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Journal of Clinical Microbiology, October 2001, p. 3768-3771, Vol. 39, No. 10
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.10.3768-3771.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Simple and Rational Approach to the Identification
of Mycobacterium tuberculosis, Mycobacterium
avium Complex Species, and Other Commonly Isolated
Mycobacteria
Derek A.
Wong,1,*
Peter C. W.
Yip,1
Danny T. L.
Cheung,2 and
Kai Man
Kam1
Tuberculosis Reference
Laboratory1 and Public Health
Laboratory, Kowloon Hospital,2 Pathology
Service, Department of Health, HKSAR Government, Hong Kong
Received 6 March 2001/Returned for modification 18 June
2001/Accepted 23 July 2001
 |
ABSTRACT |
A novel PCR-restriction fragment length polymorphism
analysis of the hsp65 gene was developed. The
restriction patterns for Mycobacterium
tuberculosis and Mycobacterium avium
complex (MAC) species were designed to be highly distinct, and the
overall number of restriction patterns was designed to be
limited. Four hundred specimens (17 reference strains and
383 clinical isolates) were tested, of which 98 were M.
tuberculosis and 132 were MAC species. The assay was virtually
100% sensitive and specific for M. tuberculosis and MAC species. Moreover, it gave highly concordant results for other
mycobacterial species other than M. terrae complex
species. This assay can be completed in one day and is
user-friendly and robust. Therefore, it is highly suitable for
large-scale use in a clinical laboratory.
| |
TEXT |
In the past two decades,
there has been a dramatic increase in the numbers of diseases caused by
Mycobacterium tuberculosis complex and other nontuberculous
mycobacteria, in particular, members of the M. avium
complex (MAC) (2). This increase is driven mainly by the
AIDS pandemic, with both M. tuberculosis and MAC
species causing disseminated disease in AIDS patients. Therefore, there
is great pressure on clinical laboratories to rapidly and accurately
detect and identify clinically important mycobacteria. Conventionally,
identification of mycobacteria grown in culture is achieved by standard
culture and biochemical tests that are time-consuming and not always
accurate (5, 6, 15). Other methods such as
high-performance, gas-liquid, and thin-layer chromatographies and DNA
sequence analysis of the 16S rRNA gene (rDNA) region are either too
labor-intensive, difficult, or expensive for routine use (3, 9,
12, 15, 21). Rapid and simple genotypic assays for the
identification of mycobacteria, such as Accuprobe (Gen-Probe Inc., San
Diego, Calif.), are available commercially. However, the high costs of
these assays have prohibited their large-scale use in most clinical
laboratories, especially in developing areas with a high incidence of
tuberculosis (14).
PCR-restriction fragment length polymorphism analysis (PRA) is simple
to perform, rapid, and economical, features that make it highly
attractive for routine clinical laboratories. However, assays for PRA
have often been criticized as being difficult to read because of minor
differences in patterns between some species that are made worse by
gel-to-gel variations. As a result, time-consuming computer-assisted
analysis is often required. PRA techniques have been developed for
several mycobacterial genes, such as hsp65, the 16S-23S rDNA
spacer, and rpoB (7, 13, 20). Of these, the one
most investigated and validated is hsp65 (1, 8, 11,
17, 18, 19, 20). However, assays for that gene have been impeded
by difficulties such as minor differences in band sizes between some
species and the occurrence of new patterns that have not been reported
previously (1, 10). In view of this, we decided to
redesign an assay for PRA of the hsp65 gene using available
DNA sequence data so that M. tuberculosis, MAC species,
and other clinically important mycobacteria can be identified with
ease. To this end, the restriction patterns of M. tuberculosis and MAC species were designed to be highly
distinct and the overall number of possible restriction patterns
was designed to be limited; thus, the patterns are highly recognizable.
Development of assay.
Seventy-six hsp65 sequences
from 36 different mycobacterial species and subspecies were
obtained from GenBank (Table 1). hsp65 sequences for different M. kansasii and M. scrofulaceum subspecies were
obtained from other sources (10, 18). All MAC strains had
a restriction site at nucleotide position 671. Primers specific
for hsp65 were then designed so that digestion of the
product with Sau96I gave a unique and highly distinctive pattern for MAC strains. Having designed the primers, 305 other restriction enzymes were screened with Genamics Expression
software for suitability for use for PRA. Accordingly,
CfoI was chosen as the second restriction enzyme
because it gave a unique pattern for M. tuberculosis.
Moreover, the CfoI patterns for M. avium, M. intracellulare, and M. scrofulaceum were different, so these species could readily be
differentiated from each other. The sizes of the restriction fragments
produced by Sau96I and CfoI for each species were
then calculated. In all, 8 different restriction patterns were
predicted for Sau96I (Table 2)
and 10 were predicted for CfoI (Table
3) (fragments which were similar in size
were grouped together so that a range of sizes is given, and fragments smaller than 30 bp were excluded). Accordingly, an algorithm was drawn
up for PRA (Table 4).
Specimens.
A total of 400 isolates consisting of 17 American
Type Culture Collection (ATCC) reference strains and 383 clinical isolates were used in the study (Table
5). All clinical isolates were from the
Tuberculosis Reference Laboratory, Yung Fung Shee Memorial Center, Hong Kong, and were identified by standard laboratory methods
(5).
Assay conditions.
A loopful of a bacterial colony was
suspended in 400 µl of distilled water, and the suspension was boiled
for 5 min. The suspension was then centrifuged at 13,000 rpm for
5 min, and the supernatant was used for PCR amplification. A 294-bp
region of the hsp65 gene was amplified with primers
HSP-1 (5'-GCCAAGAAGACCGAYGACGT) and HSP-2
(5'-GGTGATGACGCCCTCGTTGC). PCR was carried out in a final volume of 50 µl consisting of 5 µl of the DNA preparation,
each primer at a concentration of 0.2 µM, each deoxynucleoside
triphosphate at a concentration of 200 µM, 1.5 mM
MgCl2, and 1.25 U of Taq polymerase
(all reagents were from Pharmacia-Biotech, Freiburg, Germany). The
thermal profile consisted of an initial denaturation at 94°C for 2 min, followed by 40 cycles of 94°C for 30s, 62°C for 30s, and
72°C for 1 min and a final period of extension of 6 min at 72°C.
The amplicon was digested with 1 U of Sau96I and CfoI (both from Boehringer Mannheim Biochemicals, Mannheim,
Germany) in separate reactions. The restriction digests were carried
out with 10 µl of the amplicon at 37oC for at
least 1 h. The digests were then electrophoresed in 3% Metaphor
agarose gel (FMC Bioproducts, Rockland, Maine) with ethidium bromide.
The fragment sizes were determined visually by comparison with the DNA
V marker (Boehringer Mannheim).
Selected specimens which gave discrepant results by biochemical and
genotypic tests were further investigated by sequencing
hsp65 and the hypervariable region of 16S rDNA
(
12). The
hsp65 sequences of reference
strains
M. aurum ATCC 23366,
M. flavescens ATCC 14474), and
M. szulgai ATCC
35799 (GenBank accession numbers
AF350414,
AF350413, and
AF350412, respectively) were
determined; and these species were added
to the algorithm
accordingly.
All mycobacterial isolates were amplified by the primers
without any problems. During the study, 7 of 8 predicted
Sau96I restriction
patterns and 8 of 10 predicted
CfoI restriction patterns were
seen (Fig.
1). All could readily be identified by
eye, without
computer assistance. Moreover, the patterns could readily
be recognized
in the presence of gel artifacts such as a marked
"smiling effect."
Digestion of
M. tuberculosis
amplicons with
CfoI produced a characteristic
restriction
pattern (pattern d). All
M. tuberculosis clinical
isolates and the two reference strains were correctly identified
by
PRA. None of the other 302 non-
M. tuberculosis isolates
were
identified as
M. tuberculosis by PRA. Digestion of
MAC amplicons
with
Sau96I produced a characteristic
restriction pattern (pattern
B). Digestion with
CfoI allowed
differentiation between
M. avium (pattern h),
M. intracellulare (pattern i), and
M. scrofulaceum (pattern g). Reference strain ATCC 13950 was
correctly identified
by PRA. Of the 131 clinical isolates, 28 were
identified as
M. avium and 97 were identified as
M. intracellulare. 16S rDNA sequencing
was carried out
for 6
M. avium isolates and 13
M. intracellulare isolates, and the results concurred with those of
PRA. Of the
six isolates identified as non-MAC mycobacteria by PRA, one
was
found to be
M. fortuitum, one was found to be
M. simiae, and the
other four could not be matched
definitively to any
Mycobacterium species in GenBank. Of the
268 non-MAC isolates, 2
M. scrofulaceum clinical
isolates, 1
M. terrae complex clinical isolate, and 1
M. kansasii clinical isolate were identified as
M. intracellulare
by PRA; and the results were
confirmed by 16S rDNA sequencing.
All other reference strains had PRA
patterns consistent with that
of the algorithm (Table
5). Most of the
PRA results were consistent
with the biochemical results with the
exception of those for isolates
of the
M. terrae
complex, for which the PRA profiles were very
heterogeneous.
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FIG. 1.
PRA profiles for different mycobacterial species.
(A) Sau96I digests; (B) CfoI
digests. Lanes (in the designations after the strain names, the
corresponding Sau96I patterns are presented as capital
letters and the CfoI patterns are presented as lowercase
letters): M, DNA V marker (Boehringer Mannheim); 1, M.
tuberculosis complex (Gd); 2, M.
intracellulare (Bi); 3, M. avium (Bh); 4, M. kansasii (Eb); 5, M.
gordonae (Fg); 6, M. fortuitum (Gc); 7, M. chelonae (Hi); 8, M.
abscessus (Gf); 9, M. mucogenicum (Ch); 10, M. neoaurum (Ga); 11, M. simiae
(Ag). Marker positions are indicated on the left (base
pairs).
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|
We sought to develop a molecular biology-based method for the
identification of commonly isolated mycobacteria for large-scale
use in
a routine clinical laboratory. For this purpose, the assay
should be
highly validated, economical, and easy to perform, read,
and interpret.
Accordingly, the
hsp65 gene was chosen for use
in the assay
because it is the best-investigated gene other than
16S rDNA for
taxonomic purposes. Furthermore, it is already used
by a
well-established PRA (
1,
20). One salient feature of
our
assay is the small number of bands present in different restriction
patterns, which makes the results much easier to read but which
results
in reduced discriminatory power compared to other those
of PRAs.
However, unlike other PRAs for mycobacteria, this assay
was never
intended to be a catchall assay for all mycobacteria
but was designed
so that the most commonly isolated mycobacteria,
in particular,
M. tuberculosis and MAC species, could be identified
with ease. To this end, the use of frequently cutting enzymes
such as
HaeIII was purposefully avoided because it would have
generated too many patterns. The use of
HaeIII in other PRAs
was
understandable because of the desire to identify as many species
as
possible. We believe that this approach is potentially hazardous
in
view of the fact that many species are rarely encountered in
a routine
laboratory and thus are not well investigated. The heterogeneity
of our
M. terrae complex isolates convinced us of the
correctness
of our
approach.
In use, we found this assay to be virtually 100% sensitive and
specific for
M. tuberculosis and MAC species. Although
the
numbers of isolates tested are small, the initial results for
mycobacteria other than those belonging to the
M. terrae complex
were encouraging. The heterogeneity of the
M. terrae complex isolates
was expected and had been
reported in other studies (
6,
7,
21). The PRA described
here could be completed on the same day
that specimens were received
and is cost-effective compared to
other assays (
4): its
cost when used on a regular basis is
estimated to be US$1.50 per
sample, and it requires 3.5 min of
technical time per sample (for a
batch size of 40 to 80 specimens).
It is based on openly
available DNA sequence data from a well-researched
mycobacterial
gene. Above all, it is user-friendly and robust,
and it is therefore
highly suitable for large-scale use in a routine
clinical laboratory.
Moreover, the cost can be further reduced
by the use of
CfoI
only for the identification of
M. tuberculosis.
Since
M. tuberculosis and MAC species are the most important
mycobacterial
pathogens and account for more than 90% of our
mycobacterial isolates,
early identification of these organisms is of
great clinical and
public health importance. We anticipate carrying out
this assay
with more than 10,000 specimens per
year.
| |
ACKNOWLEDGMENTS |
We thank Kent Lai, Jacky Cheng, and other colleagues at the TB
Reference Laboratory, Yung Fung Shee Memorial Center, for technical assistance with this assay. We also thank Anna Ng and Viola Tung at the
Public Health Laboratory, Kowloon Hospital, for carrying out the DNA
sequencing, and Margaret Chan, director of health, HKSAR Government,
for permission to publish the manuscript.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: TB Reference
Laboratory, 6/F, Yung Fung Shee Memorial Centre, Cha Kwo Ling Rd., Kwun Tong, Hong Kong. Phone: 852 27093742. Fax: 852 29524064. E-mail: derekwong28{at}hotmail.com.
| |
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Journal of Clinical Microbiology, October 2001, p. 3768-3771, Vol. 39, No. 10
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.10.3768-3771.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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