Download Molecular Detection of Mycobacterium tuberculosis

Clinical Chemistry 47:8
1553–1558 (2001)
Beckman Conference
Molecular Detection of Mycobacterium tuberculosis:
Impact on Patient Care
Karen L. Kaul
Background: Nucleic acid amplification technologies
such as PCR are revolutionizing the detection of infectious pathogens such as tuberculosis (TB). Amplification technology offers the potential for the diagnosis of
TB in a few hours with a high degree of sensitivity and
specificity. However, molecular assays neither replace
nor reduce the need for conventional smear and culture,
speciation, and antibiotic sensitivity assays.
Methods: We undertook prospective studies of sputum
samples to assess the performance of two PCR-based
assays for the detection of TB as well as the impact of
more rapid availability of test results on patient care.
Results: The sensitivity of both the in-house and Amplicor PCR assays was 100% for smear-positive sputa.
For smear-negative sputa (two sputum samples collected during the first 24 h of hospitalization), the
sensitivity was 85% for our in-house PCR assay and 74%
for the Roche PCR assay. Approximately 10% of the
smear- and culture-negative sputa yielded positive PCR
results; however, more than one-half of these were
positive with both the in-house and Amplicor assays,
suggesting the presence of TB DNA or organisms.
Several of these came from patients whose other samples grew Mycobacterium tuberculosis during the same
admission, and others came from patients who had
previously treated TB. Overall, the specificities of the
in-house and Amplicor PCR assays in smear-negative
patients were 86% and 93%, respectively.
Conclusions: Molecular detection of slow-growing
pathogens such as M. tuberculosis have the potential to
improve clinical care through a dramatic reduction in
the time required for detection and may provide substantial savings in the overall cost of care of a patient
compared with conventional smear, culture, and speciation alone, despite the fact that conventional assays
must still be performed for speciation of nontubercu-
Department of Pathology, Evanston Northwestern Healthcare, 2650 Ridge
Ave., Evanston, IL 60201. Fax 847-570-1938; e-mail [email protected].
Received March 2, 2001; accepted April 26, 2001.
lous mycobacteria and for full assessment of antibiotic
sensitivity.
© 2001 American Association for Clinical Chemistry
Molecular approaches to the diagnosis of disease have
begun to have a major influence on current clinical
management of various disorders. In particular, detection
of human microbial pathogens has been impacted by
nucleic acid-based techniques, primarily amplification
assays. Numerous amplification-based assays have been
developed to offer improvements in the detection of
slow-growing or unculturable organisms, or when information not provided by conventional diagnostic approaches is needed. Quantitative measurement of serum
or plasma concentrations of several viruses is becoming
increasingly useful for monitoring of disease response to
treatment. Because of the potential and widespread application of molecular testing in microbiology, several commercial methods have been developed, whereas methods
for nonmicrobiologic applications remain few.
However, there are several critical differences between
nucleic acid-based testing and conventional microbiology.
Nucleic acid-based tests generally target only a single
organism, whereas cultures generally are more broad in
coverage. Although the molecular assay might rule out a
single microbe, continued analysis in culture may be
required to detect other relevant organisms. Additionally,
antimicrobial resistance studies may be required, also
necessitating further analysis in culture. In some cases,
molecular epidemiologic studies may also be required to
investigate disease spread. Thus, with few exceptions,
molecular assays do not negate the need for performance
of routine cultures.
One of the most useful applications of molecular
testing has been the detection of Mycobacterium tuberculosis. Tuberculosis (TB) is one of the major threats to public
health worldwide, infecting more that one-third of the
world’s population (1 ). The United States has seen an
increase in incidence of TB over the past 1–2 decades,
which has been attributed primarily to the effects of HIV,
poverty, and homelessness (2 ). Although the last few
years have seen a decrease in TB incidence, the emergence
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of drug-resistant strains has generated increased concern
over control of this disease. However, M. tuberculosis is
not the only human pathogen in this genus. Several
nontuberculous mycobacteria can cause disease in immunosuppressed patients, primarily M. avium, M. intracellulare, and M. kansasii (3 ). Clinical presentation and history
can help in making a presumptive diagnosis, but ultimately, positive identification and speciation of the organism are required. Treatment regimens differ for the
various mycobacterial species, as does the need for isolation.
Conventional Laboratory Diagnosis of TB
Traditionally, diagnosis of mycobacterial infection is a
two-stage process requiring weeks for completion. For
diagnosis of pulmonary infection, patients are initially
triaged into isolation if needed based on the results of
acid-fast smears of sputum collected at the time of admission and on the next three consecutive mornings (4 ).
Alternatives to the acid-fast smear include several other
stains, such as Ziehl-Neelsen, or fluorochrome staining
with auramine or auramine/rhodamine, which may be
faster to screen. Acid-fast smears are a necessary first step
but are neither specific nor sensitive; mycobacteria can be
differentiated from other acid-fast organisms such as
nocardia, but definitive discrimination of M. tuberculosis
from other mycobacterial species can be difficult or impossible. Additionally, ⬃10 000 organisms/mL of sputum
are required to yield a positive result (5 ).
Sputum samples, as well as samples from other sites in
patients suspected of having nonpulmonary TB, are also
placed into culture. The sample is decontaminated with
N-acetyl-l-cysteine/sodium hydroxide to prevent overgrowth of the culture by bacteria. Today, the traditional
Lowenstein-Jensen solid culture medium has largely been
replaced by several liquid culture systems. These commercially available systems improve the speed of diagnosis by continuously agitating the cultures to promote
growth, along with continuous monitoring to detect
growth of the organism as early as possible. Once growth
has been detected, determination of the species is still
required. In the past, time-consuming biochemical methods were performed, although most laboratories today
have replaced this approach with a commercially available DNA probe method in which the organisms are lysed
and hybridized in solution to a species-specific fluorescently labeled nucleic acid probe. Available probes target
the most common mycobacterial species and do not
differentiate between members of the M. tuberculosis complex of organisms (M. tuberculosis, M. microti, M. africanum, and M. bovis). Using this approach, laboratories can
generally report mycobacterial culture results in ⬃2
weeks (6 ).
Nucleic Acid Amplification Assays for TB
To provide more rapid diagnosis of TB, many laboratories
have turned to nucleic acid amplification. Currently, two
Food and Drug Administration (FDA)-approved commercial methods are available for this purpose. Roche offers a
PCR-based test targeting a TB-specific portion of the 16S
ribosomal RNA gene, along with a colorimetric detection
system (7–12 ). GenProbe markets a method that utilizes
transcription-mediated amplification of 16S ribosomal
gene transcripts, with product detection performed via
chemiluminescence (7, 8, 12 ). Both of these methods have
received approval from the FDA for testing of smearpositive sputum samples, and the GenProbe assay was
approved in the summer of 2000 for use on smearnegative sputum samples as well. Other manufacturers
are developing molecular assays for TB that use other
amplification methods (13–16 ).
Several laboratories continue to use methods developed in-house for the detection of TB. Great care must be
exercised in validation of these assays and in maintaining
proper quality control. The primary advantage of these
assays, aside from familiarity with their use, includes use
on sample types other than sputum. Although commercial methods can also be used to examine nonsputum
samples, these are off-label uses with respect to FDA
approval. Some laboratories have continued to use inhouse assays validated in accordance with Clinical Laboratory Improvement Amendments of 1988 (CLIA), College of American Pathologists, and NCCLS guidelines
(17–20 ). A wide variety of nucleic acid targets have been
used in in-house assays, as summarized in Table 1.
Targets include single-copy genes (21–27 ) as well as
multicopy targets (28 –33 ) such as repeat sequences,
which offer the potential advantage of increased detection
sensitivity based on the increased number of copies of the
target at the outset of amplification. IS6110, which is
present in 10 –25 copies in organisms of the M. tuberculosis
complex group (including M. tuberculosis, M. bovis, M.
microti, and M. africanum, of which only TB is regarded as
a human pathogen) is perhaps the most commonly used
target for the molecular detection of TB in in-house assays
(28 –30 ). Current molecular assays amplify targets specific
to these organisms, although possibly a more useful
approach would be amplification of a common mycobacterial target, followed by speciation and detection via
probe hybridization.
In general, performance of molecular assays is acceptable, although variation in assay performance is observed,
attributable not only to the design of the amplification
step but also to sample preparation and the detection
method. A survey of the literature revealed sensitivities of
70 –100%, which vary to some extent based on the presence of acid-fast bacillus positivity in the sputum sediment. In acid-fast bacillus-negative samples, the detection
rate of PCR is lower (sensitivity usually 50 – 65% compared with 85–90% for smear-positive sputa) and is related to the number of organisms present in the sample
(8, 9 ). In samples other than sputum, assay sensitivity has
not been systematically studied. Nucleic acid amplification assays have become widely used, however, for the
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Clinical Chemistry 47, No. 8, 2001
Table 1. Molecular detection of M. tuberculosis.
Assay
Target
Method
Detection
Utility
Reference(s)
Roche Amplicor
GenProbe MTD
In-house
16S rRNA gene
16S rRNA
Single copy
MPB64
MPB70
MTB40
65-kDa antigen
32-kDa antigen
Protein B
HSP 65
Multicopy
IS6110
IS686
rRNA genes
PCR
TMAa
Generally PCR
Colorimetric
Chemiluminescent
Detection variable
Smear-positive sputum
Smear-negative and -positive sputum
Respiratory/nonrespiratory assays
(7–10 )
(7,8 )
a
(21 )
(22 )
(23 )
(24 )
(25 )
(26 )
(27 )
(28–30 )
(31 )
(32 )
TMA, transcription-mediated amplification.
detection of tuberculous meningitis, with generally superior performance compared with conventional culture
(33, 34 ).
The specificity of molecular assays for the detection of
TB is dependent on several factors. Primer design is, of
course, important, and assays are implemented using
primers and probes that show no cross-reactivity with
other species. Unacceptable specificity is more likely to be
the result of false-positive results. As with all molecular
assays, laboratories must guard against false-positive results by use of appropriate laboratory practices, such as
spatial separation of work areas, unidirectional workflow,
and chemical inactivation of amplicons. However, there
are other potential causes of false-positive results, such as
contamination of the sample with TB organisms or DNA.
Cross-contamination during DNA preparation is a concern, as is contamination with viable or nonviable organisms during the decontamination and processing of sputum samples. Contamination has been observed in
mycobacteriology laboratories (35 ) and could certainly
lead to DNA false positives.
The risk of a false-positive PCR result in a TB assay
includes two other situations unique to molecular assays.
One situation involves potential amplification of DNA
from nonviable organisms present in samples during or
after antimycobacterial treatment. We have observed positive results in patients up to 2.5 years after completion of
a directly observed therapy program (unpublished results), and others have reported similar findings (13 ).
Thus, communication with the clinicians involved in a
patient’s care and clinical history is essential in avoiding
overcalling a positive PCR result.
A second situation in which nonviable organisms can
lead to a positive PCR result is in samples obtained via
instrumentation, such as bronchoscopy. After collection of
sterile prewashes of sterile bronchoscopes (to reflect material remaining in the barrel of the scope from previous
patients), we have observed amplifiable human DNA in
20% and amplifiable TB DNA in 3.6% of samples, a
number that exceeds our rate of false positives and the
rate of bronchoscopy samples that are culture positive for
TB at our institution (36 ). Similar results have been
reported for Helicobacter pylori DNA in sterile gastroscopes (37 ). It is our policy to request a saline prewash of
bronchoscopy samples to analyze in parallel with the
actual patient sample.
Performance of a PCR Assay
Despite these potential problems, molecular studies for
the detection of TB can provide enormous potential
benefits to patients. Previous studies have all been retrospective in nature, focusing primarily on determination of
assay performance. We undertook a prospective study to
examine the impact of molecular testing for TB in an effort
to better determine the benefits and impact on patient
care. Working with colleagues at Cook County Hospital in
Chicago, we assayed multiple sputum samples from 85
consecutive consenting patients who were admitted to the
pulmonary service with a presumptive diagnosis of TB
(38 ). We performed PCR on each sputum sample, using
both our in-house assay targeting IS6110 and the Roche
Amplicor assay. PCR results were compared to conventional smear, culture, and speciation (using the nonamplified GenProbe Accuprobe method) results generated in
the Cook County Hospital Mycobacteriology Laboratory.
In general, the performance of both our in-house and
the Roche assays was good. In smear-positive patients,
both assays showed 100% sensitivity and specificity, reassuring values because this is the primary application for
which the commercial assays have received FDA approval.
However, the results in smear-negative sputa were also
acceptable. Our in-house assay yielded a sensitivity of
89%. The Roche assay, which uses a somewhat smaller
volume of sputum, had a lower sensitivity (85%) than our
IS6110 method. However, the Roche method has a con-
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current specificity that was better (93% compared with
86% for the IS6110 assay).
The implementation of PCR testing of the first two
sputum samples collected during the first 24 h of hospitalization would allow a more rapid diagnosis of TB in
these patients. Our in-house and the Roche Amplicor
assays yielded PCR sensitivities (compared with culture)
of 85% and 74%, respectively, along with specificities of
88% and 93%. In this study, if patients with paucibacillary
(⬍20 colonies on solid medium) were excluded, the PCR
sensitivity increased to 100% and 95% for the in-house
and Roche Amplicor assays, respectively (38 ), illustrating
that the performance of PCR is related to the burden of
organisms present in the sample.
False-positive PCR results in comparison with culture
were observed for both assays. Although initially disappointing, many of these results were seen to be positive in
both assays. Because these assays target different fragments of DNA, this suggests that actual organisms or TB
DNA could be present in the sample. In fact, when full
clinical data were examined on these patients, two were
found to have been previously treated for TB infection,
but before the 1-year study exclusion period we were
using. Because these false-positive results occurred in
smear-negative patients who would have been discharged from isolation (and from the hospital were they
otherwise healthy), this would have led to unneeded
continuation of isolation and medication had the PCR
results been used to dictate treatment. Thus, false-positive
molecular results represent a major problem in the widespread implementation of these assays. On the other
hand, our assay still had a very high negative predictive
value, so that patients could have been confidently released from isolation as a result of a negative PCR.
Impact of PCR Detection on Patient Care
Possible outcomes on patient care and clinical management as a result of PCR testing are summarized in Table
2. In essence, in smear-positive patients, a positive PCR
would indicate that the mycobacterial species was in fact
TB, and patients would be appropriately retained in
isolation and treated until their sputum samples became
smear negative. A negative result would indicate the
presence of a nontuberculous mycobacterial infection, and
the patient could be released from isolation and treated
more appropriately. The potential for positive impact on
patient care with a reduction in the cost of care is
apparent.
In summary, nucleic acid amplification assays are
warranted on smear-positive sputum samples and potentially on smear-negative samples if the clinical presentation is highly suggestive of TB. Recent guidelines from the
Centers for Disease Control support this approach (12 ). In
smear-negative patients, the PCR may also be of value,
although it should be used with more caution. Most
positive PCR results will be indicative of a real TB
infection. If clinical suspicion is high, treatment could be
begun, but in the face of uncertainty, waiting until the
culture results become available before beginning treatment is perhaps the best course. Because the patient is
smear negative, there is presumably less risk of infecting
others than in the smear-positive situation. Additionally,
PCR may be useful in the diagnosis of extrapulmonary TB
infections, particularly for meningitis (33, 34 ).
Future Directions
Thus, the rapid availability of results obtained by molecular assays can be a considerable advantage to patient
management in certain situations. More rapid and appropriate treatment can lead to fewer ultimate complications
and hospitalization in patients who have the disease, and
avoidance of unnecessary treatment and isolation of those
who do not have TB. Similar pressures for more rapid
diagnostic capabilities have driven the development of
other molecular assays, particularly those for slow-growing viruses, and particularly for the diagnosis of infections
of the central nervous system.
There is, however, substantial room for further advancement. At the present time, most molecular assays
are fairly work-intensive, requiring analysis of amplicons
by one or more gels and probes. Colorimetric, chemiluminescent, and fluorescent detection methods have been
put to use, primarily in the commercial systems, but more
rapid methods as well as the automated equipment to
perform these techniques are needed. New real-time PCR
instrumentation is expected to have a very positive impact on the laboratory performance of PCR, but additional
changes in the practice of molecular pathology will still be
Table 2. Potential clinical outcomes of patients undergoing TB detection by PCR.
Smear
PCR
Culture (Speciation)
Outcome
Nega
Neg
Neg
Neg
Pos
Pos
Pos
Pos
Pos
Pos
Neg
Neg
Pos
Pos
Neg
Neg
Pos (TB)
Neg
Pos (TB)
Neg
Pos (TB)
Neg (MOTT)
Pos
Neg (MOTT)
Maintenance in isolation and on treatment
Unnecessary isolation/treatment until culture results available
No change in management; TB not detected early
Earlier discontinuation of treatment and isolation
No change in management; earlier certainty of diagnosis
Unnecessary continuation of isolation/treatment
Incorrect discontinuation of treatment and isolation
Earlier discharge and change in treatment
a
Neg, negative; Pos, positive; MOTT, mycobacteria other than tuberculosis.
Clinical Chemistry 47, No. 8, 2001
needed. Rather than detection of single microbes, in the
future molecular assays will need to be aimed at multiple
organisms. Multiplex amplification reactions may be useful for the simultaneous detection of multiple pathogens
in a single sample. Alternatively, consensus primers may
be used for amplification of related microbes, such as
mycobacteria, with species-specific detection performed
using probes or DNA arrays.
It is likely that future detection systems may be sufficiently sensitive to detect small numbers of pathogens
without any amplification. Whatever the format and
instrumentation for the assays, the impact of molecular
testing on patient care will be substantially greater in the
future.
Collaborators in our PCR studies include Dr. Robert
Cohen, Shirin Muzaffar, and David Schwartz at Cook
County Hospital in Chicago. Scott Luke provided expert
technical assistance. I would also like to acknowledge the
financial support of the American Association of Clinical
Chemistry and the Agency for Health Care Policy and
Research. Additionally, Dr. Lemuel Bowie provided invaluable professional inspiration and advice on outcome
studies in the clinical laboratory.
References
1. Anonymous. Tuberculosis morbidity—United States, 1994.
MMWR Morb Mortal Wkly Rep 1995;44:387–9, 395.
2. Hamburg MA, Freiden TR. Tuberculosis transmission in the
1990 s. New Engl J Med 1994;332:1071– 6.
3. Falkinham JO. Epidemiology of infection by nontuberculous mycobacteria. Clin Microbiol Rev 1996;9:177–215.
4. Huebner RER, Good C, Tokars JI. Current practices in mycobacteriology: results of a survey of state public health laboratories.
J Clin Microbiol 1993;31:771–5.
5. Yeager H Jr, Lacy J, Smith LR, Le Maistre CA. Quantitative studies
of mycobacterial populations in sputum and saliva. Am Rev Resp
Dis 1967;95:998 –1004.
6. Anonymous. Laboratory practices for diagnosis of tuberculosis—
United States, 1994. MMWR Morb Mortal Wkly Rep 1995;44:
587–90.
7. Abe C, Hirano K, Wada M, Kazumi Y, Takahashi M, Fukasawa Y, et
al. Detection of Mycobacterium tuberculosis in clinical specimens
by polymerase chain reaction and Gen-Probe amplified Mycobacterium tuberculosis direct test. J Clin Microbiol 1993;31:3270 – 4.
8. Dalovisio JR, Montenegro-James S, Kemmerly SA, Genre CF,
Chambers R, Greer D, et al. Comparison of the Amplified Mycobacterium tuberculosis (MTB) direct test, Amplicor MTB PCR, and
IS6110-PCR for detection of MTB in respiratory samples. Clin
Infect Dis 1996;23:1099 –106.
9. Nolte FS, Metchock B, McGowan JE Jr, Edwards A, Okwumabua O,
Thurmond C, et al. Direct detection of Mycobacterium tuberculosis
in sputum on polymerase chain reaction and DNA hybridization.
J Clin Microbiol 1993;71:1777– 82.
10. Shawar RM, El-Zaatari FAK, Nataraj A, Clarridge JA. Detection of
Mycobacterium tuberculosis in clinical samples by amplification of
DNA. J Clin Microbiol 1993;29:712–7.
11. Schirm J, Oostendorp LAB, Mulder JG. Comparison of Amplicor,
in-house PCR, and conventional culture for detection of Mycobac-
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
1557
terium tuberculosis in clinical samples. J Clin Microbiol 1995;33:
3221– 4.
Centers for Disease Control and Prevention. Update: nucleic acid
amplification tests for tuberculosis. MMWR Morb Mortal Wkly Rep
2000;49:593– 4.
Hellyer TJ, Fletcher TW, Bates JH, Stead WW, Templeton GL, Cave
MD, Eisenach KD. Strand displacement amplification and the
polymerase chain reaction for monitoring response to treatment in
patients with pulmonary tuberculosis. J Infect Dis 1996;173:
934 – 41.
DesJardin LE, Chen Y, Perkins MD, Teixera L, Cave MD, Eisenach
KD. Comparison of the ABI 7700 system (TaqMan) and competitive PCR for quantification of IS6110 DNA in sputum during
treatment of tuberculosis. J Clin Microbiol 1998;36:1964 – 8.
Rantakokko-Jalava K, Marjamaki M, Marttila H, Makela L, Valtonen V, Viljanen MK. LCx Mycobacterium tuberculosis assay is
valuable with respiratory specimens, but provides little help in the
diagnosis of extrapulmonary tuberculosis. Ann Med 2001;33:55–
62.
O’Conner TM, Sheehan S, Cryan B, Brennan N, Bredin CP. The
ligase chain reaction as a primary screening tool for the detection
of culture positive tuberculosis. Thorax 2000;55:955–7.
Medicare, Medicaid, and CLIA programs: regulations implementing the Clinical Laboratory Improvement Amendments of 1988
(CLIA). Fed Regist 1992;57:7137– 86.
Molecular pathology checklist. Northfield, IL: College of American
Pathologists, 1999.
National Committee for Clinical Laboratory Standards. Molecular
diagnosis methods for infectious disease; approved guideline
MM3-A. Villanova PA: NCCLS, 1995.
Whittaker MH, Farkas DH, Kaul KL, Shrimpton AE, Leonard DGB.
Recommendations for in-house development and operation of
molecular diagnostic tests. Am J Clin Pathol 1999;111:449 – 63.
Shankar P, Manjunath N, Lakshmi R, Aditi B, Seth P, Shriniwas.
Identification of Mycobacterium tuberculosis by polymerase chain
reaction. Lancet 1990;335:423.
Cousins DV, Wilton SD. Francis BR. Use of polymerase chain
reaction for the rapid diagnosis of tuberculosis. J Clin Microbiol
1992;30:225–58.
Del Portillo P, Murillo LA, Patarroyo ME. Amplification of a speciesspecific DNA fragment of Mycobacterium tuberculosis and its
possible use in diagnosis. J Clin Microbiol 1991;29:2163– 8.
Brisson-Noel A, Lecossier D, Nassif X, Gicguel B, Levy-Frebault V,
Hance AJ. Rapid diagnosis of tuberculosis by amplification of
mycobacterial DNA in clinical samples. Lancet 1989;ii:1069 –71.
Soini H, Skurnik M, Lippo K. Detection and identification of
mycobacteria by amplification of a segment of the gene coding for
the 32-kilodalton protein. J Clin Microbiol 1992;30:2025– 8.
Sjobring U, Mecklenburg M, Anderson AB. Polymerase chain
reaction for detection of Mycobacterium tuberculosis. J Clin
Microbiol 1990;28:2200 – 4.
Da Silva Rocha A, da Costa Leite C, Torres H, de Miranda AB, Pires
Lopes MQ, Degrave WM, Suffys PN. Use of PCR-restriction fragment length polymorphism analysis of the hsp65 gene for rapid
identification of mycobacteria in Brazil. J Microbiol Methods
1999;37:223–9.
Eisenach KD, Cave MD, Bates JH, Crawford JT. Polymerase chain
reaction amplification of a repetitive DNA sequence specific for
Mycobacterium tuberculosis. J Infect Dis 1990;161:977– 81.
Kent L, McHugh TD, Billington O, Dale JW, Gillespie SH. Demonstration of homology between IS6110 of Mycobacterium tuberculosis and DNA of other Mycobacterium spp. J Clin Microbiol
1995;33:2290 –3.
Mulcahy GM, Kaminsky ZC, Albanese EA, Sood R, Pierce M.
1558
31.
32.
33.
34.
35.
Kaul: Molecular Detection of Tuberculosis
IS6110-based PCR. Methods for the detection of Mycobacterium
tuberculosis. J Clin Microbiol 1996;34:1348 –9.
Hermans PWM, van Soolingen D, Dale JW. Schuitema ARJ,
McAdam RA, Catty D, van Embden JDA. Insertion element IS986
from Mycobacterium tuberculosis: a useful tool for diagnosis and
epidemiology of tuberculosis. J Clin Microbiol 1990;28:2051– 8.
Boddinghaus R, Rogall T, Flohr T, Blocker H, Bottger ET. Detection
and identification of mycobacteria by amplification of rRNA. J Clin
Microbiol 1990;28:1751–9.
Shankar P, Manjunath N, Mohan KK, Prasad K, Behari M, Shriniwas, Ahuta GK. Rapid diagnosis of tuberculous meningitis by
polymerase chain reaction. Lancet 1991;227:5–7.
Donald PR, Victor TC, Jordaan AM, Schoeman JG, Van Helden PD.
Polymerase chain reaction in the diagnosis of tuberculous meningitis. Scand J Infect Dis 1993;25:613–7.
Wurtz R, Demarais P, Trainor W, McAuley J, Kocka F, Mosher L,
Dieterich S. Specimen contamination in mycobacteriology laboratory detected by pseudo-outbreak of multi-drug resistant tuberculosis: analysis by routine epidemiology and confirmation by molecular technique. J Clin Microbiol 1996;34:1017–9.
36. Kaul KL, Luke S, McGurn C, Snowden N, Monti C, Fry WA.
Amplification of residual DNA sequences in sterile bronchoscopes
leading to false-positive PCR results. J Clin Microbiol 1996;34:
1949 –51.
37. Roosendaal RE, Kuipers EJ, Van Den Brule AJC, Pena AS, Uyterlinde AM, Walboomers JMM. Importance of the fiber endoscopic
cleaning procedure for detection of Helicobacter pylori in gastric
biopsy specimens. J Clin Microbiol 1994;32:1123– 6.
38. Cohen RA, Muzaffir S, Schwartz D, Luke S, Kaul KL. Diagnosis of
pulmonary tuberculosis using PCR assays on sputum collected
within 24 h of hospital admission. Am J Respir Crit Care Med
1998;157:156 – 61.