Diagnosis of Pulmonary Tuberculosis Using

PCR Assays on Sputum Collected within 24 Hours

of Hospital Admission
ROBERT A. COHEN, SHIRIN MUZAFFAR, DAVID SCHWARTZ, SHAHID BASHIR, SCOTT LUKE,
LAURA P. MCGARTLAND, and KAREN KAUL
Department of Pulmonary Medicine/Critical Care and Department of Infectious Diseases, Cook County Hospital, and Rush Medical
College, Chicago; Department of Pathology and Molecular Biology, Evanston Hospital, Evanston, Illinois

There have been few studies evaluating the efficacy of polymerase chain reaction (PCR) testing in
front-line clinical practice. We assessed the diagnostic yield of PCR prospectively in a blinded study of
patients admitted to rule out tuberculosis and compared PCR results to a culture and clinical diagno-
sis of tuberculosis. Specimens were sent for routine smear, culture, and PCR analysis. Sputum sedi-
ments were submitted for PCR amplification of IS6110 sequences by an in-house assay and also the
Roche Amplicor PCR assay targeting 16s ribosomal RNA genes. Eighty-five patients were enrolled: 27
patients had cultures positive for tuberculosis; 12 were smear-positive. PCR by both assays on the first
specimen picked up all patients smear-positive on any specimen. A positive PCR on at least one of
two specimens collected in the first 24 h was 85 and 74% sensitive and 88 and 93% specific for tuber-
culosis by the in-house and Roche techniques, respectively. Sensitivity in smear-negative patients was
73 and 53%, respectively. The in-house PCR detected 100% and Roche detected 95% of patients with
more than paucibacillary (greater than 20 colonies) tuberculosis. We conclude that PCR may be a use-
ful tool to evaluate patients for tuberculosis within the first hospital day. Cohen RA, Muzaffar S,
Schwartz D, Bashir S, Luke S, McGartland LP, Kaul K. Diagnosis of pulmonary tuberculosis us-
ing PCR assays on sputum collected within 24 hours of hospital admission.
AM J RESPIR CRIT CARE MED 1998;157:156–161.

The recent resurgence of tuberculosis complicated by the
AIDS epidemic has refocused attention on the need for more
rapid and accurate diagnostic tests, particularly those using
molecular techniques. Such tests would facilitate early isola-
tion of potentially infectious patients and prompt institution
of antituberculosis chemotherapy and contact investigation. A
valid negative rapid test would free up expensive and scarce
respiratory isolation facilities in hospitals burdened with large
populations of high-risk patients.
Currently available radiometric culture methods for tuber-
culosis diagnosis (1, 2) with nucleic acid probes for identifica-
tion still require from 10 d to 3 wk, and conventional techniques
require 3 to 6 wk. Sputum smears are rapid but insensitive, and
they are not specific for tuberculosis. Therefore, decisions re-
garding respiratory isolation and institution of therapy are still
based largely on clinical grounds. Polymerase chain reaction
(PCR) and other nucleic acid amplification methods may dra-
(Received in original form June 10, 1997 and in revised form August 19, 1997)
Supported by a grant from the Agency for Health Care Policy and Research and
the American Association of Clinical Chemists.
Correspondence and requests for reprints should be addressed to Robert A.
Cohen, M.D., Division of Pulmonary Medical/Critical Care, Cook County Hospi-
tal, 1835 West Harrison, Chicago, IL 60612.
Am J Respir Crit Care Med Vol 157. pp 156–161, 1998
matically increase the sensitivity and specificity of laboratory
tests aimed at detecting Mycobacterium tuberculosis. New au-
tomated commercial systems may be rapid enough to enable
clinicians to make important decisions within hours of admis-
sion (3).
Previous studies of PCR have focused mainly on cultured
specimens obtained selectively from microbiology laborato-
ries, and they have reported specimen-specific yields (4–7).
Some studies have been carried out with large proportions of
acid-fast bacilli (AFB) smear-positive specimens than com-
monly found in clinical populations (5, 6, 8, 9). In addition,
many studies have used culture positivity as the reference and
left out clinical tuberculosis. (3, 6–12). To our knowledge, no
study has prospectively enrolled patients, collected at least
two sputum specimens within 24 h, and studied the feasibility
of PCR as a rapid tool to diagnose or rule out tuberculosis at
the time of first patient contact.
Many amplification targets for M. tuberculosis have been
reported. One of the more common PCR targets is the repeti-
tive sequence IS6110, specific for M. tuberculosis complex. An
alternative approach utilizes the FDA-approved Roche Am-
plicor M. tuberculosis kit, which targets the 16S ribosomal
RNA gene for amplification with subsequent detection of the
products using an M. tuberculosis specific probe.
We performed this prospective study to compare utility of
both techniques with that of AFB smears, culture, and clinical
diagnosis of pulmonary tuberculosis.
Cohen, Muzaffar, Schwartz, et al.: Utility of PCR in Ruling Out PTB 157

METHODS positive-displacement pipettors, and unidirectional work flow. In ad-

dition, dUTP was incorporated into the amplicons so that they could
Population be inactivated by UNG nuclease to prevent carryover contamination.
From December 1994 through June 1995 all patients referred from Positive and negative controls for both the sample preparation and
the emergency room, wards, or clinics of Cook County Hospital who PCR processes were utilized in each experiment, and they included
required admission to isolation rooms to rule out pulmonary tubercu- both known positive and negative sputa as well as a dilution of tuber-
losis were referred to the pulmonary service for evaluation. History, culosis DNA as a positive control and water blanks as negative con-
physical examination, and chest radiography results were reviewed. trols. All samples also underwent amplification of a fragment of the
Chest radiographs were scored by two pulmonologist investigators human p53 gene as a control for amplification inhibitors. The in-house
(R.A.C. and S.M.). The chest radiographs were divided into four assay was based on the work of Eisenach and colleagues (9, 16) and
zones: above the clavicles, and upper, middle, and lower zones. Chest has been previously reported. Two hundred fifty microliters of decon-
radiographs demonstrating nodular, alveolar, or interstitial infiltrates taminated sputum was used for the in-house PCR assay, 100 ml for the
predominantly affecting the zones above the clavicles or upper zones, Amplicor assay and the remaining specimen frozen at 2708 C for re-
or a miliary pattern, were classified as “typical,” whereas those with peat testing if needed, and quality control. The in-house assay con-
other patterns were classified as “atypical.” Normal radiographs or in- sisted of mycobacterial lysis, performed according to the method of
active processes (i.e., calcified granuloma, old rib fracture) were clas- Eisenach (16), amplification, and amplicon detection using hybridiza-
sified as “negative.” Patients with typical chest radiographs were auto- tion in solution to a radioactively labeled oligonucleotide probe spe-
matically isolated and offered enrollment. Symptomatic patients (cough cific for the IS6110 product, as previously described (17). PCR amplifi-
for more than a week, 10-lb. weight loss, night sweats, or fever) with cation was also performed using the Roche Amplicor PCR amplification/
atypical chest radiographs were also isolated and offered enrollment. detection kits, following the directions provided by the manufacturer.
Symptomatic patients with known infection caused by HIV were iso-
lated and offered enrollment even if their chest radiographs were neg- Follow Up
ative. Patients who were in respiratory failure, required ICU admission, The city of Chicago Department of Health maintains a data base of all
were prisoners, or in whom smear- or culture-positive tuberculosis patients suspected to have, or diagnosed with, tuberculosis. This data
had been diagnosed within the previous year were excluded. All pa- base was reviewed 1 and 2 yr after study completion. Information was
tients gave informed consent to participate in the study, which was ap- retrieved for smear, culture, and final diagnosis for all patients evalu-
proved by the institutional review boards of Cook County Hospital ated for this study.
and the University of Illinois.
Face-to-face interviews were conducted by trained research nurses Statistical Methods
using a standardized questionnaire eliciting information on symptoms Univariate statistics were generated for all demographic, clinical, and
of tuberculosis, risk factors, social history, history of previous tubercu- laboratory variables. Frequencies and cross-tabulations were per-
losis, tuberculosis treatment, and tuberculin skin testing. Tuberculin formed on categorical data grouped according to smear, culture, and
skin testing was performed intradermally with 5 TU according to the PCR results using SPSS statistical software package (18). The sensitiv-
Mantoux technique. All patients were offered HIV testing. ity and specificity of the two methods for PCR were compared using
Tuberculosis was defined by a positive culture for M. tuberculosis the weighted least-squares method for the analysis of categorical
on any specimen, including specimens obtained outside the study pro- data (19).
tocol. Patient medical and pharmacy records were reviewed by the
two pulmonologist investigators (R.A.C. and S.M.) to determine if
they met criteria for clinical tuberculosis. They were so classified if
RESULTS
they had resolution of symptoms and infiltrates on chest radiographs Six hundred five patient referrals were made to the pulmonary
after 3 mo of antituberculosis chemotherapy and no alternative etiol- service during the study period. Four hundred nine were iso-
ogy was identified. lated to rule out tuberculosis. Of these, 26 patients had a his-
tory of a positive smear or culture for tuberculosis within the last
Sputum Collection
Trained research assistants collected sputum specimens every 4 h for a
total of three specimens within the first 12 h of arrival to the hospital.
These research assistants were available on call 24 h daily during the TABLE 1
study period, exclusive of weekends and holidays. Eligible patients PATIENT DEMOGRAPHICS
presenting during weekends and holidays were not enrolled. One
specimen was then collected every morning for the next 3 d, for a total Nonenrolled Enrolled
of six specimens per patient. Normal saline induction was performed (n 5 73) (n 5 85)
for patients unable to expectorate spontaneously. Specimens were de- Characteristics (n) (%) (n) (%)
contaminated by the N-acetyl-L-cysteine-NaOH method and concen-
trated by centrifugation (13). Smears for microscopic examination were Sex
prepared from the concentrated specimens and stained using aura- Male 58 79 73 86
mine-rhodamine and examined using fluorescence microscopy. Smears Female 15 21 12 14
demonstrating acid-fast bacilli were reviewed after staining by the Race/ethnicity
Kinyoun staining method (14, 15). A portion of sputum sediment was Black 59 81 67 79
then inoculated onto two slants of Lowenstein-Jensen and Middle- White 5 7 2 2
brook 7H10 (Difco Laboratories, Detroit, MI) media; smear-positive Hispanic 7 8 12 14
Asian Indian 2 3 4 5
specimens were also inoculated in a BACTEC vial containing 7H12
Tuberculosis risk factors
medium. Cultures were incubated for a total of 8 wk. Nucleic acid
HIV positive NA NA 30 35
probes were used to identify M. tuberculosis. Nontuberculous myco- HIV negative NA NA 22 26
bacteria were identified by conventional methods (13). The remaining HIV unknown NA NA 33 39
portion of the pellet was frozen at zero degrees centigrade and then Alcohol abuse NA NA 36 42
transported to the molecular biology laboratory at Evanston Hospital Injection drug use NA NA 13 15
for PCR analysis, which was performed 1 to 4 wk after patient enroll- Homeless NA NA 12 14
ment. PCR results were not made available to clinicians. History of incarceration NA NA 14 16
Tuberculosis, total cases 12 16 27 32
PCR Assays Culture positive 10 14 27 32
Reagent and sample preparation, PCR amplification, and product de- Smear-positive tuberculosis 7 10 12 14
tection were performed in separate rooms using dedicated equipment, Clinical tuberculosis 1 1 0 0
158 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 157 1998

year and were ineligible for the study. Sixty-one patients were TABLE 3
ineligible because they were referred from the Cook County PCR VERSUS CULTURE IN SMEAR-NEGATIVE PATIENTS
Jail. Of these, six patients proved to have tuberculosis. Of the Sensitivity Specificity PV1 PV2
remaining 322 patients, 162 were not felt to be at very high Technique (%) (%) (%) (%)
risk based on a pulmonologist’s review of their history and
In-house 73 88 61 93
chest radiograph. Their work-up proceeded according to usual Roche 53 93 67 89
hospital routine. Only one of these patients proved to have tu- In-house (2 specimens)* 27 93 50 83
berculosis, culture-positive only, on a specimen obtained 4 mo Roche (2 specimens)* 7 98 50 80
after his evaluation.
* Calculations done with the requirement that two specimens collected in the first 24 h
This left a possible 160 patients who were eligible for the be positive.
study. Sixty-eight of those eligible were not enrolled because
they were admitted on holidays and weekends, four patients
refused, and one patient was transferred to ICU. Nine of the
eligible, but nonenrolled, patients proved to have newly diag- nique on all specimens. No patient in our study had smear-
nosed tuberculosis or a rate of 16%. Characteristics of these positive MOTT infection.
groups are shown in Table 1. Although the rate of tuberculosis Overall, the in-house technique was more sensitive (85 ver-
was less in the nonenrolled patients, this difference was not sus 74%) but less specific (88 versus 93%) than the Roche
statistically significant. Amplicor for a final diagnosis of tuberculosis, although these
Two of the 87 patients did not have adequate specimen vol- differences were not statistically significant. As expected, on
ume for PCR processing. Specimens from the remaining 85 smear-negative patients the sensitivity fell (73 and 53%) for
patients were analyzed by PCR. Five-hundred ten specimens the in-house and Roche assays, respectively. The specificity
(6.0 per patient) were processed for routine mycobacteriol- for this group of patients was unchanged (see Table 3).
ogy, and 316 (average, 3.7 per patient; range, 2 to 6) were pro- The specificity of the in-house technique could be im-
cessed for PCR. proved by 5% if both of the two sputum specimens were re-
Our population was mainly black men, and it is reflective quired to be PCR-positive. However, this requirement re-
of our tuberculosis population as a whole (see Table 1). About sulted in a substantial fall in the overall sensitivity for both
one-third of the patients were infected with HIV, one-third techniques. The requirement of two positive PCR tests re-
were negative, and the remainder had unknown status and re- sulted in an even more dramatic drop in sensitivity for smear-
fused HIV testing. Twenty-seven patients had culture-positive negative patients (see Table 3).
tuberculosis (32%). Two of these patients were classified as
having tuberculosis based on positive cultures from nonstudy Incremental Yield of Two Specimens for Tuberculosis
specimens obtained during their admission. Twelve of these The first specimen was positive in 70% of all patients with tu-
had at least one smear positive for tuberculosis and were thus berculosis by the in-house technique and 55% using the Roche
considered to have smear-positive tuberculosis. Cultures from kit (see Table 4). The diagnostic yield increased with the addi-
four patients, all smear-negative, yielded mycobacteria other tional specimen to 85 and 74%, respectively, The yield for
than tuberculosis (MOTT) from sputum specimens, including smear-negative patients was 47% by the in-house and 20% by
M. avium, M. fortuitum, and M. gordonae. the Roche assay; this increased to 73 and 53% with the addi-
tion of a second specimen. We then excluded patients with
PCR and Conventional Mycobacteriology
paucibacillary disease, defined retrospectively as those with 20
The results of PCR as compared with conventional mycobac- colonies or less on final culture, from the analysis. The in-
teriology are presented in Table 2. The PCR results from the house method detected 100%, and Roche detected 95% of pa-
specimens obtained within the first 24 h identified nearly all tients with more than paucibacillary disease.
patients positive with PCR results on any specimen. Therefore
the results of the first two PCR specimens collected within the Analysis of Discrepant Samples
first 24 h of admission became the focus of this analysis. A total of seven patients had apparent false positive PCR re-
For the 12 patients with smear-positive tuberculosis, PCR sults with the in-house assay; three of these seven were also
was positive by either the in-house or the commercial tech-

TABLE 4
TABLE 2
INCREMENTAL YIELD OF PCR ON FIRST TWO SPECIMENS
PCR VERSUS SMEAR AND CULTURE RESULTS BY PATIENT
Specimen 1 Specimen 1 or 2*
Smear (2)
All Tuber- Tuber- Nontuber- (n) (%) (n) (%)
culosis culosis culosis* All tuberculosis, n 5 27
(n 5 27) (n 5 15) (n 5 58) In-house 19 70 23 85
PCR Results (n) (%) (n) (%) (n) (%) Roche 15 55 20 74
Smear-negative tuberculosis, n 515
Either of first two in-house (1)† 23 85 11 73 7 12
In-house 7 47 11 73
Either of first two Roche (1)† 20 74 8 53 4 7
Roche 3 20 8 53
Both of first two in-house (1) 16 59 4 27 4 7
Excluding paucibacillary disease, n 5 20†
Both of first two Roche (1) 13 48 1 7 1 2
In-house 17 85 20 100
Any of 6 in-house (1) 23 85 11 73 7 12 Roche 14 70 19 95
Any of 6 Roche (1) 22 81 10 67 4 7
* If either Specimen 1 or 2 was positive, the patient was considered PCR-positive for
* Includes patients with nontuberculosis mycobacteria. this calculation.
†
p . 0.2 using weighted least-squares method. †
Paucibacillary disease is defined as less than 20 colonies on mycobacterial culture.
Cohen, Muzaffar, Schwartz, et al.: Utility of PCR in Ruling Out PTB
positive by the Roche assay. Repeat PCR analysis of these
samples yielded repeatedly positive results. Three of these pa-
tients actually had a history of pulmonary tuberculosis, two
occurring 2 yr or more prior to their current admission. The
third had a history of tuberculosis diagnosed at another hospi-
tal 4 mo prior to the study; had this history been clear he
would not have been included. The positive PCR results of
these patients (all by the in-house assay, two by the Roche as-
say) indicates the potential for residual nucleic acid in nonvia-
ble organisms to generate a positive PCR result. The false pos-
itive results of the remaining four patients are unexplained,
but they are felt to most likely be cross-contamination of neg-
ative samples with a strongly positive sample during the sam-
ple preparation process. Another possibility is contamination
of the samples with nonviable organisms prior to sample prep-
aration for PCR. Amplicon carryover has been a very rare
event in our laboratory, and periodic testing of the UNG en-
zyme has not revealed failure.
Two of the five patients who had cultures that yielded
MOTT (M. gordonae and M. fortuitum) gave false positive re-
sults on PCR. One of these was positive by both PCR assays,
the other by the in-house method only. In studies by this lab-
oratory and others, both PCR targets are quite specific for
M. tuberculosis complex organisms only, so there is no clear
explanation of these positive results. These isolates were un-
available for follow-up PCR testing.
A final two patients were positive by PCR with negative
smears, cultures, PPDs, histories, and chest radiographs. Again,
no clinical explanation for these results was found.
Five patients, all of whom had smear-negative tuberculosis,
were false negative by in-house and Roche techniques. One
additional patient was false negative by Roche only. The five
patients false negative using both techniques had only one or
two out of six cultures positive for fewer than 20 colonies of
M. tuberculosis. One patient, false negative by Roche, had
only one out of six cultures 11 positive for M. tuberculosis.
None of these patients had cavitary disease on chest radio-
graph.
DISCUSSION
Our study attempted to provide some answers to the ques-
tions of diagnostic yield of PCR prospectively in a blinded
study comparing PCR results with cultures and clinical diagno-
sis of tuberculosis. We performed PCR on as many as six spec-
imens per patient and found that the first two specimens ob-
tained within the first 24 h of hospital arrival identified nearly
all patients with positive PCR results on any specimen. Test-
ing beyond two specimens therefore did not appear to en-
hance the results meaningfully, and it is probably not clinically
or economically practical.
The sensitivity of two PCR specimens obtained within the
first 24 h of arrival to the hospital was 100% for patients with
smear-positive tuberculosis. In addition, the sensitivity of PCR,
although less than 100% for patients with smear-negative tu-
berculosis, increased to 100% when patients with paucibacil-
lary disease are not considered false negative. Because pa-
tients with paucibacillary disease (defined as < 20 colonies on
all cultures) were less likely to have been infectious, the nega-
tive predictive value of two PCR specimens was 100% for the
identification of patients most likely to be infectious. This in-
formation may be useful to clinicians attempting to make diffi-
cult decisions about the need for isolation.
The clinical interpretation of positive PCR results is less
clear in patients with negative sputum smears. Although PCR
159
identified 53 to 73% of smear-negative patients with tubercu-
losis, the clinical utility of these findings is reduced by the
presence of substantial number of false positives, resulting in
positive predictive values of only 61 to 67%. Our results were
derived from a highly selected patient population with a high
prevalence of tuberculosis. If PCR were applied to a popula-
tion with lower prevalence, the positive predictive values
would likely decline further; however, the negative predictive
value would increase.
Because of financial constraints precluding sample collec-
tion on weekends and holidays, we were only able to enroll
approximately half of the eligible patients. There were no sta-
tistically significant differences in patient characteristics be-
tween the enrolled and nonenrolled groups (see Table 1). The
lower rate of tuberculosis in the nonenrolled group may be ex-
plained by the less vigorous work-up these patients received,
possibly reducing the number of cases of tuberculosis.
Financial constraints also necessitated the storage of sam-
ples for batch analysis, rather than immediate analysis as they
came into the laboratory. As commercial kits and automated
amplification/detection equipment becomes available, it will
become more practical and cost-effective for laboratories to
perform nucleic acid amplification for tuberculosis detection
on a more rapid basis. Institution-based cost effectiveness
studies would need to be performed to evaluate the expense
of running PCR assays 7 days a week compared with possible
savings of isolation days and hospital days. Additionally, auto-
mation will make such testing available in a wider number of
laboratories, and standardization will reduce the interlabora-
tory variation currently found (20).
We evaluated two PCR methods, an in-house technique,
and a commercially available kit. There was a tendency for the
commercial kit to be less sensitive but more specific than our
in-house assay; however, these differences did not prove to be
statistically significant. Similar differences have been reported
elsewhere (21). The differing sensitivities between in-house
and Roche tuberculosis assays may be attributable to a num-
ber of factors, including sample preparation methods, the
choice of PCR primers and the PCR assays themselves, the
differing detection methods, and the use of a large sample vol-
ume (22) in the in-house assay.
A strength of our study is that it assesses the potential role
of PCR as it might be used in a high volume clinical setting on
specimens collected within 24 h of patient arrival to the hospi-
tal. There have been few studies evaluating the efficacy and
yield of PCR testing in such a context (23). Many studies re-
lied on laboratory specimens only and did not prospectively
evaluate the diagnostic utility of direct PCR on sputum on pa-
tients newly admitted to the hospital with suspected pulmo-
nary tuberculosis (4–7). Other studies (6, 11, 24–27) have com-
pared PCR using in-house and commercial systems with culture
results on individual sputum samples and not the diagnostic
yield among patients. They have looked at clinical data only to
resolve cases that were false positive. Unlike our study, the spec-
imens have come from mixed populations of newly diagnosed
and treated patients. Protocols for specimen collection were not
controlled, and most clinical information was obtained retro-
spectively.
Prospective studies of similar design to ours compared PCR
results with a clinical diagnosis of tuberculosis with careful fol-
low-up to determine the patient’s final tuberculosis status.
Beige and colleagues (28) prospectively studied 103 non-HIV-
infected patients admitted to rule out tuberculosis. They com-
pared PCR using a DNA-based in-house technique with a pa-
tient-based diagnosis of tuberculosis classified according to
the American Thoracic Society (29) criteria. They found PCR
160 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE
98% sensitive and 70% specific. They had quite a few false
positives in patients who were PPD positive and no evidence
of active tuberculosis. Bradley and colleagues (30) studied res-
piratory tract specimens from 421 patients with a broad range
of risk factors for pulmonary tuberculosis. They found the
RNA-based Gen-Probe M. tuberculosis Direct Test to be
93.6% sensitive, 70% sensitive in smear-negative sputum sam-
ples. In contrast to the work of Beige and colleagues, they found
a specificity of 96.8% overall. Compared with our population,
only 5% of their study population had tuberculosis and nearly
half of these patients were receiving treatment. Chin and col-
leagues (31) evaluated the utility of PCR in respiratory tract
specimens from 227 patients and compared the results with a
rigorous clinical diagnosis of tuberculosis. Like most investiga-
tors they found PCR 100% sensitive in smear-positive patients
but only 50% sensitive in smear-negative patients. They had a
very low false positive rate of less than 1%. The variation in
amplification assays and current lack of standardization among
in-house molecular tests may have contributed to the varia-
tion in sensitivity and specificity results reported.
The potential for false positives results in amplification as-
says is a great concern. We had a rate of 7 to 12% among our
patients. These occurred despite the use of proper laboratory
technique and chemical means to inactivate amplicons, which
should reduce the danger of amplicon carryover and reampli-
fication as a cause of false positives. It seems most likely that
cross-contamination of samples during sample preparation may
have occurred, as samples yielding false positive results con-
tinued to be positive on repeat testing. The multistep nature of
sample preparation used in the in-house assay may have con-
tributed to the higher number of false positives compared with
the Roche kit and is likely to be the basis of the enhanced sen-
sitivity of the in-house assay. Another possible source of error
is contamination of samples with nonviable organisms. PCR
assays directed at DNA targets cannot distinguish viable from
nonviable organisms and therefore cannot differentiate pa-
tients with clinically active disease from those who have been
treated for tuberculosis or have resolved their tubercular dis-
ease without treatment. The observance of PCR-positive sputa
from patients long after successful treatment of tuberculosis is
well known (20). Furthermore, contamination of samples with
nonviable organisms within the laboratory could present an-
other cause for false-positive results that needs to be recog-
nized; amplifiable tuberculosis DNA has been recovered from
sterilized bronchoscopes (17).
In summary, our findings indicate that PCR testing of two
sputum samples obtained within 24 h of arrival to the hospital
may prove diagnostic in all patients who have smear-positive
tuberculosis and 53 to 73% of smear-negative pulmonary in-
fections. PCR testing on two specimens, rather than just one,
allowed the detection of 75 to 85% of patients, missing only
those patients with paucibacillary disease. Further study with
large numbers of patients would be required before this ap-
proach could be adopted widely in clinical practice. The rela-
tively low positive predictive value in smear-negative patients
makes interpretation of a positive test less certain. These find-
ings are consistent with a recent comprehensive review (32),
and the current recommendations of the Centers for Disease
Control and Prevention (33).
Acknowledgment : The writers would like to thank Maureen Gallagher, R.N.,
Carole Schmitz, R.N., Phyllis Price, R.N., Delia DeGuzman, R.N., Jean Van
Voorhis, R.N., Maria Francona, R.R.T., and Oscar Romero, M.D., for their
hard work in specimen and data collection and Tzyy-Chyn Hu, R.N., M.S.,
for data entry. A special thanks to Robert J. Anderson, Ph.D., for providing
help in statistical analysis.
VOL 157 1998
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