DIAGN MICROBIOLINFECT DIS 1991;14:75-77
75
In vitro Activity of Ceftibuten Against Haemophilus influenzae and
Branhamella catarhallis Gary V. Doern
The in vitro activity of ceftibuten, a new orally administered cephalosporin, was assessed against clinical isolates of Haemophilus influenzae and BranhameUa catarrhalis. The activity of ceftibuten was compared to that of ampicillin, amoxiciUin-clavulanic acid, and three oral cephalosporins, cefaclor, cefuroxime, and cefixime. With the exception of rare [3-1actamase-negative ampicillin-resistant strains of H. influenzae, resistance to ceftibuten was not observed with any of the study
isolates. Ceftibuten was more active than amoxicillin/clavulanic acid for AS-lactamase-positive and -negative strains of H. influenzae: it was less active than this combination for B. catarrhalis. Ceftibuten was essentially equivalent in activity to cefixime against both Haemophilus and Branhamella but more active than cefaclor and cefuroxime against these two organisms.
INTRODUCTION
as acute ofitis media, sinusitis, and community-acquired bronchopulmonary infections, its in vitro activity was assessed against two of the most common bacterial causes of these diseases, Haemophilus influenzae and Branhamella catarrhalis. For purposes of comparison, five other oral, [3-1actam antimicrobial agents were also examined: ampicfllin, amoxicillin/clavulanic acid, cefaclor, cefuroxime, and cefixime.
Ceftibuten, is a new orally administered cephalosporin with an extended spectrum of activity that includes enteric Gram-negative bacilli such as Escherichia coli, Citrobacter, Enterobacter, Klebsiella, Morganella, Proteus mirabilis and Proteus vulgaris, Providencia, and Serratia (Jones and Barry, 1988). Its chemical name is (6R, 7R)-7-[(2)-2-(2-aminothiazol4-yl)-4-carboxy-2-butenoylamino]-8-oxo-5-thia-1azabicyclo-[9,2,0]-oct-2-ene-2-carboxylic acid (Yoshida T et al., Abstra. 26th Intersci Conf Antimicrob Agents Chemother, no. 589, 1986). Peak plasma levels of 10 ~g/ml are achieved in healthy humans 2.5 hr after oral administration of 200 mg of ceftibuten (Nakashima M, et al., 26th ICAAC, no. 591, 1986). The elimination half-life of this compound is 1.5 hr. Because of the potential utility of ceffibuten in the management of outpatient infectious diseases such From the Department of Clinical Microbiologyand Division of Infectious Disease, Universityof Massachusetts Medical Center, Worcester, Massachusetts. Address reprint requests to: Gary V. Doern, Ph.D., Department of Clinical Microbiology,Universityof Massachusetts Medical Center, 55 Lake Avenue, North, Worcester, MA 01655. ReceivedMay 29, 1990; revised and accepted May 29, 1990. © 1991Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010 0732-8893/91/$3.50
Strains Tested A total of 150 clinical isolates of H. influenzae were examined in this study. Among these, 56 (37.3%) produced 13-1actamase when assessed by using a nitrocefin-chromogenic cephalosporin assay (Montgomery et al., 1979); 94 (62.7%) did not. Twentythree of the 56 [3-1actamase-producing strains (41.1%) produced type-B capsular antigen; 29 of the 94 [3lactamase-negative strains (30.1%) were capsular serotype B. A disproportionately high percentage of 13-1actamase-negative strains of H. influenzae with high minimal inhibitory concentrations (MICs) to ampicillin were intentionally included in this study. Specifically, eight ~-lactamase-negative strains with ampicillin MICs of ---4.0 ~g/ml were tested; all lacked type-B capsular antigen. In addition, 100 recent clinical isolates of B. catarrhalis were tested. Among these, 81 (81.0%) produced ~-lactamase.
76
Antimicrobial Agents Ceftibuten was provided by Schering-Plough (Bloomfield, NJ). All other agents tested were obtained from their respective manufacturers as laboratory-grade powders.
Broth Microdilution Tests Broth microdilution trays containing serial twofold concentration increments of antimicrobial agents, ranging from 0.004 to 128 ~g/ml, were prepared, using 96-well plastic trays with a Quick Spence II multichannel reagent dispenser (Sandy Springs Instrument Company, Germantown, MD). Haemophilus test medium broth, prepared in-house as described previously (Jorgensen et al., 1989) was used for tests with H. influenzae. Cation-adjusted MuellerHinton broth (Difco Laboratories, Detroit MI), adjusted to contain final concentrations of 25 p~g/ml calcium and 12.5 p~g/ml magnesium, was used for tests with B. catarrhalis (NCCLS, 1988). Stock cultures were thawed and subcul~red twice to chocolate agar (H. influenzae) or sheep blood agar plates (B. catarrhalis). Suspensions of test organisms were prepared in Mueller-Hinton broth to a turbidity equivalent to that of a 0.5 McFarland standard with the aid of a nephelometer (A-Just Meter, Abbott Diagnostics, North Chicago, IL). Appropriate dilutions of initial suspensions were made in MuellerHinton broth to achieve a final organism concentration of 5 x 10 CFU/ml. Trays were sealed with Mylar film and incubated for 20-24 hr at 35°C in ambient air prior to visual determinations of MICs. The MIC was defined as the lowest concentration of antimicrobial agent tested that yielded no visual evidence of growth of the test organism. All MICs were determined twice. The assigned MIC for a given organism-antimicrobial agent combination was defined as the mean of the duplicate MIC determinations. When the two MICs varied by one twofold concentration increment, the higher MIC was taken as the assigned value. Finally two quality control strains of H. influenzae and B. catarrhalis with known MICs to all six antimicrobial agents tested were examined twice daily on each day of testing, once at the beginning and once at the end of each test day's run. In no case did a QC strain yield on MIC greater than two concentration increments above or below the expected MIC for that given antimicrobial agent.
RESULTS A N D DISCUSSION Among the total of 900 paired MIC determinations with H. influenzae, 91.6% of the two MICs were identical; in 72 instances (8.7%), the paired MICs varied
G.V. Doern
by one twofold concentration increment; and the remaining four cases (0.4%) the duplicate MICs differed by twofold concentration increments. With the 600 duplicate organism-antimicrobial agent comparisons with B. catarrhalis, these values were 561 (93.5%), 37 (61.7%), and 2 (0.3%), respectively. The results of this study are depicted in Table 1. With ~qactamase-producing strains of H. influenzae, ceftibuten and cefixime were the most active of the agents tested. Based on NCCLS definitions of interpretive categories, 3 of 56 [3-1actamase-positive strains of H. influenzae (5.4%) were found to be intermediate to ampicillin; the remaining 53 strains were resistant. A total of 45 [3-1actamase-positive strains (80.4%) had cefaclor MICs of -32 ~g/ml and would have been classified as resistant. All 56 [3-1actamase-producing strains of H. influenzae were susceptible to amoxicillin-clavulanic acid, ceftibuten, cefuroxime, and cefixime. With [3-1actamase-negative strains of H. influenzae, ceftibuten and cefixime were again the most active of the compounds tested. Among 94 such strains included in this study, 8 had ampicillin MICs of ->4.0 ~g/ml and, thus, would be considered resistant to ampicillin despite lack of [3-1actamase production. The amoxicillin-clavulanic acid MICs of these eight strains were 1.0 p~g/ml (two strains), 2.0 p~g/ml (five strains), and 4.0 p~g/ml (one strain). These were the same eight strains that had ceftibuten MICs of ---0.5 ~g/ml. Their cefaclor, cefuroxime, and cefixime MICs were also higher than those noted for ~-lactamasenegative strains of H. influenzae with low ampicillin MICs. Amoxicillin-clavulanic acid was 10-fold more active against [3-1actamase-producing strains of B. catarrhalis than the other five antimicrobial agents examined in this study, including ceftibuten. However, with the exception of ampicillin, resistance was not observed with any agent. The most active cephalosporin tested was cefixime followed by ceftibuten, cefador, and cefuroxime. The superior activity of cefaclor for ~-lactamase-positive strains of B. catarrhalis in comparison to cefuroxime has been noted previously. So also was the existance of a large number of strains with low ampicillin MICs despite [3lactamase production. All six of the antimicrobial agents examined in this study were highly active against [3-1actamasenegative strains of B. catarrhalis. With these strains, the three most active agents were ampicillin, amoxicillin-clavulanic acid, and cefixime. Ceftibuten and cefaclor were essentially comparable but more active than cefuroxime. The results of this investigation clearly demon-
Ceftibuten against Haemophilus and Branhamella
T A B L E 1.
Test Organism (no. strains)
77
I n vitro A c t i v i t y of C e f t i b u t e n a n d Five O t h e r O r a l A n t i m i c r o b i a l A g e n t s for Haemophilus influenzae a n d Branhamella catarrhalis Cumulative Percentage of Strains with MIC (p,g/ml) of Antimicrobial Agent
40.06
0 . 1 2 0.25
0.5
1
2
4
8
16
32
64
128
20 100
45
63
80
91
89
5 98
34
17 100 4 55
9 91
30 96
55 98
80 100
82
96
100
95 82 96 59 82 99
99 88 97 67 86 100
100 96
H. influenzae
Ampicillin 13-1actamase Amox/clav~ + (56) Ceftibuten Cefaclor Cefuroxime Cefixime
2 98
91
95
4 98
5 100
7
Ampicillin Amox/clav~ Ceftibuten Cefaclor Cefuroxime Cefixime
20
50 69 13
77 49 95 18 36
86 68
66 12 1 84
68 7 92 15 6 96
Ampicillin f3-1actamase Amox/clav~ + (81) Ceftibuten Cefaclor Cefuroxime Cefixime
6 48 1
14 80 31 1
69
91
100
48 79 41 100
72 98 93
100 100 99
100
42
40 100 44 30 14 98
49
27
32 99 42 12 1 58 99 68 74 11 95
100 74 100 63 100
H. influenzae ~-lactamase (94) -
B. catarrhalis
B. catarrhalis
Ampicillin f~-lactamase Amox/clav~ - (19) Ceftibuten Cefaclor Cefuroxime Cefixime
100 79
68
94
90 63
79
28 69
84
95
89
100
100
81 89
100 100 95
95 97
98 100
100
Geometric Mean 1>256 MIC 100
26.0 1.0 0.07 5.6 0.7 0.06 0.3 1.1 0.1 2.7 1.4 0.08 1.0 0.07 0.8 0.9 1.5 0.2 0.06 0.06 0.3 0.3 0.7 0.09
aAmoxicillin-clavulanic acid was tested at a fixed ratio of 2:1; the concentration listed refers to the concentration of arnoxicillin.
strate t h a t t h e i n vitro activity of c e f t i b u t e n w a r r a n t s its c o n s i d e r a t i o n i n t h e m a n a g e m e n t of p a t i e n t s w i t h n o n - l i f e - t h r e a t e n i n g localized i n f e c t i o n s d u e to H. influenzae or B. catarrhalis, i r r e s p e c t i v e of ~ - l a c t a m a s e p r o d u c t i o n . W i t h t h e e x c e p t i o n of rare a m p i c i l l i n r e s i s t a n t s t r a i n s of H. influenzae w i t h a m e c h a n i s m
of r e s i s t a n c e o t h e r t h a n p r o d u c t i o n of a t r a n s m i s s i o n e l e c t r o n m i c r o s c o p y ( T E M ) - t y p e ~ - l a c t a m a s e , ceftib u t e n r e s i s t a n c e w a s n o t o b s e r v e d w i t h e i t h e r of these organisms. Ceftibuten was essentially comp a r a b l e to cefixime b u t m o r e active t h a n cefaclor a n d cefuroxime against these two pathogens.
REFERENCES Jones RN, Barry AL (1988) Antimicrobial activity spectrum, and recommendations for disk diffusion susceptibility testing of ceftibuten (7432-5; SCH 39720), a n e w orally administered cephalosporin. Antimicrob Agents Chemother 32:1576-1582. Jorgensen JH, Redding JS, Maher LA, Howell AW (1989) Improved m e d i u m for antimicrobial susceptibility testing of Haemophilus influenzae. J Clin Microbiol 25:21052113.
Montgomery K, Raymondo L, Drew WL (1979) Chromogenic cephalosporin spot test to detect beta-lactamase in clinically significant bacteria. J Clin Microbiol 9:205207. National Committee for Clinical Laboratory Standards (NCCLS) (1988) Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Approved standard M7-T2. Villanova, PA: NCCLS.
75
In vitro Activity of Ceftibuten Against Haemophilus influenzae and
Branhamella catarhallis Gary V. Doern
The in vitro activity of ceftibuten, a new orally administered cephalosporin, was assessed against clinical isolates of Haemophilus influenzae and BranhameUa catarrhalis. The activity of ceftibuten was compared to that of ampicillin, amoxiciUin-clavulanic acid, and three oral cephalosporins, cefaclor, cefuroxime, and cefixime. With the exception of rare [3-1actamase-negative ampicillin-resistant strains of H. influenzae, resistance to ceftibuten was not observed with any of the study
isolates. Ceftibuten was more active than amoxicillin/clavulanic acid for AS-lactamase-positive and -negative strains of H. influenzae: it was less active than this combination for B. catarrhalis. Ceftibuten was essentially equivalent in activity to cefixime against both Haemophilus and Branhamella but more active than cefaclor and cefuroxime against these two organisms.
INTRODUCTION
as acute ofitis media, sinusitis, and community-acquired bronchopulmonary infections, its in vitro activity was assessed against two of the most common bacterial causes of these diseases, Haemophilus influenzae and Branhamella catarrhalis. For purposes of comparison, five other oral, [3-1actam antimicrobial agents were also examined: ampicfllin, amoxicillin/clavulanic acid, cefaclor, cefuroxime, and cefixime.
Ceftibuten, is a new orally administered cephalosporin with an extended spectrum of activity that includes enteric Gram-negative bacilli such as Escherichia coli, Citrobacter, Enterobacter, Klebsiella, Morganella, Proteus mirabilis and Proteus vulgaris, Providencia, and Serratia (Jones and Barry, 1988). Its chemical name is (6R, 7R)-7-[(2)-2-(2-aminothiazol4-yl)-4-carboxy-2-butenoylamino]-8-oxo-5-thia-1azabicyclo-[9,2,0]-oct-2-ene-2-carboxylic acid (Yoshida T et al., Abstra. 26th Intersci Conf Antimicrob Agents Chemother, no. 589, 1986). Peak plasma levels of 10 ~g/ml are achieved in healthy humans 2.5 hr after oral administration of 200 mg of ceftibuten (Nakashima M, et al., 26th ICAAC, no. 591, 1986). The elimination half-life of this compound is 1.5 hr. Because of the potential utility of ceffibuten in the management of outpatient infectious diseases such From the Department of Clinical Microbiologyand Division of Infectious Disease, Universityof Massachusetts Medical Center, Worcester, Massachusetts. Address reprint requests to: Gary V. Doern, Ph.D., Department of Clinical Microbiology,Universityof Massachusetts Medical Center, 55 Lake Avenue, North, Worcester, MA 01655. ReceivedMay 29, 1990; revised and accepted May 29, 1990. © 1991Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010 0732-8893/91/$3.50
Strains Tested A total of 150 clinical isolates of H. influenzae were examined in this study. Among these, 56 (37.3%) produced 13-1actamase when assessed by using a nitrocefin-chromogenic cephalosporin assay (Montgomery et al., 1979); 94 (62.7%) did not. Twentythree of the 56 [3-1actamase-producing strains (41.1%) produced type-B capsular antigen; 29 of the 94 [3lactamase-negative strains (30.1%) were capsular serotype B. A disproportionately high percentage of 13-1actamase-negative strains of H. influenzae with high minimal inhibitory concentrations (MICs) to ampicillin were intentionally included in this study. Specifically, eight ~-lactamase-negative strains with ampicillin MICs of ---4.0 ~g/ml were tested; all lacked type-B capsular antigen. In addition, 100 recent clinical isolates of B. catarrhalis were tested. Among these, 81 (81.0%) produced ~-lactamase.
76
Antimicrobial Agents Ceftibuten was provided by Schering-Plough (Bloomfield, NJ). All other agents tested were obtained from their respective manufacturers as laboratory-grade powders.
Broth Microdilution Tests Broth microdilution trays containing serial twofold concentration increments of antimicrobial agents, ranging from 0.004 to 128 ~g/ml, were prepared, using 96-well plastic trays with a Quick Spence II multichannel reagent dispenser (Sandy Springs Instrument Company, Germantown, MD). Haemophilus test medium broth, prepared in-house as described previously (Jorgensen et al., 1989) was used for tests with H. influenzae. Cation-adjusted MuellerHinton broth (Difco Laboratories, Detroit MI), adjusted to contain final concentrations of 25 p~g/ml calcium and 12.5 p~g/ml magnesium, was used for tests with B. catarrhalis (NCCLS, 1988). Stock cultures were thawed and subcul~red twice to chocolate agar (H. influenzae) or sheep blood agar plates (B. catarrhalis). Suspensions of test organisms were prepared in Mueller-Hinton broth to a turbidity equivalent to that of a 0.5 McFarland standard with the aid of a nephelometer (A-Just Meter, Abbott Diagnostics, North Chicago, IL). Appropriate dilutions of initial suspensions were made in MuellerHinton broth to achieve a final organism concentration of 5 x 10 CFU/ml. Trays were sealed with Mylar film and incubated for 20-24 hr at 35°C in ambient air prior to visual determinations of MICs. The MIC was defined as the lowest concentration of antimicrobial agent tested that yielded no visual evidence of growth of the test organism. All MICs were determined twice. The assigned MIC for a given organism-antimicrobial agent combination was defined as the mean of the duplicate MIC determinations. When the two MICs varied by one twofold concentration increment, the higher MIC was taken as the assigned value. Finally two quality control strains of H. influenzae and B. catarrhalis with known MICs to all six antimicrobial agents tested were examined twice daily on each day of testing, once at the beginning and once at the end of each test day's run. In no case did a QC strain yield on MIC greater than two concentration increments above or below the expected MIC for that given antimicrobial agent.
RESULTS A N D DISCUSSION Among the total of 900 paired MIC determinations with H. influenzae, 91.6% of the two MICs were identical; in 72 instances (8.7%), the paired MICs varied
G.V. Doern
by one twofold concentration increment; and the remaining four cases (0.4%) the duplicate MICs differed by twofold concentration increments. With the 600 duplicate organism-antimicrobial agent comparisons with B. catarrhalis, these values were 561 (93.5%), 37 (61.7%), and 2 (0.3%), respectively. The results of this study are depicted in Table 1. With ~qactamase-producing strains of H. influenzae, ceftibuten and cefixime were the most active of the agents tested. Based on NCCLS definitions of interpretive categories, 3 of 56 [3-1actamase-positive strains of H. influenzae (5.4%) were found to be intermediate to ampicillin; the remaining 53 strains were resistant. A total of 45 [3-1actamase-positive strains (80.4%) had cefaclor MICs of -32 ~g/ml and would have been classified as resistant. All 56 [3-1actamase-producing strains of H. influenzae were susceptible to amoxicillin-clavulanic acid, ceftibuten, cefuroxime, and cefixime. With [3-1actamase-negative strains of H. influenzae, ceftibuten and cefixime were again the most active of the compounds tested. Among 94 such strains included in this study, 8 had ampicillin MICs of ->4.0 ~g/ml and, thus, would be considered resistant to ampicillin despite lack of [3-1actamase production. The amoxicillin-clavulanic acid MICs of these eight strains were 1.0 p~g/ml (two strains), 2.0 p~g/ml (five strains), and 4.0 p~g/ml (one strain). These were the same eight strains that had ceftibuten MICs of ---0.5 ~g/ml. Their cefaclor, cefuroxime, and cefixime MICs were also higher than those noted for ~-lactamasenegative strains of H. influenzae with low ampicillin MICs. Amoxicillin-clavulanic acid was 10-fold more active against [3-1actamase-producing strains of B. catarrhalis than the other five antimicrobial agents examined in this study, including ceftibuten. However, with the exception of ampicillin, resistance was not observed with any agent. The most active cephalosporin tested was cefixime followed by ceftibuten, cefador, and cefuroxime. The superior activity of cefaclor for ~-lactamase-positive strains of B. catarrhalis in comparison to cefuroxime has been noted previously. So also was the existance of a large number of strains with low ampicillin MICs despite [3lactamase production. All six of the antimicrobial agents examined in this study were highly active against [3-1actamasenegative strains of B. catarrhalis. With these strains, the three most active agents were ampicillin, amoxicillin-clavulanic acid, and cefixime. Ceftibuten and cefaclor were essentially comparable but more active than cefuroxime. The results of this investigation clearly demon-
Ceftibuten against Haemophilus and Branhamella
T A B L E 1.
Test Organism (no. strains)
77
I n vitro A c t i v i t y of C e f t i b u t e n a n d Five O t h e r O r a l A n t i m i c r o b i a l A g e n t s for Haemophilus influenzae a n d Branhamella catarrhalis Cumulative Percentage of Strains with MIC (p,g/ml) of Antimicrobial Agent
40.06
0 . 1 2 0.25
0.5
1
2
4
8
16
32
64
128
20 100
45
63
80
91
89
5 98
34
17 100 4 55
9 91
30 96
55 98
80 100
82
96
100
95 82 96 59 82 99
99 88 97 67 86 100
100 96
H. influenzae
Ampicillin 13-1actamase Amox/clav~ + (56) Ceftibuten Cefaclor Cefuroxime Cefixime
2 98
91
95
4 98
5 100
7
Ampicillin Amox/clav~ Ceftibuten Cefaclor Cefuroxime Cefixime
20
50 69 13
77 49 95 18 36
86 68
66 12 1 84
68 7 92 15 6 96
Ampicillin f3-1actamase Amox/clav~ + (81) Ceftibuten Cefaclor Cefuroxime Cefixime
6 48 1
14 80 31 1
69
91
100
48 79 41 100
72 98 93
100 100 99
100
42
40 100 44 30 14 98
49
27
32 99 42 12 1 58 99 68 74 11 95
100 74 100 63 100
H. influenzae ~-lactamase (94) -
B. catarrhalis
B. catarrhalis
Ampicillin f~-lactamase Amox/clav~ - (19) Ceftibuten Cefaclor Cefuroxime Cefixime
100 79
68
94
90 63
79
28 69
84
95
89
100
100
81 89
100 100 95
95 97
98 100
100
Geometric Mean 1>256 MIC 100
26.0 1.0 0.07 5.6 0.7 0.06 0.3 1.1 0.1 2.7 1.4 0.08 1.0 0.07 0.8 0.9 1.5 0.2 0.06 0.06 0.3 0.3 0.7 0.09
aAmoxicillin-clavulanic acid was tested at a fixed ratio of 2:1; the concentration listed refers to the concentration of arnoxicillin.
strate t h a t t h e i n vitro activity of c e f t i b u t e n w a r r a n t s its c o n s i d e r a t i o n i n t h e m a n a g e m e n t of p a t i e n t s w i t h n o n - l i f e - t h r e a t e n i n g localized i n f e c t i o n s d u e to H. influenzae or B. catarrhalis, i r r e s p e c t i v e of ~ - l a c t a m a s e p r o d u c t i o n . W i t h t h e e x c e p t i o n of rare a m p i c i l l i n r e s i s t a n t s t r a i n s of H. influenzae w i t h a m e c h a n i s m
of r e s i s t a n c e o t h e r t h a n p r o d u c t i o n of a t r a n s m i s s i o n e l e c t r o n m i c r o s c o p y ( T E M ) - t y p e ~ - l a c t a m a s e , ceftib u t e n r e s i s t a n c e w a s n o t o b s e r v e d w i t h e i t h e r of these organisms. Ceftibuten was essentially comp a r a b l e to cefixime b u t m o r e active t h a n cefaclor a n d cefuroxime against these two pathogens.
REFERENCES Jones RN, Barry AL (1988) Antimicrobial activity spectrum, and recommendations for disk diffusion susceptibility testing of ceftibuten (7432-5; SCH 39720), a n e w orally administered cephalosporin. Antimicrob Agents Chemother 32:1576-1582. Jorgensen JH, Redding JS, Maher LA, Howell AW (1989) Improved m e d i u m for antimicrobial susceptibility testing of Haemophilus influenzae. J Clin Microbiol 25:21052113.
Montgomery K, Raymondo L, Drew WL (1979) Chromogenic cephalosporin spot test to detect beta-lactamase in clinically significant bacteria. J Clin Microbiol 9:205207. National Committee for Clinical Laboratory Standards (NCCLS) (1988) Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Approved standard M7-T2. Villanova, PA: NCCLS.
