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Journal of Clinical Microbiology, March 1999, p. 879-880, Vol. 37, No. 3
0095-1137/99/$00.00+0
LETTERS TO THE EDITOR
Disc Testing of Meningococci
 |
LETTER |
In a recent article (1), Block et al. compared the disc diffusion
test with MIC determination by the E test for studying penicillin
susceptibility of Neisseria meningitidis, concluding that
the disc diffusion test was unreliable. Although the authors used the
definitions of reduced susceptibility proposed by us (2, 3), it is not
clear whether they also followed our disc susceptibility procedure; if
this procedure is not folllowed, our zone diameter breakpoints cannot
be applied. They mention that the "disc diffusion testing was
performed by using 2-U penicillin and 1-µg oxacillin discs ... according to National Committee for Clinical Laboratory Standards
recommendations" (1). To the best of our knowledge, the National
Committee for Clinical Laboratory Standards (NCCLS) has not recommended
such a procedure. To support their conclusions, the authors refer to a
previous study (8) in which our breakpoints, generated in
Mueller-Hinton agar (MH) plus 5% blood, were found to be of little
use. They do not mention that in that previous study, another growth
medium was used: chocolate agar with a GC base (8), a much richer
medium for which we never proposed breakpoints.
In our opinion, the data reported by Block et al. (1) lack both
internal and external validation. The authors apparently did not use
internal quality control procedures, which are mandatory, such as
reference strains, blinded determinations by several independent observers, or checking internal reproducibility of the E test and the
disc test. The E test is a commercial method, not a "gold standard," for MIC determination; strains for which MICs are between 0.064 and 0.094 µg/ml may already be low-level resistant. The rare
zone diameter distribution Block et al. observed could be explained by
inconsistency of the low-charge disc potency, but this factor was
neither taken into account nor ruled out. No attempt was made to study
ampicillin susceptibility, although the oxacillin disc was clearly
described as detecting both the Penr Ampr
and the Pens Ampr phenotypes (3).
None of the strains, particularly the ones giving conflicting results,
were sent to a reference laboratory to obtain an authoritative external
validation. Distinguishing between penicillin-susceptible and low-level
penicillin-resistant strains can be very difficult, even for
experienced laboratories (5); the NCCLS had to modify their initial
criteria for determining MICs for meningococci by recommending addition
of 5% blood to MH (7). We obtained consistent results by combining
susceptibility data, penicillin PBP2 affinity data, and data on
penA gene modifications (4, 6, 9).
| |
REFERENCES |
| 1.
|
Block, C.,
Y. Davidson, and N. Keller.
1998.
Unreliability of disc diffusion test for screening for reduced penicillin susceptibility in Neisseria meningitidis.
J. Clin. Microbiol.
36:3103-3104[Abstract/Free Full Text].
|
| 2.
|
Campos, J.,
P. M. Mendelman,
M. U. Sako,
D. O. Chaffin,
A. L. Smith, and J. A. Saez-Nieto.
1987.
Detection of relatively penicillin G-resistant Neisseria meningitidis by disk suceptibility testing.
Antimicrob. Agents Chemother.
31:1478-1482[Abstract/Free Full Text].
|
| 3.
|
Campos, J.,
G. Trujillo,
T. Seuba, and A. Rodriguez.
1992.
Discriminative criteria for Neisseria meningitidis isolates that are moderately susceptible to penicillin and ampicillin.
Antimicrob. Agents Chemother.
36:1028-1031[Abstract/Free Full Text].
|
| 4.
|
Campos, J.,
M. C. Fusté,
G. Trujillo,
J. A. Saez-Nieto,
J. Vázquez,
J. G. Loren,
M. Viñas, and B. G. Spratt.
1992.
Genetic diversity of penicillin-resistant Neisseria meningitidis.
J. Infect. Dis.
166:173-177[Medline].
|
| 5.
|
Jackson, L. A.,
F. C. Tenover,
C. Baker,
B. D. Plikaytis,
M. W. Reeves,
S. A. Stocker,
R. E. Weaver, and J. D. Wenger.
1994.
Prevalence of Neisseria meningitidis relatively resistant to penicillin in the United States, 1991.
J. Infect. Dis.
169:438-441[Medline].
|
| 6.
|
Mendelman, P. M.,
J. Campos,
D. O. Chaffin,
D. A. Serfass,
A. L. Smith, and J. A. Sáez-Nieto.
1988.
Relative penicillin G resistance in Neisseria meningitidis and reduced affinity of penicillin-binding protein 3.
Antimicrob. Agents Chemother.
32:706-709[Abstract/Free Full Text].
|
| 7.
|
National Committee for Clinical Laboratory Standards.
1997.
Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 4th ed. Approved standard. Document M7-A4.
National Committee for Clinical Laboratory Standards, Wayne, Pa.
|
| 8.
|
Perez-Trallero, E.,
N. Gómez, and J. M. Garcia-Arenzana.
1994.
E test as susceptibility test for evaluation of Neisseria meningitidis isolates.
J. Clin. Microbiol.
32:2341-2342[Free Full Text].
|
| 9.
|
Woods, C. R.,
A. L. Smith,
B. L. Wasilauskas,
J. Campos, and L. Givner.
1994.
Invasive disease caused by Neisseria meningitidis relatively resistant to penicillin in North Carolina.
J. Infect. Dis.
170:453-456[Medline].
|
| | | | |
Jose Campos
Centro Nacional Microbiologia 28220 Majadahonda Madrid, Spain
|
| |
AUTHORS' REPLY |
Dr. Campos raises several points regarding our study (1) which need to
be addressed and which further highlight the problematic nature of
susceptibility testing of meningococci.
Regarding the methodological issues raised, while we admittedly did not
plan our study precisely according to the procedure of Campos et al.
(3, 4), the medium and inocula used for the disc diffusion tests were
identical. Major deviations were the use of 90-mm instead of
150-mm-diameter plates and that incubation was carried out in a 5%
CO2 atmosphere. In a previous study, the need to test
spiramycin in air had shown us that 27% of clinical isolates and 41%
of carrier strains of N. meningitidis required CO2 for adequate growth (2). We fully acknowledge that the NCCLS has not recommended the 2-U penicillin or the 1-µg oxacillin discs. We had hoped that it would be clear that we were referring to
inoculum size, media, culture conditions, and reading of zone diameters. The use of GC base by others (6) may certainly be criticized.
Our brief note was not intended as a standard methodological work which
was meant to lay foundations for other laboratories' routine
procedures. It was a report of an evaluation of a reference laboratory's application of recommended criteria (3, 4) in day-to-day
service to the country. Our quality control procedures are those
performed in regular clinical laboratory work. E tests and disc
diffusion assays alike are controlled by using ATCC strains recommended
by the NCCLS for the relevant drugs, on the media used for the testing.
From our results and those of others, it appears that further studies
under meticulously controlled conditions, with subsequent validation in
the field, would contribute to resolving some of the issues.
We agree that the E test should not be considered a gold standard, but
rather an acceptably accurate and convenient method in situations where
the balance of cost and convenience are favorable. Before adopting the
E test, the Israel National Center for Meningococci (INCM) evaluated
the device in-house (unpublished data) and relied on peer-reviewed
publications, such as that of Hughes et al. (5). Israel fortunately has
a very modest meningococcal disease problem, and the resources
available for sophisticated reference work are limited. The need for
simple and inexpensive methods to monitor the worrying upward trend in
penicillin MICs in much of the world is nevertheless obvious, and an
inexpensive (would that it were) E test might be a simply applied
method suitable for many developing countries. Furthermore, clinical
laboratories in relatively less-regulated health systems frequently
report "routine" disc test results for meningococci to clinicians,
which most will probably agree are inappropriate. Clinical isolates in
individual laboratories are usually quite infrequent, so for those
wishing to report susceptibility results, a simple and reasonably
accurate diffusion method would be a welcome alternative to broth or
agar dilution MIC estimations.
We indeed made no attempt to study ampicillin, which has never been
included among the antibiotics tested against meningococci at the INCM.
Campos et al. (4) recommended the oxacillin disc partly because of its
excellent precision in indicating strains with reduced ampicillin
susceptibility. We agree with Tenover (7) that the clinical
implications of identifying penicillin-susceptible-relatively ampicillin-resistant strains of N. meningitidis are
unknown. Indeed, the clinical relevance even of elevated penicillin
MICs remains something of a mystery.
The question of which MICs truly represent reduced susceptibility to
penicillin has, in our view, not yet been satisfactorily settled. The
terminology, too ("low-level," "reduced susceptibility," "intermediate," etc.), needs a measure of consensus. And to date, there is no international consensus as to the optimal procedures and
definitions for susceptibility testing of N. meningitidis.
| |
REFERENCES |
| 1.
|
Block, C.,
Y. Davidson, and N. Keller.
1998.
Unreliability of disc diffusion test for screening for reduced penicillin susceptibility in Neisseria meningitidis.
J. Clin. Microbiol.
36:3103-3104.
|
| 2.
|
Block, C.,
Y. Davidson,
E. Melamed, and N. Keller.
1993.
Susceptibility of Neisseria meningitidis in Israel to penicillin and other drugs of interest.
J. Antimicrob. Chemother.
32:166-168[Free Full Text].
|
| 3.
|
Campos, J.,
P. M. Mendelman,
M. U. Sako,
D. O. Chaffin,
A. L. Smith, and J. A. Saez-Nieto.
1987.
Detection of relatively penicillin G-resistant Neisseria meningitidis by disk susceptibility testing.
Antimicrob. Agents. Chemother.
31:1478-1482.
|
| 4.
|
Campos, J.,
G. Trujillo,
T. Seuba, and A. Rodriguez.
1992.
Discriminative criteria for Neisseria meningitidis isolates that are moderately susceptible to penicillin and ampicillin.
Antimicrob. Agents Chemother.
36:1028-1031.
|
| 5.
|
Hughes, J. H.,
D. J. Biedenbach,
M. E. Erwin, and R. N. Jones.
1993.
E test as susceptibility test and epidemiologic test for evaluation of Neisseria meningitidis isolates.
J. Clin. Microbiol.
31:3255-3259[Abstract/Free Full Text].
|
| 6.
|
Perez-Trallero, E.,
N. Gomez, and J. M. Garcia-Arenzana.
1994.
E test are susceptibility test for evaluation of Neisseria meningitidis isolates.
J. Clin. Microbiol.
32:2341-2342.
|
| 7.
|
Tenover, F. C.
1993.
Antimicrobial susceptibility testing of Neisseria meningitidis.
Clin. Microbiol. Newsl.
15:37-38.
|
| | | | |
Colin Block
Department of Clinical Microbiology and Infectious Diseases Hadassah
University Hospital Jerusalem, Israel
|
| | | | |
Yehudit Davidson
Nathan Keller
Israel National Center for
Meningococci Tel Hashomer, Israel
|
Journal of Clinical Microbiology, March 1999, p. 879-880, Vol. 37, No. 3
0095-1137/99/$00.00+0
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