Susceptibility testing of H. influenzae
air or CO2. Results obtained with a 15 ꢀg disc incubated in
Discussion
CO2 would indicate that this method was the most practical
for day-to-day use. A cut-off zone of ꢂ10 mm diameter
denoting susceptibility would still be indicated for use but
only 5% of isolates tested gave this value; most were ꢂ14
mm diameter.
It is important that we address problems of susceptibility
testing of any organism on a dynamic continuing basis and
that reports to clinicians reflect the in vivo response as
accurately as possible. We would therefore recommend
that for routine susceptibility testing of H. influenzae from
respiratory sources a 15 ꢀg disc be used. Incubation should
be in 4–6% CO2, a zone diameter of ꢂ10 mm denoting
sensitivity and ꢄ9 mm diameter denoting resistance. If the
breakpoint method is used the value should be 16 mg/L
with incubation in CO2 overnight.
In our laboratory we formerly tested respiratory isolates of
H. influenzae for susceptibility to clarithromycin by agar
breakpoint method. A breakpoint value of 16.0 mg/L was
used and incubation was in 4–6% CO2 incubated overnight.
This value is thought to reflect concentrations of clarithro-
mycin at sites of respiratory infection rather than in serum.1
The resistance rate determined by this method was ꢃ5%
during the period 1996–1998.
We have adopted the recent BSAC (British Society for
Antimicrobial Chemotherapy) guidelines for disc suscepti-
bility testing of H. influenzae.6 Employing this method it
was found that susceptibility testing with clarithromycin
employing IST agar with added NAD at 20 mg/L and a 5 ꢀg
disc incubated in 4–6% CO2 overnight increased our
resistance rate to approximately 25%.
The BSAC guidelines suggest that clarithromycin MIC
values of 1–16 mg/L be categorized as intermediate suscep-
tibility to this drug, with values below and above this range
denoting susceptibility and resistance, respectively. The
zone diameter for disc susceptibility testing in 4–6% CO2
overnight suggests a diameter of 10–24 mm denoting inter-
mediate sensitivity, with values below and above this range
suggesting sensitivity and resistance, respectively. It should
be noted, however, that these guidelines for clarithromycin
are tentative and have not been confirmed by a BSAC field
trial.
References
1. Honeybourne, D. & Baldwin, D. R. (1992). The site concentra-
tions of antimicrobial agents in the lung. Journal of Antimicrobial
Chemotherapy 30, 249–60.
2. Guay, D. R. & Craft, J. C. (1992). Comparative safety and effic-
acy of clarithromycin and ampicillin in the treatment of out-patients
with acute bacterial exacerbation of chronic bronchitis. Journal of
International Medicine 3, 295–301.
3. Olsson-Liljequist, B. & Hoffman, B. M. (1991). In-vitro activity of
clarithromycin combined with its 14-hydroxy metabolite A62671
against Haemophilus influenzae. Journal of Antimicrobial Chemo-
therapy 27, Suppl. A, 11–17.
Etest strips were used as the reference method for our
study as they have been shown to be reliable for macrolides
and have good correlation with the NCCLS reference 4. Dibb, W. L., Digranes, A. & Bottosfen, K. L. (1986). Effects of
microdilution method.5
carbon dioxide upon the in vitro activity of erythromycin. Acta Patho-
logica, Microbiologica et Immunologica Scandinavica (B) 94, 173–6.
The most accurate results in the study were with the
5. National Committee for Clinical Laboratory Standards. (1995).
Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria
that Grow Aerobically—Third Edition: Approved Standard M100-S6,
M7-A3. NCCLS, Villanova, PA.
15 ꢀg disc incubated overnight in air; however, this tech-
nique suffered from the inability of 24% of the isolates
to grow without incubation in CO2. Testing under these
conditions would suggest a cut-off zone of ꢂ14 mm dia-
meter to indicate susceptibility. This would greatly aid
interpretation of the results. The ꢂ10 mm zone diameter
recommended at present with a 5 ꢀg disc incubated in
4–6% CO2 is only 4 mm larger than the disc size itself and is
a difficult value to measure. Clearly, these results show that
6. British Society for Antimicrobial Chemotherapy Working Party.
(1998). BSAC Standardized Disc Sensitivity Testing Method. News-
letter of the British Society for Antimicrobial Chemotherapy,
Summer 1998.
Received 6 July 1999; returned 8 November 1999; revised 25
the use of a 5 ꢀg disc is unacceptable whether incubated in November 1999; accepted 6 December 1999
531