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JID 1997;176 (October)
Correspondence
were intervening cannot be answered at present. The absolute proof
of the etiologic role of any microorganism in respiratory infections
in very difficult to produce, since the presence of the microorganism in the inflammatory focus can only exceptionally be documented.
In our study, we could conclude that M. pneumoniae was responsible for 13.3% of lower respiratory tract infections (bronchopneumonia and lobar pneumonia) in children, whereas it was found in
only 2.1% of all other clinical presentations. This difference is
significant (P õ .01). Although little is known about the prevalence
of colonization with M. pneumoniae in healthy children, we would
expect to find a much higher prevalence of M. pneumoniae in
children with, for example, upper respiratory tract infections if
colonization is high.
For the 16S-rDNA PCR, the annealing temperature was lowered
from 607C to 527C because no satisfactory amplification signal
was obtained with an annealing temperature of 607C with the
thermocycler we used (which is not the same as the cycler that
was used for the original article).
We did not conclude that signals produced by contamination
are detected only by hybridization. Contaminations were detected
through this more sensitive procedure, but it is certainly not a
general rule.
Although two positive results in two PCRs do not absolutely
rule out contamination, they make it most improbable. Indeed,
the specimens were divided between the two laboratories, each
performing one extraction procedure. Extracted samples were
exchanged and two PCRs performed in each laboratory — one
with its ‘‘own’’ and one with the ‘‘second’’ sample. In these
circumstances, two false-positive results in the two different
laboratories would suppose two independent contaminations in
the tubes containing samples of the same specimen. This chance
is extremely small (and therefore practically zero). Moreover,
the division of the specimens into two parts (the only source of
contamination left) was performed according to what is considered good laboratory practice in the laboratory of Antwerp,
where the 16S-rDNA PCR was never performed.
In using this procedure of confirming a positive PCR result
by another PCR, the second PCR should be used sparingly to
avoid amplicon build-up in the laboratory. The frequent use of
two different PCRs in the same laboratory makes contamination
with both amplicons possible and likely as stated by Tjhie et
al. [1].
In the conclusion and summary of our paper, we stated that,
although visual inspection of the gel can be recommended for
routine diagnosis, maximal results are obtained after hybridization. In some cases, the appearance of many aspecific bands can
be a problem in the interpretation of gel results and without
hybridization a definite conclusion is impossible.
In our experience, this is often the case in samples containing
a lot of foreign DNA, such as biopsy samples, but in general, we
do not have this problem with nasopharyngeal aspirates. For this
application, hybridization would thus be applied for sensitivity
rather than for specificity. However, in a routine laboratory for
clinical microbiology, one always has to compromise between
rapid diagnosis and maximal sensitivity. It is in view of these
arguments, labor intensity of the procedure used and rapidity of the
answer, that we recommend this procedure for routine diagnosis of
M. pneumoniae infections, while remembering that maximal re-
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sults can be obtained with extensive sample preparation and hybridization.
M. Ieven, D. Ursi, H. G. M. Niesters, and H. Goossens
Department of Microbiology, University Hospital Antwerp, Belgium, and
Department of Virology, University Hospital Dijkzigt, Rotterdam,
The Netherlands
Reference
1. Tjhie JHT, Savelkoul PHM, Vandenbroucke-Grauls CMJE. Polymerase
chain reaction evaluation for Mycoplasma pneumoniae. J Infect Dis 1997;
176:1124.
Reprints or correspondence: M. Ieven, Dept. of Microbiology, University
Hospital Antwerp, Wilrijkstraat 10, 2650 Edegem, Belgium.
The Journal of Infectious Diseases 1997;176:1124–5
q 1997 by The University of Chicago. All rights reserved.
0022–1899/97/7604–0045$02.00
Lack of Clinical Significance for the Common Arginine-toLeucine Substitution at Codon 463 of the katG Gene in
Isoniazid-Resistant Mycobacterium tuberculosis in Singapore
To the Editor—In a recent report, Musser et al. [1] sequenced
the katG gene and detected alterations at residue Arg463. Alterations in the katG gene, encoding catalase-peroxidase, can result
in resistance to isoniazid, which is widely used as one of the firstline anti-tuberculous agents [2].
The most common alteration in the katG gene is an arginineto-leucine substitution at codon 463 (R463L) [3,4,5]. However, the
significance of this mutation is unclear. Site-directed mutagenesis
experiments have demonstrated that the R463L substitution does
not significantly alter the level of expression of katG or the peroxidase activity of the Mycobacterium tuberculosis catalase-peroxidase [4]. The R463L substitution is located in the C-terminal domain and does not alter either enzyme activity or heat stability
[5]. In addition, the R463L substitution has been detected even in
catalase-positive isolates [4].
Musser et al. [1] detected the R463L substitution in 12 of 34
isoniazid-resistant strains but not in 12 isoniazid-susceptible
strains. In 61 other strains cultured from patients in the Netherlands, alterations at codon 463 were identified in 11 of 51 isoniazidresistant strains and in 1 of 10 isoniazid-susceptible strains [1].
Using the polymerase chain reaction–restriction fragment length
polymorphism (PCR-RFLP) method, we screened 68 consecutive
isoniazid-resistant isolates and 50 isoniazid-sensitive Mycobacterium tuberculosis isolates from patients in Singapore for the R463L
mutation. Resistance to isoniazid was confirmed by the radiometric
method, using the BACTEC 460 system. Primers for PCR were
primer pair 7, as previously described [5]. The PCR products were
then restricted with MspI, producing fragments of 187 bp, 65 bp,
and 48 bp when no mutation was present and fragments of 187
bp and 113 bp when the R463L substitution was present. Precautions taken to avoid cross-contamination of the PCR products were
the inclusion of negative controls, the use of aerosol-resistant pipette tips, and the physical separation of pre- and post-PCR areas.
Direct cycle sequencing of PCR products was also done on 49
UC: J Infect
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Correspondence
isoniazid-resistant isolates, and results were completely concordant
with those from the PCR-RFLP analysis.
To confirm that the isolates were from different strains, DNA
RFLP analysis was done on 57 of the 68 isoniazid-resistant
isolates, using standardized methodology [6]. In brief, genomic
DNA was restricted with PvuII, and Southern blots were probed
with the insertion sequence IS6110. Of the 57 isolates, only two
had identical patterns, and these isolates were from different
patients.
PCR products were detected in 63 of the 68 isoniazid-resistant
strains. The lack of PCR product in 5 strains was possibly due to
deletion of the katG gene. In the remaining 63 isoniazid-resistant
strains, the R463L mutation was present in 46 isolates (73%).
However, the R463L mutation was also present in a high proportion (56%) of the isoniazid-sensitive isolates from Singapore,
which were collected over a similar time period.
Our findings are in contrast to those of Musser et al. [1], who
detected no R463L alterations in 12 isoniazid-susceptible strains.
In that study, although the R463L substitution was identified in
12 of the isoniazid-resistant strains, concurrent mutations at other
codons in the katG gene were also present in 11 of those strains.
These other concurrent mutations, not the R463L substitution, may
be responsible for conferring resistance to isoniazid.
Our results suggest that the R463L alteration may not confer
resistance to clinically significant concentrations of isoniazid but
may in fact be a common polymorphism present in Mycobacterium
tuberculosis isolates from Singapore. To determine the significance
of this alteration, further studies on the frequency of the R463L
substitution in both isoniazid-susceptible and isoniazid-resistant
isolates from different geographic locales around the world need
to be conducted.
Ann Siew-Gek Lee, Lynn Lay-Hoon Tang,
Irene Hua-Khim Lim, Moi-Lin Ling, Leng Tay,
and Sin-Yew Wong
Department of Clinical Research, Ministry of Health; Communicable
Disease Centre, Tan Tock Seng Hospital; and Department of Pathology,
Singapore General Hospital, Singapore
References
1. Musser JM, Kapur V, Williams DL, Kreiswirth BN, van Soolingen D, van
Embden JDA. Characterization of the catalase-peroxidase gene (katG)
and inhA locus in isoniazid-resistant and -susceptible strains of Mycobacterium tuberculosis by automated DNA sequencing: restricted array of
mutations associated with drug resistance. J Infect Dis 1996; 173:196 –
202.
2. Snider DE Jr, Roper WL. The new tuberculosis. N Engl J Med 1992; 326:
703 – 5.
3. Morris S, Bai GH, Suffys P, et al. Molecular mechanisms of multiple drug
resistance in clinical isolates of Mycobacterium tuberculosis. J Infect Dis
1995; 171:954 – 60.
4. Rouse DA, Li ZM. Bai GH, Morris SL. Characterization of the katG and
inhA genes of isoniazid-resistant clinical isolates of Mycobacterium tuberculosis. Antimicrob Agents Chemother 1995; 39:2472 – 7.
5. Heym B, Alzari PM, Honore N, Cole ST. Missense mutations in the catalase-peroxidase gene, katG, are associated with isoniazid resistance in
Mycobacterium tuberculosis. Molec Microbiol 1995; 15:235 – 45.
6. van Embden DJA, Cave MD, Crawford JT, et al. Strain identification of
Mycobacterium tuberculosis by DNA fingerprinting: recommendations
for a standardized methodology. J Clin Microbiol 1993; 31:406 – 9.
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JID 1997;176 (October)
Informed consent was obtained from the patients when sputum samples were
collected.
Financial support was obtained from the National Medical Research Council,
Singapore.
Reprints or correspondence: Dr. Ann Siew-Gek Lee, Dept. of Clinical Research, Ministry of Health, Block 6, Level 6, Singapore General Hospital,
Singapore 169608.
The Journal of Infectious Diseases 1997;176:1125–6
q 1997 by The University of Chicago. All rights reserved.
0022–1899/97/7604–0046$02.00
Reply
To the Editor—We appreciate the interest of Lee et al. in our
paper [2]. The central issue raised in their letter concerns the
importance in isoniazid resistance of the polymorphism (leucine
or arginine) found at position 463 of the gene (katG) encoding
catalase-peroxidase in Mycobacterium tuberculosis [1]. Our work
was based in part on an earlier report by Cockerill et al. [3] that
a disproportionate number of isoniazid-resistant strains had KatG
463L rather than 463R. Neither our report nor that of Cockerill
et al. claimed that KatG with 463L directly conferred isoniazid
resistance. Rather, there was a nonrandom association with drug
resistance. Of importance, with a single exception, all isoniazidresistant organisms in our data set and that of Cockerill et al. having
the Kat 463L polymorphism contained other missense changes or
structural alterations that could confer resistance. For example,
many isolates had an amino acid substitution at KatG position 315
(SjT) that has been shown directly to confer isoniazid resistance
[4]. Our data, interpreted in the light of the findings of Cockerill
et al. [3] and other recently published studies [4, 5], along with
the results reported by Lee et al. [1], suggest that the R463L
polymorphism does not directly affect isoniazid susceptibility in
vitro. However, unambiguous assessment of the role of R463L
structural variants in isoniazid resistance and host-pathogen interaction requires use of isogenic strains of M. tuberculosis strict
sense. These organisms have not been described in the literature.
It is also important to understand the polymorphism located at
KatG 463 in the broader context of M. tuberculosis evolutionary
biology. We reported that all isolates of Mycobacterium bovis,
Mycobacterium microti, and Mycobacterium africanum studied
contained KatG 463L rather than 463R. The most likely interpretation of these data, as noted in our publication, is that KatG 463L
is the ancestral condition in the M. tuberculosis complex. This
means it is more appropriate to think of the KatG 463 polymorphism as a leucine-to-arginine substitution. Recent work has shown
that only 15%–20% of M. tuberculosis strict sense isolates from
the United States and Europe have Leu at this position (regardless
of isoniazid susceptibility phenotype), whereas the great majority
of organisms recovered from patients in China and Southeast Asia
contain Leu at this position (unpublished data). Sampling of isolates from Mexico, Honduras, Guatemala, Peru, and several other
areas of Latin America revealed that ú85% of organisms have
KatG 463R (unpublished data). It is clear there are striking geographic differences in the frequency of occurrence of the Arg
versus Leu polymorphism. This important point is highlighted by
the occurrence of 463R in 74 (65%) of 113 of all organisms in
the Singapore sample studied by Lee et al. Inasmuch as there is
UC: J Infect