Journal of Clinical Microbiology, February 1998, p. 604-605, Vol. 36, No. 2
0095-1137/98/$00.00+0
LETTERS TO THE EDITOR
Evaluation of a Rapid Air Thermal Cycler for Detection
of Mycobacterium tuberculosis
 |
LETTER |
Chapin and Lauderdale (1) describe their evaluation of
a rapid air thermal cycler (ATC) (Idaho Technology, Idaho Falls, ID)
for the detection of Mycobacterium tuberculosis by PCR. We also have compared the use of the ATC to that of a more conventional heat block thermocycler (HBTC) (GeneAmp PCR System 9600; Perkin Elmer,
Cheshire, England) for the direct detection of M. tuberculosis in clinical samples. Our findings support the general
conclusions of Chapin and Lauderdale but differ significantly in regard
to inhibitors.
Samples were prepared by sonication using glass beads, and we used a
123-bp sequence of IS6110 as the target for DNA
amplification, as previously described (4). However, we
adapted the PCR cycling parameters for use with both the ATC and HBTC
(Table 1). Since PCR of clinical samples
has been hindered by inhibitors present in 3 to 20% of specimens
(2, 5), each PCR mixture included an internal amplification
control of 169 bp (Novocastra Laboratories Ltd., Newcastle, England)
which underwent coamplification. To date, we have tested 26 clinical
samples (including 24 sputum and 2 bronchoalveolar lavage samples) from
18 patients by PCR using both cyclers and compared the results with
those of microscopy and culture (Table 2).
On initial testing, inhibitors (as evidenced by nonamplification of the
internal control) were detected in 1 sample (3.9%) with the ATC and 10 samples (38.5%) with the HBTC. Following further purification by
simple chloroform extraction (3), 2 samples still had
evidence of inhibitors with the HBTC. Both of these samples were
therefore nonevaluable by using the HBTC, and both were M. tuberculosis culture positive.
By using culture as the "gold standard" for M. tuberculosis, of 14 culture-positive samples studied, 10 (71.4%)
were PCR positive with the ATC and 8 (57.1%) were PCR positive with
the HBTC, following chloroform purification where necessary. All the
culture-positive but PCR-negative samples were smear negative,
suggesting a low bacillary load, although 2 of the 10 samples found to
be PCR positive with the ATC were also smear negative, as was one of
the 8 successfully amplified samples with the HBTC.
Of 12 culture-negative samples examined (2 of which contained M. malmoense), all were negative with both cyclers.
We therefore agree with Chapin and Lauderdale that the ATC is an
excellent alternative to the HBTC in decreasing both overall cost and
total assay time (3 versus 4.5 h). However, in our limited series,
and in contrast to what Chapin and Lauderdale found, despite the
smaller sample input for the ATC (1 versus 5 µl), it not only provided an assay as sensitive as the HBTC but also had fewer problems
with inhibitors.
| |
REFERENCES |
| 1.
|
Chapin, K., and T.-L. Lauderdale.
1997.
Evaluation of a rapid air thermal cycler for detection of Mycobacterium tuberculosis.
J. Clin. Microbiol.
35:2157-2159[Abstract].
|
| 2.
|
Forbes, B. A., and K. E. Hicks.
1996.
Substances interfering with direct detection of Mycobacterium tuberculosis in clinical specimens by PCR: effects of bovine serum albumin.
J. Clin. Microbiol.
34:2125-2128[Abstract].
|
| 3.
|
Miller, N.,
S. G. Hernandez, and T. J. Cleary.
1994.
Evaluation of Gen-Probe amplified Mycobacterium tuberculosis direct test and PCR for direct detection of Mycobacterium tuberculosis in clinical specimens.
J. Clin. Microbiol.
32:393-397[Abstract/Free Full Text].
|
| 4.
|
Tan, M. F.,
W. C. Ng,
S. H. Chan, and W. C. Tan.
1997.
Comparative usefulness of PCR in the detection of Mycobacterium tuberculosis in different clinical specimens.
J. Med. Microbiol.
46:164-169[Abstract/Free Full Text].
|
| 5.
|
Vahaboglu, H.,
V. Avkan,
S. Dodanli,
M. Uzun,
I. Yildirim, and L. Mulazimoglu.
1997.
Comparison of two DNA extraction methods on inhibitory sputum samples.
Clin. Microbiol. Infect.
3:144-145.
[Medline] |
| | | | |
A. M. Kearns
R. Freeman
Public Health Laboratory
Newcastle General Hospital
Westgate Rd.
Newcastle upon Tyne NE4 6BE, England
|
| | | | |
M. Steward
Novocastra Laboratories Ltd.
24 Claremont Pl.
Newcastle upon Tyne NE2 4AA, England
|
| |
AUTHORS' REPLY |
We are encouraged to see that other investigators have had the success
with the air thermal cycler (ATC) that we have had in our laboratory.
In response to the greater inhibition that Kearns et al. had with the
heat block thermocycler (HBTC), we would respond with the following
comments. In our laboratory, we perform chloroform purification on all
of our samples. It is not clear from the letter of Kearns et al.
whether they performed chloroform purification only after noting a
problem with inhibition or whether further chloroform purification was
performed in addition to an initial extraction. In our case, initial
chloroform purification on all samples saves the majority of
"headaches" of uninterpretable PCRs. However, we did not fully
explain in our paper the following: if no internal control was seen
with a specific specimen, the PCR was repeated with DNA samples either
undiluted or in a 1:10 dilution. There were 11 samples overall for both
the ATC and HBTC that showed no internal control with the first PCR (11 of 270 [4%]). Nine of our samples with the ATC and seven with the
HBTC showed no presence of inhibitors on repeat of the PCR. There were two samples with the ATC that showed inhibition on repeat and four with
the HBTC that showed inhibition on repeat, <1 and 1.5%, respectively.
Thus, we did not have the inhibition rate that Kearns et al. reported
for initial samples (11 of 26 samples [42%]), nor did we find that
the HBTC samples showed far greater inhibition than the ATC samples.
Other differences between our procedure and that of Kearns et al. to
which the differences in the inhibition rate may be attributed are the
primer sequence being targeted and the cycling parameters. The cycling
parameters for the ATC and the HBTC in our procedure are quite similar,
yet those of Kearns et al. are quite different. One possible
explanation for the difference between the inhibition rates is the
input volume of the DNA sample and the overall ratio in the reaction
mixes for the two instruments. For our samples, the ratio of DNA input to reaction mix was always 1:10 for both the ATC (1 µl in 10 µl of
reaction mix) and the HBTC (5 µl in 50 µl of reaction mix). Neither
the input volume nor the reaction mix ratio was clear from the letter
from Kearns et al.; however, if the overall input volume of the DNA
sample for the HBTC was much greater, there would likely be more
inhibition with the samples.
Again, we are very excited to find others who have had success with
this instrument and wish the investigators the best in their future
endeavors with the ATC.
| | | | |
Kimberle Chapin
Tsai-ling Lauderdale
Department of Pathology
University of South Alabama Medical Center
2451 Fillingin St.
Mobile, Alabama 36617-2293
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Journal of Clinical Microbiology, February 1998, p. 604-605, Vol. 36, No. 2
0095-1137/98/$00.00+0
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