
0.0
o
Phase 2: Clarithromycin
in the two groups during Phase 1 did not reach statistical significance. During Phase 2, the patients treated with placebo and four drugs had an increase in fever (0.4 ± 1.1)and worsening of Karnofsky score (-12.9 ± 21.4). Conversely, the patients treated with clarithromycin and four drugs had a decrease in fever ( - 0.7 ± 0.3) and a small change in Karnofsky score (- 3.5 ± 5.8). Statistical evaluation was not done during Phase 2 because the groups were no longer comparable. Of the 11 patients who underwent at least one audiogram, one had hypoacusis before treatment was started, one developed it during Phase 1 placebo treatment, and one developed it after clarithromycin treatment. In all cases, decrease in hearing was mild. One patient had hypothermia (34 0 C) while receiving placebo during Phase 1. In one patient receiving clarithromycin during Phase 1, treatment was stopped because of an increase in SOOT (25 to 55 U/L) and alkaline phosphatase (65 to 266 U/L). While receiving placebo during Phase 1, another patient had increases in SOOT (51 to 645 U/L); the level rose further during administration of clarithromycin and the four-drug combination (as much as 1,130 U/L), but because of clinical improvement the patient was continued in the trial. No difference in gastrointestinal tolerance was observed between the groups. Only two patients spontaneously complained of nausea during Phase 1, one receiving clarithromycin and the other receiving placebo. Discussion The main finding of our trial, based on blood cultures, was that clarithromycin has definite activity against M avium in patients with AIDS. All eight patients who were treated with clarithromycin during Phase 1 had steep decreases in their M avium CFU counts in blood. In contrast, all seven patients treated with placebo had persistent positive cultures and gradually increasing CFU counts. The effectiveness of clarithromycin was also strongly suggested by the results of blood cultures during Phase 2 of the trial. Patients who initially received placebo and were crossed over to clarithromycin plus the four-drug combination (rifampin, isoniazid, ethambutal, and clofazimine) had marked declines in their CFU levels. We could not attribute this effectiveness to the four-drug combination during Phase 2 because four
568
of the patients initially treated with clarithromycin and then crossed over to placebo plus the four-drug combination either had cultures that converted from negative to positive during Phase 2 or had increases in their CFU counts. Therefore, our results suggest that the four-drug combination was not effective in affectmg the M avium load in blood of patients with AIDS. Although our data indicate that clarithromycin shows definite activity against M avium within 6 wk of treatment, this interval is definitely not enough to cure all patients with AIDS of the infection. Blood cultures reflect only the concentration of organisms released from the tissue and circulating in the blood. A much longer period of treatment is probably required to eradicate M avium from both tissues and blood. Our clinical observations are consistent with encouraging results from studies of disseminated M avium infection in beige mice treated with clarithromycin (50 or 100 mg/kg daily) (20) and from experiments using human macrophages infected with M avium and cultured in a medium containing clarithromycin (4 ug/ml) (15). However, we found a discrepancy between clinical activity and our in vitro data: in our study, the MIC 9 0 of clarithromycin against M. avium was 8 ug/rnl, whereas the peak serum level reported in pharmacokinetic studies in humans was only about 4 J,1g/ ml after administration of 1,000mg twice daily. Taking into account the fact that M. avium is located primarily inside the cells of patients with AIDS, it may be that the discrepancies between MIC 9 0 values in vitro and plasma levelsachieved clinically could be due to concentration of clarithromycin by the cells. Indeed, in vitro studies have demonstrated an liE ratio (intracellular to extracellular concentration) of 9.2 (34). Alternatively, clarithromycin may be inactivated by the medium used to determine MIC, i.e., 7Hll at pH 6.6 on agar medium (Difco). In agar most drugs are absorbed, and macrolides are partially inactivated at low pH (35). It is likely that the MICs of clarithromycin against M avium would be lower if determined at pH equivalent to that of blood. Wewere unable to demonstrate more clinical improvement because the sample sizesof the two groups were small and the overallsymptoms of our patients were moderate (no high fever, etc.). Side effects after clarithromycin ad-
DAUTZENBERG. TRUFFOT, LEGRIS, MEYOHAS, BERLlE, MERCAT, CHEVRET, AND GROSSET
ministration weremild when they did occur. Transient hypoacusis, although observed after clarithromycin treatment, was also found before treatment or during placebo treatment. Elevation of SGPT and/or alkaline phosphatase occurred in two patients, one receiving clarithromycin and the other receiving placebo. In summary, our data suggest that clarithromycin has clear activity against M avium and may benefit patients with AIDS and disseminated M. avium infection. However, we must emphasize that our findings are based on a short-term trial involving only small numbers of patients. Moreover, the assessment of antimicrobial activity was based mainly on blood cultures. Future studies must be designed to obtain information about optimal dosage, length of treatment required to achieve the best therapeutic results, and the potential risk of acquired resistance during longer periods of clarithromycin monotherapy. Therefore, the search for active companion drugs that will lessen or prevent the risk of acquired resistance is of the utmost importance. Acknowledgment The writers thank the following physicians who participated in the protocol and entered patients into the study: V. Averous, D. Terminassian, G. Arlet, J. Modai', and J. M. Decazes, Hopital Saint Louis, Paris; J. Merrer and M. Le Neveu, Hopital de Poissy; D. Quinsat and T. Fosse, Hopital d'Antibes, Nice; P. Boudes and L. Desforges, Hopital Henri Mondor, Creteil; M. Siffert and C. Perez, Centre Medico Chirurgical Gui de Chauliac, Montpellier; G. Guermonprez, D. Tosoni, A. Coutelier, and F. Antoun, Groupe Hospitalier Pitie-Salpetriere, Paris; P. Brion and S. Duborgel, Hopital de la Tronche, Grenoble; J. Lacronique and G. Paul, Hopital Cochin, Paris; M. Eliazewitch and C. Kristetter, Hopital Saint-Antoine, Paris; Y. Laurian, Hopital Bicetre, Kremlin-Bicetre; J. P.Chauvin, Abbott France, Rungis; Cl. Chastang, Biostatistician, DBIM Hopital St. Louis, Paris; and P. Prokocimer, Abbott Laboratories, USA. The writers also thank Barbara Cox, Biomedical Editor, for her assistance in preparing the manuscript for publication. References 1. Truffot-Pernot C, Lecoeur H, Maury L, Dautzenberg B, Grosset J. Results of blood cultures for detection of mycobacteria in AIDS patients. Tubercle 1989; 70:187-91~ 2. Wilkes MS, Fortin AH, Felix JC, Godwin TA, Thompson WG. Valueof necropsy in acquired immunodeficiency syndrome. Lancet 1988; 2:85-8. 3. Wallace JM, Hannah JB. Mycobacterium avium complex infection in patients with the acquired immunodeficiency syndrome. A clinicopathologic study. Chest 1988; 93:926-32.
4. Yajko DM, Nassos PS, Hadley WK. Broth microdilution testing of susceptibilities to 30 antimicrobial agents of Mycobacteriumavium strains from patients with acquired immune deficiency syndrome. Antimicrob Agents Chemother 1987; 31: 1579-84. 5. Gangadharam PRJ, Kesavalu L, Rao PNR, Perumal VK, Iseman MD. Activity of amikacin against Mycobacterium avium complexunder simulated in vivo conditions. Antimicrob Agents Chemother 1988; 32:886-9. 6. Cynamon MH, KlemensSP. Newantimycobacterial agents. Clin Chest Med 1989; 10:355-64. 7. Heifets LB, Lindholm-LevyPJ, Flory MA. Bactericidal activity in vitro of various rifamycins against Mycobacterium avium and Mycobacterium tuberculosis. Am Rev Respir Dis 1990; 141: 626-30. 8. Fenlon CH, Cynamon MH. Comparative in vitro activities of ciprofloxacin and other 4-quinolones against Mycobacterium tuberculosis and Mycobacterium intracellulare. Antimicrob Agents Chemother 1986; 29:386-8. 9. Inderlied CB, Young LS, Yamada JK. Determination of in vitro susceptibility of Mycobacterium avium complex isolates to antimycobacterial agentsby various methods. Antimicrob Agents Chemother 1987; 31:1697-702. 10. Khardori N, Rolston K, Rosenbaum B, Hayat S, BodeyGP. Comparative in vitro activity of twenty antimicrobial agents against clinical isolates of Mycobacterium avium complex. J Antimicrob Chemother 1989; 24:667-73. 11. Banks J, Jenkins PA. Combined versus single antituberculosis drugs on the in vitro sensitivity patterns of non-tuberculous mycobacteria. Thorax 1987; 42:838-42. 12. Heifets LB, Iseman MD, Lindholm-Levy PJ. Combination of rifampin or rifabutine plus ethambutol against Mycobacterium avium complex: bactericidal synergistic, and bacteriostatic additive or synergistic effects. Am Rev Respir Dis 1988; 137: 711-5. 13. Yajko DM, Kirihara J, Sanders C, Nassos P, Hadley WK. Antimicrobial synergism against Mycobacterium avium complex strains isolated from patients with acquired immune deficiencysyndrome. Antimicrob Agents Chemother 1988; 32: 1392-5. 14. Heifets LB. Synergistic effect of rifampin, streptomycin, ethionamide, and ethambutol on Mycobacterium intracellulare. Am Rev Respir Dis 1982; 125:43-8. 15. Perrone C, Gikas A, Truffot-Pernot C, Grosset J, Pocidalo J J, Vilde JL. Clarithromycin, sulisoxazoleand rifabutin activity against Mycobacterium avium complex multiplication within human macrophages. Antimicrob Agents Chemother 1990; 34:1508-11. 16. Yajko DM, Nassos PS, Sanders CA, Hadley WK. Killing by antimycobacterial agents of AIDSderived strains of Mycobacterium avium complex inside cells of the mouse macrophage cell line J 774. Am Rev Respir Dis 1989; 140:1198-203. 17. Crowle AJ, Elkins N, May MH. Effectiveness of ofloxacin against Mycobacterium tuberculosis and Mycobacterium avium, and rifampin against M tuberculosis in cultured human macrophages. Am Rev Respir Dis 1988; 137:1141-6. 18. Bermudez LEM, Young LS. Activities of amikacin, roxithromycin, and azithromycin alone or in combination with tumor necrosis factor against Mycobacterium avium complex. Antimicrob Agents Chemother 1988; 32:1149-53. 19. Perumal VK, Gangadharam PRJ, Iseman MD. Effect of rifabutin on the phagocytosis and intracellular growth of Mycobacterium intracellulare in
ACTIVITY OF CLARITHROMYCIN AGAINST M. AV/UM INFECTION IN AIDS
mouse resident and activated peritoneal alveolar macrophages, Am Rev Respir Dis 1987; 136:334-7. 20. Fernandes PB, Hardy DJ, McDaniel D, Hanson CW, Swanson RN. In vitro and in vivo activities of ciarithromycin against Mycobacterium avium. Antimicrob Agents Chemother 1989; 33: 1531-4. 21. Gangadharam PRJ, Peru mal VK, Jairam BT, et al. Activity of rifabutin alone or in combination with ciofazimine or ethambutol or both against acute and chronic experimental Mycobacterium intracellulare infections. Am Rev Respir Dis 1987; 136:329-33. 22. Saito H, Saito K. Activity of rifabutin alone and in combination with ciofazimine, kanamycin and ethambutol against Mycobacterium intracellulare infections in mice. Tubercle 1989; 70:201-5. 23. Gangadharam PRJ, Perumal VK, Podapati NR, Kesavalu L, Iseman MD. In vivo activity of amikacin alone or in combination with ciofazimine or rifabutin or both against acute experimental Mycobacterium avium complex infection in beige mice. Antimicrob Agents Chemother 1988; 32: 1400-3. 24. Inderlied CB, Kolonoski PT, Wu M, Young LS. Amikacin, ciprofloxacin, and imipenem treat-
ment for disseminated Mycobacterium avium complex infection in beige mice. Antimicrob Agents Chemother 1989; 33:176-80. 25. Cynamon MH, Swenson CE, Palmer GS, Ginsberg RS. Liposome-encapsulated amikacin therapy of M avium complex infection in beige mice. Antimicrob Agents Chemother 1989; 33: 1179-83. 26. Agins BD, Berman DS, Spicehandler D, El Sadr W, Simberkoff MS, Rahal J J. Effect of combined therapy with ansamycin, clofazimine, ethambutol and isoniazid for Mycobacterium avium infection in patients with AIDS. J Infect Dis 1989; 159:784-7. 27. American Thoracic Society and Centers for Disease Control. Mycobacterioses and the acquired immunodeficiency syndrome. Joint Position Paper. Am Rev Respir Dis 1987; 136:492-6. 28. Hoy J, Mijch A, Sandland M, Grayson L, Lucas R, Dwyer B. Quadruple-drug therapy for Mycobacterium avium-intracellulare bacteremia in AIDS patients. J Infect Dis 1990; 161:801-5. 29. Chieu J, Nussbaum J, Bozzerre S, et al. Treatment of disseminated Mycobacterium avium complex infection in AIDS with amikacin, ethambutol, rifampin and ciprofloxacin. Ann Intern Med 1990;
569 113:358-61. 30. Hawkins CC, Gold JWM, Whimbey E, et al. Mycobacterium avium complex infections in patients with the acquired immunodeficiency syndrome. Ann Intern Med 1986; 105:184-8. 31. Young LS. Mycobacterium avium complex infection. J Infect Dis 1988; 1988; 157:863-7. 32. Berlin OGW, Young LS, Floyd-Reising SA, Bruckner DA. Comparative in vitro activity of the new macrolide A-56268 against mycobacteria. Eur J Clin Microbiol 1987; 6:486-7. 33. Canetti G, Rist N, Grosset J. Mesurede la sensibilite du bacille tuberculeaux aux drogues antibacillaires par la methode des proportions. Methodologie, criteres de resistance, resultats, interpretation. Rev Tuberc Pneumol1963; 27:217-72. 34. Anderson R, Joone G, Van Rensburg CEJ. An in vitro evaluation of the cellular uptake and intraphagocytic bioactivity of clarithromycin (A = 56268, TE-301), a new macrolide antimicrobial agent. J Antimicrob Chemother 1988; 22:923-33. 35. Barry AL, Jones RN, Thornsberry C. Disk diffusion and disk elution test with A 56268 and erythromycin. Eur J Clin Microbiol1987; 6:109-11.
