Eur J Clin Microbiol Infect Dis (2000) 19 : 859–867
Q Springer-Verlag 2000
Article
Clinical Evaluation of an In-House IS6110 Polymerase
Chain Reaction for Diagnosis of Tuberculosis
J. Almeda, A. García, J. González, L. Quintó, P.J. Ventura, R. Vidal, G. Rufí,
J.A. Martínez, M.T. Jiménez de Anta, A. Trilla, P.L. Alonso
Abstract The aim of this study was to clinically validate a heminested polymerase
chain reaction (PCR) method, based on the IS6110 insertion segment of Mycobacterium tuberculosis complex, for the diagnosis of tuberculosis. Samples of pulmonary,
extrapulmonary and blood origin were collected prospectively from 331 patients. All
samples were processed to detect acid-fast bacilli by direct stain, culture and PCR.
The gold standard comparison was a clinically based final case definition of tuberculosis corresponding to group 3 of the American Thoracic Society’s classification
system. The sensitivities of stain, culture and PCR were 41%, 65% and 59%, respectively. Overall specificity exceeded 97% for all techniques. The combination of PCR
and direct stain achieved a sensitivity similar to that of culture alone. The PCR
method detected 74 of 95 (78%) culture-positive results. In a hospital setting, PCR
could be a useful, reliable tool for diagnosis of tuberculosis and may be introduced
as a complementary routine diagnostic laboratory method.
J. Almeda, L. Quintó, P.J. Ventura, P.L. Alonso
Unitat d’Epidemiologia i Bioestadística, Institut d’Investigacions
Biomèdiques August Pi i Sunyer (IDIBAPS), Hospital Clínic,
Barcelona, Spain
A. García
Departament de Microbiologia i Parasitologia, Institut
d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS),
Facultat de Medicina, Universitat de Barcelona
J. González (Y)
Servei de Microbiologia, Hospital Clínic, Villarroel 170,
08036 Barcelona, Spain
e-mail: jgm6medicina.ub.es
Tel.: c34-93-2275522
Fax: c34-93-2275400
J. González, M.T. Jiménez de Anta
Departament de Microbiologia i Parasitologia, Institut Clínic
d’Infeccions i Inmunologia, Institut d’Investigacions
Biomèdiques August Pi i Sunyer (IDIBAPS), Hospital Clínic,
Barcelona, Spain
R. Vidal
Servei de Neumologia, Ciutat Sanitària Vall d’Hebron,
Barcelona, Spain
G. Rufí
Servei de Malalties Infeccioses, Ciutat Sanitària Universitària de
Bellvitge, L’Hospitalet del Llobregat, Barcelona, Spain
J.A. Martínez, A. Trilla
Departament de Malalties Infeccioses, Institut Clínic
d’Infeccions i Inmunologia, Institut d’Investigacions
Biomèdiques August Pi i Sunyer (IDIBAPS), Hospital Clínic,
Barcelona, Spain
Introduction
Current policy for control of tuberculosis (TB) consists
of early identification and cure of infectious cases. Most
infectious cases are detected by direct-smear microscopy examination of sputum, and treatment is usually
started before obtaining laboratory confirmation.
Microscopy for acid-fast bacilli (AFB) is a rapid, simple
diagnostic method. Although its sensitivity ranges from
22% [1] to 78% [2], its specificity remains high (99% [1]
to 100% [3]). The sensitivity of direct staining may vary
as well, depending on the clinical radiological findings,
ranging from 52% in patients with tuberculous caverns
to 32% in patients with pulmonary infiltrates only [4].
Culture is more sensitive than direct staining for detection of AFB, but it takes longer. Culture requires an
average of 21.7 days to obtain results; even the Bactec
radiometric technique (Becton Dickinson, USA)
requires 13.4 days to produce results [5]. The sensitivity
of culture ranges from 50% [6] to 81% [7], estimates
that can also vary depending on the clinical presentation. Thus, a more rapid, sensitive and specific diagnostic assay, such as PCR, could contribute to the
control of Mycobacterium tuberculosis [8–11]. PCR may
overcome some difficulties associated with conventional diagnostic techniques, such as the large number
of bacilli required for positive stained smears and the
slow growth of mycobacteria in selective media [12].
860
Several studies validating PCR as a tool for diagnosis of
TB have recently been published. Clarridge et al. [13]
reported a very high sensitivity using an IS6110targeted PCR system. Overall, the sensitivity and
specificity of PCR ranges from 50 to 100% [14–19]. It
has been suggested that this wide range may be due to
the variability of PCR results in different laboratories
[20, 21].
According to this data, the patients were classified as belonging to
group 3 of the ATS classification. Classification was determined
by two independent TB expert reviewers, based on the initial clinical history recorded, without seeing the patient, and before
confirmation by any microbiological laboratory result. Undefined
cases were established by lack of agreement between the two
reviewers.
In this study we evaluated the clinical validity and
applicability of a PCR method based on the IS6110
insertion segment for the detection of Mycobacterium
tuberculosis in HIV-negative patients. The PCR was
compared with direct staining and culture. The results
of all diagnostic methods were compared with a clinically based case definition of TB, which is our gold
standard, and sensitivity and specificity values were
calculated.
The American Thoracic Society [23] classifies people exposed to
Mycobacterium tuberculosis into six groups. Group 0 comprises
persons not exposed, not infected and tuberculin skin test (PPD)negative. Group 1 comprises those people who have been
exposed to tuberculous patients but who are PPD-negative.
Group 2 comprises infected (positive PPD test) persons who have
no clinical, radiological or microbiological signs of TB. Group 3
comprises patients with active TB who have clinical or radiological evidence of TB. Ideally, the culture should be positive. If the
culture is negative, the PPD test must be positive. Group 4
comprises persons with nonactive TB, defined as cases that are
PPD-positive, without clinical signs of active TB but with positive
microbiological data or radiological signs that suggest TB in the
past. Group 5 comprises patients suspected to have TB but for
whom confirmation is unavailable and diagnostic studies are
incomplete.
Materials and Methods
Microbiological Processing of Clinical Samples. All clinical
samples were processed at the microbiology laboratory of the
Hospital Clinic. Laboratory technicians were blinded to the group
of patients from which each clinical sample came.
Study Population. The study was conducted at the Hospital
Clinic of Barcelona from 1 March 1994 to 29 February 1996. In
order to identify all TB patients promptly, a list of those patients
admitted to the hospital who were started on isoniazid treatment
was obtained from the Pharmacy Department every day. In addition, a list of the patients being followed-up in the TB outpatient
clinic was obtained twice a week. On the basis of this information,
the patients were classified into the following categories: new
HIV-positive TB cases, new HIV-negative TB cases, TB cases
with more than 1 week of treatment, patients undergoing prophylactic treatment, or non-TB cases. Following receipt of informed
consent, all HIV-negative patients who started anti-TB treatment
were included. TB treatment was a short-course, rifampicin-based
6-month chemotherapy regimen (rifampicin, isoniazid and pyrazinamide for the first 2 months, followed by 4 months of rifampicin
and isoniazid). Pulmonary or extrapulmonary clinical samples for
diagnostic purposes were collected, according to the clinical presentation in each case, at the time of diagnosis and before treatment was initiated. Blood samples were also obtained from all
patients at the same time.
Another two control groups of volunteers, who were supposed to
be non-TB individuals, were also recruited. Group 1 comprised
third-year students attending the School of Medicine, University
of Barcelona, and group 2 comprised patients with chronic
obstructive pulmonary disease (COPD) without clinical signs of
TB who were admitted to the Pneumology Department. HIV
antibodies were determined and tuberculin skin tests performed
anonymously; signed, informed consent was obtained in all
cases.
Gold Standard and Case Definition. The following case definitions [22] were established before the study was started. The final
definition (at the end of treatment) was determined by an investigators’ consensus, taking the following criteria into consideration:
clinical picture, chest radiographic findings, tuberculin skin test,
microbiology results, other laboratory findings, computed axial
tomography scan (when available) and clinical response to antiTB drug therapy. According to this data, the cases were classified
into group 3 of the American Thoracic Society (ATS) classification described below [23]. Doubtful cases were defined by a lack
of consensus of two or more authors. Initial definition represents
the situations that clinicians face the first day of treatment.
Nonsterile samples were digested and decontaminated by the
NaOH-NALC (N-acetyl-L-cysteine) method [24] before culture.
Sterile samples were divided into two equal aliquots, one for
culture and smear and the other for amplification procedures.
The latter aliquot, was submitted to decontamination as a
nonsterile specimen in order to obtain an appropriate sample for
the amplification technique. Liquid samples such as cerebrospinal
fluid, joint fluid, abdominal fluid, pleural fluid and urine were
concentrated by centrifugation if more than a 1 ml volume was
available [24]. The tissue biopsies were ground and processed as
the remaining samples. Smears of all samples (except blood) were
prepared and stained with auramine-rhodamine fluorochrome
stain. Determination of semiquantitative grade was made in a 1 to
4 scale [24].
For all samples, aliquots of 200 ml were inoculated onto Löwenstein-Jensen slants and incubated at 37 7C for up to 8 weeks. In
addition, 500 ml of the sediment was inoculated into radiometric
Bactec 12B medium and incubated at 37 7C for 6 weeks. Blood
samples were cultured directly by adding 5 ml to a Bactec 13A
bottle and incubated following the same scheme. Solid medium
slants were read weekly and Bactec cultures were read eight times
over 6 weeks. A growth index of 200 on Bactec medium was
considered positive and was further confirmed by Ziehl-Neelsen
stain. Solid medium slants were considered positive when colonies grew; growth on positive slants was further confirmed as
mycobacteria by Ziehl-Neelsen stain.
Strains were identified using the morphological aspect on Bactec
(Becton Dickinson, USA) [25] smears and the AccuProbe (Gen
Probe, USA) method. When identification was inconclusive, gas
chromatography [26] and routine biochemical methods were used
[24].
A 0.5 ml aliquot of the pellet remaining after decontamination
was obtained for each sample except blood. Blood samples were
processed as follows: leukocytes were extracted from 3 ml of total
blood by Ficoll-Hypaque (1119) gradient, washed and resuspended in Tris-EDTA buffer (100 mM Tris-HCl, 10 mM EDTA,
pH 8.0) [27]. Two 0.5 ml aliquots were obtained. Aliquots were
stored at –30 7C until amplification procedures were performed.
861
A single-tube heminested PCR with unlabeled primers was used
for amplification [28, 29]. This method is a modification of the
tube-nested technique described by Wilson et al. [30].
Aliquots were heated at 95 7C for 30 min and concentrated by
centrifugation at 12,500!g for 15 min. The pellet was resuspended in tenfold TE buffer with 50 mg/ml of lysozyme. This
suspension was incubated for 90 min at 37 7C, after which it was
centrifuged for 10 min. Proteinase K and sodium dodecyl sulfate
(SDS) were added to the final concentrations of 250 mg/ml and
1% (w/v) and incubated for 30 min (or overnight) at 43 7C. The
suspension was extracted with phenol chloroform (25 : 24 : 1) twice
and chloroform-isoamyl alcohol (24 : 1) twice and was precipitated
with two volumes of ethanol 100% and 0.2 M NaCl and stored at
–20 7C overnight. It was then washed with 70% ethanol, dried and
resuspended in 100 ml of distilled water. Ten microliters was used
for PCR amplification.
The DNA target used was the repetitive sequence IS6110, which
is found in the members of Mycobacterium tuberculosis complex
at varying copy numbers [31, 32]. The outer primers used were
Tb294 (5b-GGACAACGCCGAATTGCGAAGGGC-3b) and
Tb850 (5b-TAGGCGTCGGTGACAAAGGCCACG-3b), which
generate a product of 580 base pairs (bp), and the inner primer
used was Tb505 (5b-ACGACCACATCAACC-3b) [30], which
generates a final fragment of 369 bp.
The outer primers Tb294 and Tb850 were both used at a concentration of 10 nM, and the inner primer Tb505 was used at a
concentration of 500 nM. The amplification was performed in
0.5 ml PCR tubes with a final volume of 50 ml using a 480 thermal
cycler (Perkin-Elmer, USA). Each reaction tube contained 2.5 U
of Taq DNA polymerase (Gibco BRL, USA), 2.5 U of uracil-Nglycosylase (UNG) (Boehringer Mannheim, Germany) in order
to avoid contamination with the products of previous reactions
[33], 200 mM each of dATP, dCTP, dGTP and 600 mM dUTP
(Boehringer Mannheim), final buffer 1!, 2 mM of MgCl2 and
20 ml of sample. After incubating for 15 min at 25 7C to allow
UNG to act, the temperature was raised to 94 7C for 5 min to
inactivate the enzyme. The thermal cycler is programmed to
perform the PCR in two stages that are distinguished by different
annealing temperatures. The first stage of amplification involves
30 cycles of denaturation at 94 7C for 45 s, with primer annealing
and extension carried out in one step at 72 7C for 1 min 30 s. The
second stage included 30 cycles of denaturation at 94 7C for 45 s,
primer annealing at 55 7C for 1 min and extension at 72 7C for
30 s, after which the reaction mixture was held at 72 7C.
To detect sample inhibitors, a duplicate tube of 50 ml of PCR mix
for each sample was spiked with 5 ml of an aqueous solution
containing 43 pg of purified DNA target. In order to maintain
reagent proportions, 5 ml of the original sample was added to
these duplicate tubes instead of 10 ml. To avoid cross-contamination, DNA purification, amplification and detection was carried
out in a separate room from that in which the buffers were
prepared. Samples were processed in lots of 20, including one
extraction-negative control and one amplification-negative
control. A positive control was also included in each lot of
samples.
Twenty microliters of amplified product was analyzed by
ethidium bromide (0.5 mg/ml; Sigma) electrophoresis in 2% (w/v)
agarose gel. The gels were visualized and photographed on an
ultraviolet transilluminator. The presence of a band in the same
position as that shown by a positive control and spiked duplicates,
namely 369 bp, was considered positive.
Specificity Testing of the Amplification Method Using Bacteria and
Mycobacterial Strains. The following 13 strains of six mycobacterial species and 18 strains of eight bacterial species were
assayed: Mycobacterium tuberculosis (np5), Mycobacterium
xenopi (np3), Mycobacterium kansasii (np2), Mycobacterium
avium (np1), Mycobacterium scrofulaceum (np1), Mycobacterium gordonae (np1), viridans group streptococci (np2),
coagulase-negative staphylococci (np3), Proteus mirabilis (np1),
Klebsiella pneumoniae (np2), Enterococcus spp. (np2), Staphylococcus aureus (np3), Escherichia coli (np2) and Pseudomonas
aeruginosa (np3). All strains were isolated previously in clinical
samples. For each strain a suspension of 5 ml of distilled water
was prepared for 1 McFarland bacterial density. Dilutions were
prepared, and 0.5 ml aliquots for several concentrations were
obtained. For Mycobacterium tuberculosis the concentrations
studied were 10 7 cfu/ml, 10 5 cfu/ml, 10 3 cfu/ml and 10 2 cfu/ml. For
the remaining species the concentrations used were 10 7 cfu/ml,
10 5 cfu/ml and 10 3 cfu/ml. The 0.5 ml aliquots were processed for
lysis, DNA extraction, amplifications and detection following the
same protocol as that used for the clinical samples.
Statistical Methods. Results were expressed as proportions, and
95% confidence intervals were calculated. Sensitivity and specificity were calculated using standard methods [34], comparing the
results of each diagnostic method with the case definitions. Statistical differences between sensitivity and specificity of different
diagnostic methods were tested by the McNemar exact binomial
test. Degree of agreement between the case definitions was
measured through Kappa statistic index. A Kappa value of 1 indicates perfect agreement and a value of zero means no agreement.
Cases not well defined due to lack of case definition agreement
were not considered in the analysis. A significance level of 5%
was chosen for all statistical tests, using Bonferroni’s correction in
multiple tests.
Results
Following the recruitment system described, a total of
553 patients started receiving isoniazid treatment
during the study period or were attended in the TB
outpatient clinic. One hundred ninety-eight (36%)
were found to be HIV-positive, 39 had previously
started anti-TB treatment, 53 were receiving TB
prophylaxis and 25 were lost to follow-up. Therefore, a
total of 238 HIV-negative TB patients were included in
the study group. The mean age of patients in the study
was 48 years (95% CI, 46–51), with a range of 17 to 91
years. One hundred sixty-six (69%) were male. The
other TB-free cohorts comprised 46 students and 47
COPD patients. The students’ mean age was 20 years,
67% being female. The mean age of the COPD patients
was 68 years, ranging from 42 to 82 years, with only
13% being female.
Clinical samples were collected from 331 study and
control individuals (Table 1). In total, 316 samples were
available for stain, 319 for culture and 314 for PCR.
Additionally, blood samples were collected from 261
individuals.
Clinical samples were of pulmonary origin (sputum,
bronchial aspirate, bronchoalveolar lavage and gastric
fluid) in 270 individuals, including all controls, and of
extrapulmonary origin (lymph nodes, feces, joint fluid,
abdominal fluid, cerebrospinal fluid, pleural effusion,
bone marrow aspiration, urine, vertebral bone biopsy
and pus) in 50 individuals; 11 samples were insufficient
862
Table 1 Results of stain, culture and PCR, according to sample origin
Type of sample
PCR
result
Negative culture
Negative
stain
Respiratory
Gastric fluid
Lymph node
CSF
Pleural effusion
Urine
Other a
Insufficient
Total
negative
positive
NA
negative
positive
NA
negative
positive
NA
negative
positive
NA
negative
positive
NA
negative
positive
NA
negative
positive
NA
negative
positive
NA
173
2
3
4
Positive
stain
Positive culture
NA
3
Negative
stain
Positive
stain
10
19
1
1
9
40
NA
No culture,
no stain
available
3
192
65
5
5
3
2
3
3
1
1
3
1
4
1
5
1
1
17
8
1
1
16
3
2
1
5
1
3
7
217
4
Total
2
1
1
40
55
7
2
1
11
12
11
331
a
Includes 2 vertebral bone samples, 1 bone marrow sample, 1 fecal sample, 1 peritoneal fluid sample, 1 joint fluid sample and 3
samples of nonspecific origin
NA, not available; CSF, cerebrospinal fluid
to perform any test. Laboratory results of stain, culture
and PCR were negative for all the students and COPD
cohort members, although one COPD patient had an
isolated positive blood sample result.
One case of cross-contamination during extraction was
detected. New aliquots of the samples affected in the
same lot were prepared and tested again, and previous
aliquots were rejected.
Thirty-four of 314 (10.8%) clinical samples (excluding
blood samples) from patients and control individuals
showed evidence of inhibition; among blood samples,
this percentage was 32.1%.
The presence of inhibitors was demonstrated in five
(22.7%) microbiologically confirmed cases for which
PCR results were negative. Likewise, 29 (13.8%) of
those with negative results by PCR and no microbiological confirmation showed the presence of inhibitors.
The specificity testing of the amplification method used
with bacteria other than mycobacteria gave the
following results: none of the 18 bacterial strains
belonging to eight different species assayed at three
different concentrations showed amplification product
in the gel electrophoresis analysis. Moreover, no
amplification was obtained from the eight strains of five
mycobacterial species included in the test. All five
Mycobacterium tuberculosis isolates amplified the
specific band in gel electrophoresis when tested at the
1!10 7 cfu/ml, 1!10 5 cfu/ml and 1!10 3 cfu/ml concentrations. However, only one of the five strains amplified
the 1!10 2 cfu/ml concentration. According to these
results, the sensitivity limit of this PCR method is
around 1,500 to 3,000 cfu/ml.
On the basis of the final case definition, there were 141
TB cases, 85 non-TB cases and 12 poorly defined cases
(Table 2). Overall, PCR was more sensitive than stain,
reaching statistically significant differences for extrapulmonary (Pp0.035) and total samples (P~0.0001)
but not for pulmonary samples. Compared with culture,
PCR had a lower non-statistically significant sensitivity.
All methods showed a high and similar specificity.
Culture appeared to be more sensitive than stain for
both pulmonary and total samples (P~0.0001), but the
difference was not significant for extrapulmonary
samples.
Table 3 shows the results of PCR and the PCR-stain
combination compared with the results of culture. A
total of 96 of 331 culture results were positive for AFB.
Overall sensitivity values were below 87%. PCR was
more sensitive than stain, reaching statistically significant differences for extrapulmonary (Pp0.024) and
Table 2 Sensitivity and specificity of each test according to sample origin, compared with the final case definition of tuberculosis
Test
Total samples
Sensitivity
(%)
Stain
57/138
(41.3)
Culture
92/140
(65.7)
PCR (main samples) c
82/139
(59.0)
Stain & PCR combined 89/137
(65.0)
PCR (blood samples)
13/104
(12.5)
Pulmonary samples
Extrapulmonary samples
95% CI
Specificity
(%)
95% CI
Sensitivity
(%)
95% CI
Specificity
(%)
95% CI
Sensitivity
(%)
33.0–50.0
75/77
(97.4)
74/78 a
(94.9)
76/76
(100)
74/76
(97.4)
52/52
(100)
90.9–99.7
53/109
(48.6)
80/110
(72.7)
68/109
38.9–58.4
57/59
(96.6)
55/59 a
(93.2)
58/58
88.3–99.6
4/29
(13.8)
12/30
(40)
14/30
57.2–73.5
50.3–67.3
56.4–72.9
6.8–20.4
87.4–98.6
95.3–100 b
(62.4)
90.8–99.7
93.2–100 b
74/108
(68.5)
63.4–80.8
52.6–71.5
(100)
58.9–77.1
56/58
(96.6)
83.5–98.1
93.8–100 b
(46.7)
88.1–99.6
32.5–70.6
(51.7)
95% CI
3.9–31.7
22.7–59.4
28.3–65.7
(100)
18/18
Specificity
(%)
95% CI
18/18
100
19/19
(100)
18/18
81.5–100 a
82.4–100 b
81.5–100 b
81.5–100 b
(100)
a
Two cases of Mycobacterium xenopi and 2 cases of Mycobacterium kansasii infection
One side 97.5% confidence interval
c
Samples other than blood are included
b
Table 3 Sensitivity and specificity of each test by sample origin, compared with the results of culture
Test
Total samples
Sensitivity
(%)
Stain
55/95
(57.9)
PCR
74/95
(77.9)
Stain & PCR combined 82/94
(87.2)
Pulmonary samples
Extrapulmonary samples
95% CI
Specificity
(%)
95% CI
Sensitivity
(%)
95% CI
Specificity
(%)
95% CI
Sensitivity
(%)
47.3–68
126/130
(96.9)
121/129
(93.8)
119/128
(93)
92.3–99.2
52/83
(62.7)
63/83
(75.9)
71/82
(86.6)
51.3–73
91/94
(96.8)
87/92
(94.6)
87/92
(94.6)
91–99.3
3/12
(25)
11/12
(91.7)
11/12
(91.7)
68.2–85.8
78.8–93.2
88.1–97.3
87.1–96.7
65.3–84.6
77.3–93.1
87.8–98.2
87.2–98.2
95% CI
5.5–57.2
61.5–99.8
61.5–99.8
Specificity
(%)
95% CI
35/36
(97.2)
34/37
(91.9)
32/36
(88.9)
85.5–99.9
78.1–98.3
73.9–96.6
863
864
total samples (P~0.047) but not for pulmonary
samples. Both methods showed a high and similar
specificity.
Based on the initial case definition (data not shown),
120 cases were classified as TB cases, 108 as non-TB
cases and 10 as undefined cases. Sensitivity, for total
samples, was as follows: stain, 35% (95% CI,
26.5–44.4); culture 59% (95% CI, 49.4–67.8), PCR, 53%
(95% CI 44.0–62.6); and combined stain-PCR, 58%
(95% CI 49.1–67.7). The difference between PCR and
stain reached statistical significance for total samples
(P~0.0001) but not for extrapulmonary and pulmonary
samples. PCR and culture had similar sensitivity. No
differences in specificity were shown for any method.
In the samples submitted to the three assayed tests, the
agreement between culture and both (initial and final)
case definitions was as follows: (i) when comparing
culture and the final case definition, there was 83.2%
agreement and a Kappa index of 0.65 (P~0.001); (ii)
when comparing culture and the initial case definition,
there was 76.7% agreement and a Kappa index of 0.49
(P~0.001); (iii) when comparing the initial and final
case definitions, there was 87.4% agreement and a
Kappa index of 0.74 (P~0.001).
Discussion
This study shows that PCR is not more sensitive than
culture, but that it is more sensitive than stain for extrapulmonary but not for pulmonary samples. Overall,
culture is the most sensitive technique, However, the
combination of PCR and stain, both of which are faster
than culture, has a sensitivity similar to that of culture.
When the non-TB groups were considered, the same
pattern was seen. By adding “healthy” individuals, the
estimates for specificity were more precise and slightly
higher, but the sensitivity parameters remained the
same as those presented in Tables 2 and 3. These
results are comparable to those published recently and
based on in-house methods or commercial kits; sensitivity rates reported ranged from 58 to 64% [14, 35–43].
Reports comparing commercial and in-house methods
[44, 45] showed in-house techniques to be more sensitive. The lack of standardization and a poor reproducibility have been argued as limitations for the routine
use of in-house techniques [14], but the same phenomenon has been demonstrated with commercial kits
[46].
We also studied the value of heminested PCR in blood
samples for diagnosis of TB in nonimmunosuppressed
individuals (as far as testing for HIV is concerned).
Thirteen of 104 blood samples collected from patients
with confirmed TB (final definition) were positive by
PCR, whereas only one of 23 classified control samples
was positive.
The presence of inhibiting substances found in this
study is similar to that reported by others, ranging from
5 to 16% [47, 48]. Blood samples showed a higher
percentage of inhibition (23%). Inhibition caused a
problem of misclassification, which was reflected in the
calculation of the sensitivity rate for PCR; the sensitivity of PCR might be higher if inhibiting substances
are avoided. Blood is considered to contain a high level
of inhibiting substances such as hemoglobin, although
recently it has been demonstrated that pretreatment
with SDS may remove the hemoglobin, at least
partially [49].
We conducted this prospective study within a hospital
setting to test the possibility of introducing PCR as part
of the routine diagnosis. It is impossible to generalize
the results for other settings. Nevertheless, these results
are similar to those found elsewhere and also correspond with those found in an epidemiological study of
TB in Barcelona [50]. In the design stage, we intended
to include some negative controls to validate the PCR
test. However, we excluded them from the primary
analysis on the basis of the likelihood of introducing
bias. Indeed, the total number of controls was arbitrary
and we found that the bigger the number, the higher
the specificity (data not shown). Nevertheless, given
the fact that specificity is, in any case, high, this
approach seems to be valid for evaluating sensitivity,
the estimates of which did not change at all. The gold
standard remains the critical issue. Our data shows that
culture and the final definition seem to have similar
value as a gold standard for validating a new diagnostic
technique. Although this study had a rather long
follow-up, its prospective design allowed us to compile
all the essential data required to determine the case
definition, on which the analysis was based.
We used a diagnostic method developed by our group
[28, 29], a one-tube heminested PCR based on a modification of a nested PCR described by Wilson et al. [21].
This technique targeted the most commonly used fragment, the insertion sequence IS6110 [16–19, 31]. In
order to test the specificity of the amplification technique described, several bacterial and mycobacterial
strains were tested at different concentrations. None of
these strains amplified the 369 bp band. This was
confirmed when samples from non-TB patients were
tested, showing the high specificity of the amplification
technique used. In addition, five Mycobacterium tuberculosis strains at four different concentrations of mycobacteria were tested in order to approximate the
threshold of this technique. The results of this test
showed a sensitivity of around 1,000 to 3,000 cfu/ml,
which may be lower than that reported by several
authors in the initial studies applying this methodology
to the diagnosis of TB [16–18]. However, the clinical
evaluation of the method described in the present study
showed a sensitivity comparable to that of other
methods that were evaluated using clinical cases,
865
including a representative proportion of smear-positive
and smear-negative cases, despite the different protocols applied in extraction, purification and amplification [14, 35–45]. This could mean that, with the current
methodology, the sensitivity of PCR techniques is
similar to that described here. This conclusion corresponds with the results obtained in a multicentric study
using artificial samples with a known number of mycobacteria [21]. On the other hand, it could be argued
that our PCR sensitivity estimates could be lower
because Mycobacterium tuberculosis strains lack the
insertion segment IS6110, as has recently been found in
Asian patients. Nevertheless, the spread of this strain
appears to be limited [51–53]: most Mycobacterium
tuberculosis strains have 2–20 copies, and only 1 to 5
copies are present in Mycobacterium bovis strains [16,
31]. In order to overcome the well-known PCR-related
sensitivity and specificity problems, the heminested
technique includes a second step that enhances the
sensitivity by amplifying a second set of primers [54]
and that enhances specificity by reducing the need for
manipulation and, therefore, decreasing the risk of
contamination. This technique has the advantage of a
much lower cost when compared with a conventional
two-stage nested PCR, which requires more reagents.
In the present study, the sensitivity and specificity of
the amplification technique used were the main focus,
and therefore, the extraction technique used was
considered a reference, despite being laborious.
However, from a theoretical point of view, other, faster
extraction methods could be applied to obtain results
the same day.
In conclusion, we consider that the PCR cannot, at
present, replace culture for the final confirmation of
clinical cases because of its lower sensitivity and the
additional need to have growth of Mycobacterium
tuberculosis colonies for susceptibility testing and other
special studies. However, the use of stain and PCR
combined greatly increases the number of cases
detected before culture results are available; therefore,
it is recommended that PCR be introduced into the
hospital laboratory as part of the standard procedures
for the diagnosis of tuberculosis.
The PCR technique is a fast, reliable method for the
diagnosis of TB, but its sensitivity, total automation and
cost should be improved in the future. In the future,
PCR results will probably need to be considered for the
classification and treatment of TB, just as the results of
stains are now considered in developed countries.
Acknowledgements We thank the Departments of Infectious
Diseases, Microbiology, Internal Medicine, Pneumology, Pharmacology and Hematology of the Hospital Clinic (Barcelona) for
their support and collaboration. Finally, we are very grateful to
all of the patients and medical students (course 1992–1998 School
of Medicine, Barcelona University) who participated in this
project. Financial support was provided by the following institutions: (i) Fondo de Investigaciones Sanitarias de la Seguridad
Social (FIS), grant 96/0028, (ii) Sociedad Española de Neumo-
logía y Cirugía Torácica (SEPAR), grant 96/444, (iii) Departament de Microbiologia i Parasitologia, Divisió de Ciències de la
Salut, Universitat de Barcelona, predoctoral grant (A.G.).
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