Peripheral-blood-based PCR assay to identify patients with active pulmonary tuberculosis

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Title: Peripheral-blood-based PCR assay to identify patients with active pulmonary tuberculosis
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THE LANCET

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Peripheral-blood-based PCR assay to identify patients with acti
pulmonary tuberculosis

Rany Condos. Amy McClune. William N Rom. Neil W Schluger

Summary
Background There is a need for rapid diagnosis of
pulmonary tuberculosis. We have previously used a PCR to
detect circulating Mycobacterium tuberculosis DNA in
blood samples from patients (mostly HIV-infected) with
pulmonary tuberculosis. We have now prospectively
investigated the role of this bloodbased PCR assay for
diagnosis of this disease in a clinical setting.

Methods Our PCR assay is specific for the IS6110 insertion
element of the M fuberculosis complex of organisms. We
used it to test peripheral blood from 88 consecutive
patients admitted to a chest ward with suspected
pulmonary tuberculosis. Personnel who carried out the
assay did not know the results of any clinical
investigations and ultimate diagnosis, and clinicians did
not know the PCR results. Results of the PCR assay were
compared with the final clinical diagnosis. A subgroup of
15 patients had blood samples assayed serially to track
the PCR signal over time.
Findings 41 patients had a final clinical diagnosis of
tuberculosis, and the cases were typical of those seen at
our hospital: HIV infection was common, and most cases
were not sputum-smear positive for acid-fast bacilli. The
PCR assay correctly identified 39 of 41 patients with
proven pulmonary tuberculosis. 26 (63%) of whom were
sputum-smear negative. There were five patients in whom a
positive PCR result did not accord with the final clinical
diagnosis, and two of the 44 negative PCR results were
classified as false negatives. The overall sensitivity and
specificity of the PCR assay for a diagnosis of tuberculosis
was 95% and 89%. respectively. In 15 patients with
pulmonary tuberculosis and a positive blood assay, the
PCR result remained positive after 1 month of therapy, but
had reverted to negative in 13 of the 15 by 4 months of
therapy.

Interpretation We conclude that peripheral-blood-based
PCR detection for the diagnosis of tuberculosis is a
technically feasible approach that has a potentially
important role in the diagnosis of pulmonary tuberculosis.
Lancet 1996; 347: 1082-85

Bellevue Chest Service and the Division of Pulmonary and
Critical Care Medicine, New York University Medical Center and
School of Medicine. New York, NY. USA IR Condos mo,
A McClune AB. W N Rom mo. N W Schluger mo)
Correspondence to: Dr Nell W Schluger, Bellevue Hospital 7N25.
27th Street and First Avenue. New York. NY 10016. USA
1082

Introduction
Diagnostic approaches to pulmonary tuberculosis lu
been virtually unchanged for many years; sputum str»
examination, sputum culture, and chest radiognffi
remain the most frequently used methods.' Howe*
there is an urgent need for rapid and accurate diagnosis
tuberculosis. HIV infection, institutional outbHM
transmission of tuberculosis, and the emergence':
multidrug-resistant disease underscore the urgency'
early identification and treatment.
Recently, developments in molecular biology hi
raised hopes about the possibilities of new strategics !
tuberculosis diagnosis.'-’ Most of these methods hr
focused on the application of PCR to sputum samp
from patients with suspected mycobacterial discs
Although these approaches have been promising, t
exact clinical role of the PCR assay as it applies to spun
analysis is unclear. The infectious nature of spun
(when handled in the laboratory and when coilcci
through induction by nebulised saline), potential preset
of PCR inhibitors in samples, and difficulty in obtain:
samples from some patients have led us to search for
accessible biological specimens that might be us
adjuncts in the diagnosis of pulmonary tubercul
Though rapid tests may not replace the need for spu
cultures generally (to look for other organisms or
susceptibility testing of mycobacterial isolates), a I
test with another type of sample may reduce the nuji
of sputum samples that need to be collected, an
negative result may allow the diagnosis of tuberculoi
be ruled out quickly and with certainty.
We have previously reported in a preliminary stu<
eight patients (mostly HIV infected) with a<
pulmonary tuberculosis that DNAJfrom Mycobocun
tuberculosis could be amplified by PCR from peripl
blood mononuclear cells.1 We now repon the results
prospective evaluation of the usefulness of a PCR-b
peripheral blood assay for the diagnosis of pulmotj.
tuberculosis.

Methods
Clinical samples
Samples of blood were taken from consecutive patients udmb
during a 6-month period to the Bellevue Chest Service who u
undergoing diagnostic evaluation for suspected tuberculosis
blood samples were obtained in the course of routine diagnp
evaluation and were assayed without knowledge of the suspa
diagnosis or the results of any other laboratory investigation*,
conventional diagnosis at least three sputum samples (range tl
to 12) were obtained and cultured. Blood samples from patir
were coded by number after collection and delivered to
laboratory with only that number as an identifier. No clip
information was available to the laboratory workers doing
BCR assay.
After initial diagnoses were estal .ished, 15 patients A
culture-proven pulmonary tuberculosis had follow-up hl

Xfol 147 • April 20,

o

active

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Number (%)•

Racial/ethnic distribution
Black
Hispanic
White
Asian
HIV serostatus
Positive

Sex (M/F)

ogy have
itegies for
ods have
i samples
disease,
sing, the
o sputum
sputum
collected
presence
’btaining
for more
e useful
rculosis.
sputum
> or for
a rapid
number
and a

40

21
13
3

Results
PCff in initial diagnosis of pulmonary tuberculosis

•Unless otherwise stated.
fThP ch1' .Dem°fraphfc characteristics of patients ad.
Imitted to
the chest service with suspected tuberculosis

forPCR RSrPlC a™ each °f 88 patients was

samples taken after 1 month and 4 months of
conventional
antituberculous therapy. These 15 patients '
were selected for
further studies because they received <
continuing care in our
to7hC’ W1i'ereaSrthC Other Patients received follow-i
---------- -up care closer
to their place of residence

ta HWe i FCVant dem0Eraphic characteristics are shown
n table 1. Four patients had received BCG vaccination
previously, and 12 (14%) had a history of p““us

44 Ze PCR LTr-’ 44 Pa"ents were PCR Positive and
nJberculosis weregpcVR Posi^LdTo” « patienZS

-“SSsX^^;

and a specificity of 8Q% tjZ
•
95/o
or ay/o. The positive and negative
predictive values were 89% and 95%, respectively
centnfugauon (700

" b^’eotX'’'

bX’

“ “ta” ^0

(70° SX 10 m,"; b"k=

tris-EDTA fTFl?

ere susPended in 40 gL of

m^w^ddedX
hm.r f 11

The ceUs were incubated at 56°C for 1

j

of aZT tub

rCR-~ P--ts, 38 had'evidence

ot active tuberculosis as shown by a positive snntum
culture one additional patient had a clinkal diagZXf
tuberculosis (without culture confirmation) Tnd he
improved both clinically and radiographically while on
antituberculous
therapy.
The
relevant
chnical

2- notablv008 °
gr°UP °f PatiCntS are shown in ^ble
2, notably, apart from the two patients with a miliary

pattern seen on chest radiograph, no patient had evidence
of extrapulmonary involvement. Five of the PGR r, v

ZX^hZ^d^1"c,,nical

PCR
fslT'o
u nA

/ •

m?COba«'rial ■“'«<>" sequence
to the
the mycobacterial insertion
d’ Th S lnsertIon element is specific for the

5 TGAACCGCCCCgSt^cTgg^CT01^'^

mi tied
o were
-is. All
inostic
aected
is. For
three
itients
o the
inical
g the

duplicate t~
products were
products
were then analysed by agaro"
All PCR recnirc ..
■
y garosc gel (2 /o) electrophoresis,
^mplcs Wre processed .X'1’"1"11 by
ObSCrVerS’ and a“

0
11

ilosis to

tudy of
active
■cterium
ipheral
Its of a
-based
nonary

bssses

37-5 years

21-30
31-40
41-50
51-60
61-70

COntrO, reaCti°nS

containing the positive control was prepared underThood in^

84/4

Age
Mean
Range (number in range)
0-20 years

ipd ,”zd ™ctions-—

fed"

consisted of buffer nudZZ

37 (42)
41(47)
10(11)

Negative
Unknown

ulosis have
turn smear
adiography
However,
iagnosis of
outbreaks,
rgence of
rgency of

primer
pair
consisted
of
the
primer
5' CGTGAGGGCATCGAGGTGGC y andsense
the antisense
pnmer 5' GCGTAGGCGTCGGTGACAAA T

44(50)
19(22)
15(17)
10(11)

Number (%)

Sputum AFB smear result
Positive
Negative

15(37)
26(33)

Chest radiographic pattern
Parenchymal infiltrate
Mediastinal lymphadenopathy
Miliary pattern
Normal

34(85)
3(5)
2(5)
2(5)

HIV status
Positive

Negative
Unimown

AFB=acid-fast bacilli.

~

18(43)
16 (40)
7(17)

with
>lood

Table 2: Clinical features of
cases of active pulmonary
tuberculosis

1996

Vol 347 • April 20, 1996

J,1r H
adenocarcinoma of the lung, and of the
remaining four three were HIV seropositive, with a mean
4 count of 293/p.L (range 89-400/jxL). Two of the
four patients (one HIV positive) had received BCG

vaccination previously, and one of the four had
diagnosis of tuberculosis made within the previous 12
months and had received antituberculous therapy before
admission to our hospital.
eiore
Of the 44 PCR-negative patients,
twoand
hadsputum
active
cXe o'8’ "
67
StatUS
the hne
hrnn h

on"?568 *n
rema,nm8 42 Were carcinoma of
z’ mVaS1Ve aSPergilIoSis (two), bronchitis/
___ i • 4
■
^^tis cannii pneumonia (ten),
tuberculosis
_ 3 infection without disease
(five), and
disease
unknown (three).

Effect of therapy on PCR detection of mycobacteria!

blood" PCR ^IfCU,tUrrPr°Ven tuber
^losis and
tuberculosis
and positive
positive
wMe

n

u 3t

thXv air^n?11

t,me Of diagn<«is were followed

theraPy-

1 month of

month
r U
p
ntS remauied PCR positive. At 4
nari^t °hf ?eurapy’ 13 had a negative PCR result. All 15
patients had been responding to therapy clinically and
sputum cultures had become nezarive tZ
L

remained PCR positive for M tuberculosis'. In one of these

1083

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THE LANCET

two patients antituberculous therapy had been
interrupted because of abdominal pain; the other patient
had a miliary pattern on his chest radiograph at the time
of diagnosis.
Discussion
We have shown that the detection of circulating
mycobacterial DNA by PCR is sensitive and specific in
die diagnosis of active pulmonary tuberculosis, the results
being available within a day. In our cohort, PCR of blood
samples in patients with suspected tuberculosis yielded a
sensitivity of 95% and a specificity of 89%. These values
compare favourably to results for diagnosis of tuberculosis
generally obtained by conventional methods such as
sputum microscopy, and they are similar to results
obtained with PCR analysis of sputum samples. The yield
of sputum microscopy for the immediate diagnosis of
tuberculosis has usually been reported in the 50-75%
range, with smears less often positive in non-cavitary
disease or in patients with HIV infection.7-8
In our series, positive PCR results were not affected
either by the radiographic presentation or by the HIV
status of the patients. In addition, recent work suggests
that the positive predictive value of sputum smears for
acid-fast bacilli may be higher in patients from whom
respiratory samples are collected by sputum induction or
bronchoalveolar lavage, since these techniques are more
likely to raise samples containing organisms from the
Al avium complex.'1 Our PCR assay is specific for
organisms of the M tuberculosis complex. Although the
clinical usefulness of sputum-based PCR remains unclear,
a recent well-done clinically oriented study using a
commercially developed PCR kit indicates a rough
equivalence between PCR and culture results, with a
clinical case definition as the gold standard for diagnosis.10
Our blood-based assay reported here has yielded similar
results.
There were five patients who were PCR positive for
Af tuberculosis DNA without culture or clinical
confirmation of tuberculosis. Further analysis of these
patients showed that one had been given a diagnosis of
tuberculosis in the months before admission to our
hospital, and had received some antituberculous therapy.
Two other patients had received BCG vaccination,
including one with HIV infection. PCR assays based on
detection of the insertion element IS0110 cannot
distinguish between Af tuberculosis and M bovis, and it is
possible, especially in HIV-infected patients in whom
clinical infection with Al bovis may occur several years
after vaccination, that this was responsible for the positive
PCR result." There was no obvious explanation for the
other two patients with positive PCR results but without
clinical or culture evidence of tuberculosis. For the
calculation of sensitivity and specificity of our assay,
however, we included all five cases as false-positive
results.
Two patients with negative PCR results had positive
sputum cultures for M tuberculosis, and we counted them
as false negatives. Although we had speculated that these
false-negative results were due to low levels of infection or
early stages of disease, both patients had cavitary disease,
and one was sputum-smear positive. It is possible that
these two patients were infected with strains of
Af tuberculosis that did not carry the insertion sequence
IS6110. Such strains are uncommon, but do exist.12

—

not available for the isolates from these two false-negativei
patients to assess whether the insertion element was!
present.
Although
sputum-based
PCR
detection
of
M tuberculosis has a similar sensitivity and specificity to
those of our blood-based PCR, the procurement and
processing of sputum samples for PCR reactions are often
cumbersome and associated with infectious risks to the
personnel collecting the specimens and doing the assay.
This problem has led many investigators to search for less
complex methods of sample preparation.” Sputum
induction, an increasingly used method of sample
collection, requires careful attention to infection control
procedures as well as the time and effort of trained
personnel." A blood-based assay potentially avoids these
difficulties. Additionally, all patients can provide blood
samples for analysis, which is not always possible with
sputum.
Sputum-based PCR assays for the diagnosis of?
tuberculosis remain positive for many months after
antituberculous therapy has started.” We attempted to
address this issue in our assay by obtaining serial blood
samples from a subset of patients in whom we
documented active pulmonary tuberculosis with a positive
peripheral blood PCR signal. In all of these cases, the
PCR assay remained positive after 1 month of therapy,
but in 13 of 15 patients the signal had reverted to negative
after 4 months of therapy. It is noteworthy, that one of the
patients with a persistently positive result had interrupted
his therapy, and the other patient initially presented with a
miliary pattern on chest radiograph, suggesting perhaps a
higher burden of bloodborne organisms.
Several caveats should be mentioned when generalising the results of this study to other populations of patients, f
We feel that the patients with proven tuberculosis in our '
study are typical of those seen in many urban hospitals in
the USA: HIV infection was common, the radiographic
presentations were typical, and the patients were not
overwhelmingly sputum-smear positive. We do not believe
that our results reflect an excess of extrapulmonary
disease or an over-representation of patients with
enormous bacterial burdens. Positive blood cultures for
M tuberculosis have been reported as positive in a relatively
small number of cases, usually in HIV-infected patients
with advanced AIDS or extrapulmonary tuberculosis,1617
but in our series only two of 56 patients tested had
mycobacteraemia as detected by blood cultures.
We emphasise that in our series the blood-based PCR
assay was used as a diagnostic adjunct in a cohort of
patients admitted to the chest ward with a high suspicion
of tuberculosis. In fact, the prevalence of active
tuberculosis in our cohort was 46%. Suspected cases of
tuberculosis included only patients whose clinical and
radiographic presentations were strongly suggestive of
tuberculosis, rather than patients involved in a contact
investigation, for example. In that sense, our cohort was
similar to that described by Gordin and colleagues,18 who
found that 48% of smear-negative patients with strongly
suggestive clinical and radiographic presentations had
active disease, in contrast to a series from Kenya in which I
disease was suspected in patients with a cough persisting ■
for 4 weeks or more.” With this less stringent definition of J
suspected tuberculosis, the prevalence of disease was only t
4-8%. We did not attempt to use the assay as a screening i
modality for active tuberculosis in a nooulation with a ’

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egative
it was

1
of
city to
it and
e often
to the
assay,
for less
putum
sample
control
trained
s these
blood
e with
isis of
; after
>ted to
blood
m we
positive
es, the
herapy,
egative
2 of the
rupted
I with a
rhaps a

ralising
atients.
in our
>itals in
graphic
:re not
believe
nonary
s with
ires for
latively
patients
osis,16,17
ed had

d PCR
hort of
spicion
active
ases of
:al and
live of
contact
ort was
;,18 who
.trongly
ns had
i which
rsisting
ition of
as only
reening
with a
•st were

used as such, we would expect the positive predictive
value to decrease substantially.
Our peripheral-blood-based PCR assay for the
diagnosis of tuberculosis is a rapid, sensitive, and specific
test that could prove useful in certain clinical situations.
The problem of product carry-over contamination may
become a serious issue if DNA amplification techniques
become widely used by less experienced laboratory
workers. We had a laboratory environment and used an
experimental protocol designed to reduce to a minimum
any contamination problems. In general use, carry-over
contamination can be avoided with various techniques,
including the use of uracil DNA glycosylase.20

Presented in part at the annual meeting of the American Thoracic Society,
1995, Seattle, Washington. Supported in part by Tuberculosis Academic
Award K07 HL03030 (NWS) from the National Institutes of Health and
the General Clinical Research Center of New York University Medical
Center (NIH grant MOI 00096).

References
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