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ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Aug. 1996, p. 1889–1892 0066-4804/96/$04.0010 Copyright q 1996, American Society for Microbiology
Vol. 40, No. 8
Efficacy of Clarithromycin Treatment of Acute Otitis Media Caused by Infection with Penicillin-Susceptible, -Intermediate, and -Resistant Streptococcus pneumoniae in the Chinchilla CUNEYT M. ALPER,* WILLIAM J. DOYLE, JAMES T. SEROKY,
AND
CHARLES D. BLUESTONE
Department of Pediatric Otolaryngology, Children’s Hospital of Pittsburgh, and Department of Otolaryngology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania Received 18 March 1996/Returned for modification 14 May 1996/Accepted 11 June 1996
Because of the increasing frequencies of recovery of penicillin-resistant Streptococcus pneumoniae from the middle ears of children with acute otitis media, non-beta-lactam antibiotics are being explored as treatment alternatives to amoxicillin. In this study, the efficacy of a 10-day course of clarithromycin was evaluated with chinchillas. After the pharmacokinetic profiles for clarithromycin were established, 180 animals were assigned to one of three susceptibility groups (n 5 60/group; penicillin-susceptible, -intermediate, and -resistant S. pneumoniae), and the right middle ear was infected with the appropriate strain of S. pneumoniae. Equal numbers of animals in each group were treated orally beginning on day 2 with a 10-day course of clarithromycin (15 mg/kg of body weight; given twice a day) or amoxicillin as a control (20 mg/kg twice a day). On days 4, 9, and 13, otomicroscopy and tympanometry were performed, and on day 13, the middle ears were cultured for bacteria. The results showed 100% eradication of the challenge organism in both treatment groups for the susceptible strains of S. pneumoniae. Cultures were negative in 87 and 74% (P > 0.05) of the animals challenged with the intermediate resistant strains and in 100 and 56% (P < 0.05) of the animals challenged with the resistant strains and treated with clarithromycin and amoxicillin, respectively. There were no differences between treatments in the diagnosis of effusion for any group. These results support the use of the chinchilla to evaluate drug efficacy in the treatment of acute otitis media and show clarithromycin to be effective in sterilizing the middle ears of animals challenged with penicillin-susceptible, -intermediate, and -resistant strains of S. pneumoniae. profiles that offer the possibility of twice daily (BID) dosing. In clinical trials, clarithromycin has been shown to be as effective as amoxicillin-clavulanate in the treatment of AOM, though in those studies, few pneumococcal isolates were resistant to penicillin (13, 15, 17). Because clinical trials of antimicrobial agents for AOM caused by resistant organisms are difficult to perform given the high natural rate of clinical cure, the incident frequency of recovery of the target organism, and at present, the rather low frequency of penicillin-resistant S. pneumoniae in AOM (19), we attempted to develop an animal model of the disease with the chinchilla (1). Previously, a chinchilla model of AOM caused by b-lactamase-producing H. influenzae has been successfully used to evaluate the efficacy of new antimicrobial agents in the treatment of that disease (8, 10, 18, 25). Therefore, the purposes of this study were as follows: (i) to develop, using the chinchilla, an animal model of AOM secondary to infection with S. pneumoniae strains of different susceptibilities to penicillin; (ii) to determine pharmacokinetic profiles for blood and MEE samples following single-dose oral administration of clarithromycin in the chinchilla; and (iii) to determine if a 10-day standard course of clarithromycin is effective in eradicating the challenge organism and resolving the MEE in chinchillas infected transbullarly with penicillinsusceptible, -intermediate, or -resistant strains of S. pneumoniae.
Acute otitis media (AOM) is a common infectious disease of infants and children. Clinical studies document a bacterial etiology for most cases of AOM, with Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis representing the species most frequently recovered from the middle ears of children with the disease (7). The results of a recent metaanalysis of clinical studies (26) confirms the efficacy of antimicrobial treatment of AOM, and empirical therapy with antibiotics remains the mainstay of treatment. Amoxicillin is the first-line treatment for AOM in the United States (6). However, increasing frequencies of recovery of S. pneumoniae strains that are resistant to penicillin have been observed in recent years (3, 9, 14, 20, 27, 28). Unlike the mechanism of resistance for H. influenzae and M. catarrhalis, that of S. pneumoniae is not associated with plasmid-mediated production of b-lactamase but rather involves structural modifications under genomic control (16). Thus, developed strategies to counter the penicillin resistance of H. influenzae and M. catarrhalis are not applicable to S. pneumoniae, and new approaches need to be explored (5, 11, 22). These approaches include development of effective pneumococcal vaccines, altered dosing regimens for the first-line antimicrobial agents (4, 22), and development of secondary agents that have broad coverage with respect to the various strains of S. pneumoniae (2, 5, 21, 24). Regarding the latter, clarithromycin is a 14-membered macrolide antimicrobial agent that has activity against the most common bacterial species cultured from middle ear effusions (MEEs), concentrates well in tissues, and has pharmacokinetic
MATERIALS AND METHODS Pharmacokinetic experiment. A total of 30 adult chinchillas were purchased and enrolled in this study. The middle ears were challenged bilaterally via a transbullar approach with 50 mg of Escherichia coli endotoxin in 0.1 ml of sterile saline to create acute inflammation and effusion. Four days after challenge, the animals were randomly assigned to one of three equal groups and then admin-
* Corresponding author. Mailing address: Department of Pediatric Otolaryngology, Children’s Hospital of Pittsburgh, 3705 Fifth Ave., Pittsburgh, PA 15213. Phone: (412) 692-6962. Fax: (412) 692-6074. 1889
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ANTIMICROB. AGENTS CHEMOTHER. TABLE 1. Summary of the results of this study Cure rate (%)a
Sample size
Group and treatment subgroup
Entry
Posttreatment (day 13)
Group S Clarithromycin Amoxicillin
28 28
Group I Clarithromycin Amoxicillin Group R Clarithromycin Amoxicillin
Mortality rate (%)
Culture
Otomicroscopy
Tympanometry
MEE recovery
8 9
71 68
100 100
38 44
88 56
63 56
30 30
16 19
47 37
87 74
50 37
38 42
50 63
30 30
19 18
37 40
100 56b
47 33
47 33
53 61
a Percentage of ears that were cured at day 13 on the basis of the results of culture, otomicroscopy, tympanometry, and effusion recovery for all surviving animals in the six categories. b Significantly different from the value obtained with clarithromycin (P , 0.05 by chi-square test).
istered a single oral dose of clarithromycin at 7.5 (group A), 15 (group B), or 30 (group C) mg per kg of body weight. Blood samples and MEEs were collected from animals in each group over a 12-h period after dosing. Two MEE samples from either side (if present), and three or four blood samples were obtained from each animal according to a random hourly schedule. Samples were processed in a microbiological assay (done in triplicate) of clarithromycin concentrations by protocols and methods supplied to our laboratories by Abbott Laboratories (12). In the analysis of these data, the median value of the triplicate measurements was used as an estimate of serum or MEE clarithromycin concentration for each animal at each time point. Efficacy experiment. A total of 180 adult chinchillas were purchased from local ranches and randomly assigned to one of three equal groups (n 5 60) (groups S, I, and R). Both ears of all animals were examined by otomicroscopy and tympanometry to document healthy middle ear status. Then, the right middle ears were inoculated via the transbullar approach with 0.1 ml of phosphate-buffered saline containing approximately 10 CFU of one of three (n 5 20/strain) penicillin-susceptible strains (group S), -intermediate-resistant strains (group I), or -resistant strains (group R) of S. pneumoniae. Two days after inoculation, animals challenged with the different strains in each group were randomly assigned to one of two equal subgroups for treatments with amoxicillin (control) or clarithromycin (experimental). Beginning on that day and continuing for a total of 10 days, antibiotics (supplied by Abbott Laboratories) were administered by mouth BID. The total daily dose of amoxicillin was 40 mg/kg/day, and the total daily dose of clarithromycin was 30 mg/kg/day. Animals were reexamined under ketamine (20 mg/kg; given intramuscularly) sedation at 4, 9, and 13 days postinoculation by tympanometry and otomicroscopy, and then the animals were killed by barbiturate overdose (324 mg of pentobarbital given intracardiac). The right middle ear bullae were opened, and effusions were recovered into sterile traps. These effusions were swabbed and cultured onto chocolate plates for detection of S. pneumoniae. Bullae lacking effusion were washed with phosphate-buffered saline, and the recovered wash fluid was cultured for S. pneumoniae by standard microbiological techniques. Culture results were coded on a five-point scale (no growth [rating of 0] to heavy growth [rating of 4]). All procedures were performed by personnel blinded to the group and subgroup assignment of the animals. The protocol was approved by the Animal Care and Use Committee at the Children’s Hospital of Pittsburgh. The nine strains of S. pneumoniae were originally isolated from the middle ears of children with AOM. These strains corresponded to three different levels of penicillin susceptibility as documented by standard microbiological assay performed by the clinical microbiology laboratory at the Children’s Hospital of Pittsburgh and included three susceptible strains (group S; MIC # 0.1 mg/ml), three intermediate strains (group I; 1.0 mg/ml $ MIC . 0.1 mg/ml), and three resistant strains (group C; MIC $ 2.0 mg/ml). At the end of the experiment and before the group and subgroup assignments of the animals were revealed, all strains were submitted to Abbott Laboratories for blinded microbiological assay of susceptibility to amoxicillin and to clarithromycin (23). Otomicroscopy was performed with a Zeiss operating microscope at 6 power. Disease status with respect to AOM was categorized as present or absent on the basis of observation of an air-fluid level and/or bulging of the tympanic membrane. Tympanometry was performed with a clinical instrument (model AE-105; American Electronics Corp.) previously validated in our laboratories. The presence of effusion was diagnosed if the middle ear pressure was less than 2100 mm H2O and/or the recorded compliance of the tympanic membrane was less than or equal to 0.5 cm3 (1). The primary outcome measure was sterility of the middle ear after a 10-day course of treatment. Secondary outcomes included mortality, disease course, and
resolution of effusion. Analysis of the data was performed independently for each of the three groups. The frequencies for the outcomes related to sterility, mortality, and presence of effusion were compared between subgroups by a chisquare test evaluated at a 5 0.05. Data summarizing the results of this study are presented in Table 1.
RESULTS Pharmacokinetic experiment. Measurable serum and MEE clarithromycin concentrations were observed following administration of all three clarithromycin doses. For the 7.5-mg/kg dose, a peak average serum clarithromycin concentration of 0.33 6 0.22 mg/ml was observed at 2 h with no measurable concentrations documented at 6 h after dosing. Concentrations in MEE as high as 0.55 mg/ml were measurable for up to 9 h after dosing. The 15-mg/kg dose resulted in a peak level in serum at 2 h of 1.88 6 .55 mg/ml and a peak level in MEE of 0.78 mg/ml at 9 h after dosing. Detectable antibiotic concentrations in these fluids could be measured over the 12-h follow-up period. Doubling that dose to 30 mg/kg did not have an appreciable effect on the measured serum drug concentrations (peak 1.6 6 1.7 mg/ml at 7 h) but did appear to increase the peak effusion drug concentration (2.75 mg/ml at 10 h). Comparable concentrations in serum to those documented for children following a single oral clinical dose of 7.5 mg/kg were achieved at the 15-mg/kg dose in the chinchillas. Therefore, in the efficacy experiment, clarithromycin was administered BID at a dose of 15 mg/kg. Efficacy experiment. (i) Mortality. In developing the protocol, we estimated from past experience with S. pneumoniae infection, a mortality rate of between 30 and 40% by day 13, and this was approximated in the groups challenged with the penicillin-intermediate (group I, 42%) and -resistant (group R, 39%) S. pneumoniae strains. There were no significant differences in mortality between these groups or between treatments within these groups. However, for group S, both treatment subgroups experienced a high mortality rate (clarithromycin subgroup, 71%; amoxicillin subgroup [control], 68%). This mortality rate was significantly greater than those for groups R and I and was heterogeneous with respect to the challenge strain (mortalities of 60, 85, and 50% for groups S, R, and I, respectively). The data analysis reported below considers only the animals that survived to study day 13. (ii) Culture results. Table 1 reports, for each group and subgroup, the sample size (day 13) available for analysis of bacterial cultures and the percentage of surviving animals in
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which the challenge organism was not recovered. All ears (100%) in both treatment subgroups of group S animals were sterile for the challenge organism on day 13. For group I animals, 14 ears (87%) in the clarithromycin-treated group and 14 ears (74%) in the amoxicillin-treated subgroup were culture negative for S. pneumoniae, while in group R animals, 19 ears (100%) in the clarithromycin-treated subgroup and 10 ears (56%) in the amoxicillin subgroup were culture negative. Between-treatment differences in culture negativity were significant for group R. Both ears with recoverable S. pneumoniae on day 13 in the clarithromycin-treated subgroup were challenged with the same strain of intermediate resistant S. pneumoniae. The colony count rating of the two samples representing clarithromycin treatment failures was 4 (heavy growth). The clarithromycin susceptibility (MIC) of this strain was 4 mg/ml, suggestive of resistance to the test drug. In contrast, clarithromycin susceptibilities of the other eight strains were less than 0.015 mg/ml. Amoxicillin susceptibilities for the strains were as follows: 8, 2, and 0.5 mg/ml for the penicillin-resistant strains; 0.5, 0.06, and 0.03 mg/ml for the penicillin-intermediate-resistant strains, and 0.03, ,0.015 and ,0.015 mg/ml for the penicillin-susceptible strains. The colony count ratings were 4 in three ears and 1 in two ears of group I animals and 4 in six ears and 1 in two ears of group R animals. Subdividing this data set on the basis of measured MICs for amoxicillin showed positive cultures in 1 of 21 ears (5%) for strains with MICs of ,0.06 mg/ml, 5 of 10 ears (50%) for strains with MICs of 0.5 mg/ml, 2 of 8 ears (25%) for strains with MICs of 2.0 mg/ml, and 5 of 7 ears (71%) for strains with MICs of 8 mg/ml. (iii) Clinical cure. Clinical cure was defined on the basis of fluid recovery on day 13, and on the results of otomicroscopic and tympanometric examinations for that day. The percentages of ears cured for the two treatment subgroups of each group are reported in Table 1. MEE was recovered from between 50 and 63% of the ears in the various groups, with no obvious differences noted between groups or between treatment subgroups of any group. Similar results are reported for cure rates on the basis of otomicroscopic examinations and tympanometric measurements. The percentage of ears with disease as diagnosed by otomicroscopy and tympanometry in each group and subgroup on each day of observation was examined. On treatment day 2 (study day 4), otomicroscopy diagnosed disease in almost all of the animals (94 to 100%) in the different groups. There was no significant heterogeneity in the temporal pattern of disease expression, as diagnosed by either otomicroscopy or tympanometry that was attributable to group or subgroup assignment. DISCUSSION S. pneumoniae remains the major pathogen in AOM, and antimicrobial therapy should target this microorganism (7). Amoxicillin is recommended as the primary drug of choice for this disease (6), but recent reports from a number of centers and geographical areas have described an alarming increase in the frequency of recovery of penicillin-resistant strains of S. pneumoniae (3, 9, 14, 20, 27, 28). To control the infections caused by these resistant organisms, a variety of options have been suggested including increasing the dose of the first-line amoxicillin therapies or including alternative antibiotics for primary treatment failures (4, 5, 11, 22). In that regard, some evidence supports the usefulness of the first approach (4, 22), while current clinical practice includes the second approach (5). Evaluations of the clinical efficacy of the various treatment options would clearly benefit from the development of an an-
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imal model of AOM caused by penicillin-resistant strains of S. pneumoniae that mimics the human condition (19). Previously, the chinchilla has been used to evaluate antibiotic efficacy with regard to AOM caused by b-lactamase-producing H. influenzae, and therefore, one of the purposes of this study was to extend that model to penicillin-resistant S. pneumoniae (8, 10, 18, 25). All nine challenge strains used in this experiment resulted in acute infection and the development of AOM in the chinchilla. One of the penicillin-susceptible strains of S. pneumoniae chosen for study was unexpectedly virulent and associated with a high mortality rate (85%), while the mortality rates for the other strains were lower and varied between 35 and 60%. On the basis of the behavoral signs exhibited by the animals prior to death which included head tilt, abnormal gait, labored breathing, and lethargy, the mortality documented in this study was attributed to disseminated infection causing labrynthitis, meningitis, and sepsis. Previously, differences in mortality using the chinchilla as a model of AOM were reported for different S. pneumoniae strains and mortalities in untreated control groups were unacceptably high for all strains tested (1). The high rates of mortality secondary to S. pneumoniae infection of the middle ear documented in the chinchilla are not representative of the human condition and represent a significant limitation of the model. This limitation may be obviated by a careful screening in the chinchilla of a large number of S. pneumoniae strains to identify strains that induce disease expression in the absence of significant mortality. However, for ethical reasons, the use of a placebo control group in this model is not justified because the high expected mortality in the untreated control group would significantly limit the power of any comparisons between treatment groups. Prior to evaluating the efficacy of clarithromycin in treating the AOM caused by the different strains of S. pneumoniae, the pharmacokinetics of clarithromycin was studied in chinchillas with acute middle ear inflammation caused by endotoxin. The data showed that single-dose oral administration of clarithromycin at 15 mg/kg to the chinchilla resulted in good serum levels and appreciable MEE drug levels. The sustained concentrations documented for up to 12 h suggested that BID dosing would result in higher MEE levels than those documented following a single dose. Thus, these data show that clarithromycin rapidly penetrates the inflamed middle ear in this animal model. For reasons discussed above, a strict negative-control group was not included in the efficacy study design. Rather, the results for animals treated with clarithromycin were compared with those for animals treated with amoxicillin. To maintain the researcher’s ignorance of the group and subgroup assignment of animals in the present study, an amoxicillin dose of 20 mg/kg was administered BID and at the same times as the clarithromycin doses. In a previous study, amoxicillin administered three times a day for 10 days to chinchillas at a comparable total dose (39 mg/kg/day) resulted in negative culture rates of 76, 68, and 60% compared with negative culture rates of 21, 38, and 25% for placebo treatments in middle ears infected with a penicillin-susceptible, -intermediate, and -resistant S. pneumoniae strain, respectively. Assay of amoxicillin concentration in the MEEs at different times following administration of the final drug dose showed mean (6 standard deviation) concentrations of 0.7 6 1.0 (n 5 3), 8 6 12 (n 5 5), 5.4 6 2.2 (n 5 2), 3.1 6 4.1 (n 5 5), 9.5 6 9.5 (n 5 2), and 0 (n 5 1) mg/ml for effusions collected at 0.5, 1, 2, 3, 4, and 6 h, respectively (1). These data show that 10 days of treatment with a total daily amoxicillin dose of 39 mg/kg achieves signif-
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icant concentrations of the antibiotic in the MEEs and is more effective than the placebo in sterilizing the middle ear cleft. The efficacy data from the present study show that both antibiotic treatments were equally effective in eradicating penicillin-sensitive strains of the challenge organism. For the intermediate resistant strains, clarithromycin treatment was associated with fewer treatment failures (positive middle ear cultures) than amoxicillin treatment, but the difference between treatment subgroups was not significant. In contrast, none of the animals challenged with resistant strains of S. pneumoniae and treated with clarithromycin was considered a treatment failure compared with approximately 46% of the animals treated with amoxicillin. For both antibiotics, a relationship between drug susceptibility and effectiveness was supported by the data. However, the clinical significance of the higher cure rate for clarithromycin than for amoxicillin should be interpreted with caution. Specifically, the serum and effusion amoxicillin concentrations achieved at the dosing schedule utilized in this study were not measured and may not be comparable to those achieved in humans administered clinically effective doses of that antibiotic. However, the data from the earlier study discussed above showing efficacy in the chinchilla model of comparable doses of amoxicillin suggest that the amoxicillin dose utilized represents a reasonable positive control for demonstrating clarithromycin efficacy. Thus, these results support the use of clarithromycin in treating AOM caused by S. pneumoniae. As reported previously, effective eradication of the challenge organism was not associated with a resolution of the middle ear inflammation as judged by presence of effusion and tympanometric and otomicroscopic evaluations of signs (8, 10, 18, 25). Thus, there were no apparent differences between treatment subgroups in the resolution of the inflammation at the end of the 10-day course of treatment. This may be attributable to the relatively short follow-up period for animals in this study. Resolution of inflammation may occur over a period of weeks following eradication of the infection, and this possibility should be evaluated in a future study. In summary, the results of this study show that the chinchilla offers a reasonable animal model for evaluating treatment strategies for AOM caused by penicillin-resistant S. pneumoniae. In the model, cure rates defined on the basis of sterilization of the middle ear cleft correlated with the measured susceptibilities of the challenge strains to each of the two antibiotics tested. The primary disadvantage of the model is the relatively high mortality rate documented for these infections, an outcome not reported for the disease in humans. Accepting that limitation, clarithromycin treatment was demonstrated to be effective in sterilizing the middle ears of animals infected with different strains of S. pneumoniae that were sensitive, intermediate, and resistant to penicillin. However, the documented resistance to clarithromycin of 1 in 9 (11%) randomly chosen pneumococcal strains used in this study may reflect a relatively high rate of clarithromycin resistance of S. pneumoniae strains in children with otitis media with effusion. This rate should be determined for a significant number of S. pneumoniae strains recovered from MEEs from children with AOM to establish the potential limitations of clarithromycin therapy. ACKNOWLEDGMENTS We thank Brenda Kerber, Jean Betch, and Melissa Garret for providing technical assistance with this study. This study was supported in part by Abbott Laboratories. REFERENCES 1. Alper, C. M., W. J. Doyle, J. T. Seroky, and C. D. Bluestone. 1996. Ampicillin and amoxicillin treatment of acute otitis media caused by penicillin sensitive,
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