Vol.1, No.2, 63-66 (2009)
doi:10.4236/health.2009.12011
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Copyright © 2009 http://www.scirp.org/journal/HEALTH/
Health
Openly accessible at
Direct real-time PCR examination for Mycobacterium
tuberculosis in respiratory samples can be cost effective
Bryan Joseph Renton1, Patricia Denise Morrell2, Richard Peter Davidson Cooke2, Peter David
Owen Davies1
1Department of Respiratory Medicine, University Hospital Aintree, Liverpool; Correspondence author:
2Department of Clinical Microbiology, University Hospital Aintree, Liverpool
Received 5 June 2009; revised 15 July 2009; accepted 18 July 2009.
ABSTRACT
Aim: To assess whether the use of direct real-
time polymerase chain reaction (PCR) on
smear-positive sputa can be cost-effective, by
speciating mycobacteria earlier than current
methods and thereby preventing unnecessary
screening tests as part of the contact tracing
process.
Methods: A retrospective study of all patients
with smear-positive sputa in a Liverpool teach-
ing hospital between 2004 and 2007. All the
PCRs performed on these patients were re-
viewed and compared them with their myco-
bacterial culture results. Unit costs for PCR,
chest X-ray (CXR), tuberculin skin test (TST),
interferon-gamma (IFN-γ) and medical/nursing
time were conservatively estimated at £50, £11,
£10, £40 and £30 respectively. The total PCR
costs were compared with the costs of unnec-
essary follow up of patients, negative for My-
cobacterium tuberculosis (MTB) by PCR, sub-
sequently confirmed to be MTB culture nega-
tive.
Results: 203 smear-positive patients under-
went direct PCR testing. 126 (62%) patients grew
Mycobacterium tuberculosis (MTB), 74 (37%)
had environmental mycobacterial infection (EMI)
and 3 (1%) were culture negative. Of the 126
patients’ culture positive MTB patients, 123 were
PCR positive and 3 PCR negative. Of the 77 pa-
tients that were culture negative for MTB, 75
were PCR negative and 2 PCR positive The
sensitivity, specificity, positive and negative
predictive values for direct PCR versus MTB
culture were 98%, 96%, 98% and 97% respec-
tively. Total costs of all PCRs performed
amounted to £10,150. The cost of contact pro-
cedures for PCR-negative and MTB culture-
negative index cases was estimated at £19,650.
This equated to a total saving of £9,500 in con-
tact tracing costs.
Conclusions: Direct PCR examination testing
of smear-positive patients can be cost-effective
in areas where there is a high incidence of EMI.
Keywords: Tuberculosis; PCR and Cost Effective
1. INTRODUCTION
Current national guidelines for diagnosing active pul-
monary tuberculosis (TB) recommends performing a
posterior-anterior chest X-ray (CXR) and obtaining at
least 3 sputum samples (with one early morning sample),
which are then sent for TB microscopy and culture. [1]
These sputum smears are initially stained for acid-fast
bacilli (AFB), but as Mycobacterium tuberculosis (MTB)
and environmental mycobacteria have a similar micro-
scopic appearance, it is not possible to differentiate be-
tween them at this stage. Mycobacterial culture and sub-
sequent speciation is therefore required but is often not
available until several weeks after specimen collection.
In patients suspected of active TB, which is usually
based on a smear-positive sputum or bronchoalveolar
lavage (BAL), a contact tracing process is initiated early
to prevent potential further spread. Screening is usually
offered to household and other close contacts. Typically,
most contacts will have a chest X-ray and a tuberculin
skin test (TST), with a significant proportion of contacts
also going on to have an interferon-gamma (IFN-γ) test.
However, in areas where there is a high incidence of
environmental mycobacterial infection (EMI), this can
result in a large proportion of contacts being unnecessar-
ily screened. Indeed, in the study carried out by Corless
et al., 51% of smear positive patients did not have TB.
This resulted in 31% of contacts of patients being
screened who either cultured environmental organisms,
or had negative cultures. The median number of contacts
traced per index case was 11 for patients culturing MTB
and 4 for patients culturing environmental organisms or
with negative cultures. This report highlighted the cur-

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64
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rent inefficiencies that exist in the contact tracing pro-
cedures. The authors felt that there are clear grounds for
using rapid tests to identify and type mycobacteria more
quickly than current solid or liquid media methods;
thereby avoiding extra unnecessary screening costs. [2]
This would certainly seem to be applicable to areas with
a high incidence of lung disease caused by EMI, but
unfortunately there still remains no published national
database on this topic. However, a further review of
mycobacterial isolates cultured from respiratory samples
in Merseyside 2000 – 2008 has again confirmed the high
proportion (approximately 50%) of smear positive spu-
tum that yield environmental mycobacteria.
Polymerase chain reaction (PCR) allows for more
rapid identification of MTB, but is not routinely recom-
mended, principally due to cost implications. Current
national guidance advises its use only if rapid confirma-
tion of a TB diagnosis in a sputum smear-positive person
would alter their care (e.g. exclusion of non-tuberculosis
mycobacteria in immunocompromised patients), or before
conducting a large contact tracing initiative (e.g. in a
school or hospital). [3] In this study, we aimed to assess
whether the use of direct PCR on smear-positive sputa
can be cost-effective, by speciating mycobacteria earlier
than current methods, and therefore preventing unneces-
sary screening tests as part of the contact tracing process.
2. MATERIALS AND METHODS
This retrospective study was carried out at a large teach-
ing hospital in Liverpool (University Hospital Aintree).
All AFB smear-positive respiratory specimens under-
went direct PCR testing for MTB and Mycobacterium
avium-intracellulare complex. PCR testing on smear-
positive sputa has been routine practice in this hospital
for the last 4 years, initially on a weekly basis, and due
to increasing clinical demand, is now performed twice-
weekly. On receiving the smear-positive sample, the
average time to issue a PCR result is between 48-72
hours. Once this result is available, the responsible clini-
cian is notified immediately.
All the direct PCRs performed on patients with
smear-positive sputa between 2004 and 2007 were re-
viewed and compared with their mycobacterial culture
results.
A real-time PCR assay (Real ArtTM Mycobac. Diff.
LC PCR kit, artus Biotech USA) was used for the detec-
tion of MTB complex (human MTB, M. bovis, M. afri-
canum, M. microti, M. cannetti) and Mycobacterium
avium-intracellulare complex. A region of the mycobac-
terial 16S DNA, conserved in all members of MTB com-
plex, was amplified and detected by the specific melting
point temperature of the flurogenic probe used [4].
The assay system contains, in one master mix, all re-
agents and enzymes for the specific amplification and
detection of a 163 base pair region of the MTB genome.
The kit also provides a dilution series of external posi-
tive MTB controls for precise quantification of the MTB
complex load. PCR inhibitors are also to be detected to
prevent the generation of false negative results. An in-
ternal control, co-amplified with the specific target DNA
in the same capillary, is therefore included in the assay
kit. This internal control does not influence the sensitiv-
ity or specificity of the MTB PCR. If the internal control
is added to the sample before the nucleic acid isolation
process, it can also be used to control the efficiency of
the DNA extraction procedure.
Unit costs for PCR, CXR, TST and IFN-γ were esti-
mated at £50, £11, £10 and £40 respectively. We also
conservatively estimated a further minimum cost of £30
per patient, which incorporated district nurse, TB control
nurse and medical time. We aimed to compare the total
PCR costs with the costs of unnecessary follow up of
PCR negative patients, who subsequently proven to be
MTB culture negative.
The cost of contact procedures for PCR-negative and
MTB-culture negative index cases was based on the as-
sumption that there were 4 contacts traced per index case
for patients with EMI. [2]
3. RESULTS
A total of 203 smear-positive patients underwent direct
PCR testing. 126 (62%) patients grew MTB, 74 (37%)
had EMI and 3 (1%) were culture negative. Of the 126
patients that were culture positive for MTB, 123 were
PCR positive and 3 PCR negative. Of the 77 patients
that were culture negative for MTB, 75 were PCR nega-
tive and 2 PCR positive (Table 1). The sensitivity, speci-
ficity, positive and negative predictive values for direct
PCR versus MTB culture were 98%, 96%, 98% and 97%
respectively.
Table 1. Comparison of PCR and culture results for 203, AFB
smear-positive sputa.
MTB Culture
+ -
+
MTB*
PCR
-
123 2
3 75
125
78
126 77 203
MTB: Mycobacterium tuberculosis
PCR: Polymerase chain reaction
*Sensitivity: 98%
Specificity: 96%
Positive predictive value: 98%
Negative predictive value: 97%
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65
Table 2. Breakdown of estimated costs for PCR tests and contact tracing procedures for PCR / M. tuberculosis
culture negative index cases.
TST CXR INF-γ test Medical/nursing Contact
tracing
PCR Savings
£3,000 £1,650 £6,000 £9,000 £19,650 £10,150 £9,500
TST : Tuberculin skin test
CXR : Chest xray
INF-y : Interferon-gamma
PCR : Polymerase chain reaction
The following costs per index case was identified:
Number of PCR-negative/MTB culture-negative cases
(i.e. unnecessarily screened) = 75
Number of contact cases per index case with EMI = 4
Total cost for TST = 75 x 4 x £10 = £3,000
Total cost for INF-γ (approximately 50% of contacts
go on to get this) = 75 x 2 x £40 = £6,000
Total cost for CXR (at least 50% of contacts require
this) = 75 x 2 x £11 = £1,650
Total cost for medical/nursing time = 75 x 4 x £30 =
£9,000
Total costs of all PCRs performed amounted to
£10,150 (£50 x 203). The cost of contact procedures for
PCR-negative and MTB culture-negative index cases
was estimated at £19,650. This equated to a total saving
of £9,500 in contact tracing costs (Table 2).
4. DISCUSSION
Previous studies looking at the cost effectiveness of PCR
have focused primarily on the savings due to averted
isolation, drug treatment, in-patient beds saved and fur-
ther investigations. [5-7] To our knowledge, there have
been no studies that have specifically looked at the
cost-effectiveness of PCR in avoiding unnecessary con-
tact tracing costs. We estimated that direct PCR use in
this population of patients with smear-positive sputa,
resulted in an overall net saving of £9,500 in contact
tracing costs. Whilst this seems a relatively modest
amount over a 3-year time period, it is nevertheless, a
saving, and justifies the expenditure on PCR (£10,150).
We also should not underestimate the other benefits that
an early diagnosis will have. In our study, over a third of
patients who had smear-positive sputa did not have MTB,
which is in keeping with previous data in this area. [2]
Currently, all smear-positive patients should be com-
menced on anti-tuberculosis treatment. In a population
such as ours, this will result in a significant number of
patients being incorrectly treated. Also, there are sig-
nificant adverse effects that are associated with anti-TB
therapy, [8] so these decisions should not be taken lightly.
Prompt diagnosis using direct PCR may therefore also
save on unnecessary treatment costs and adverse events
which were not included in the costing model. Further-
more, from a psychological perspective, an early diag-
nosis will save a lot of patients’ close contacts going
through the stress of being subjected to a number of un-
necessary tests to establish that they don’t have MTB.
Whilst our study showed a saving in contact tracing
costs of £9,550, this is likely to be a very conservative
estimate. Once a person has been diagnosed with active
TB, the diagnosing physician should inform relevant
colleagues so that the need for contact tracing can be
assessed without delay. [1] This contact tracing process
usually begins with a TB health visitor interviewing the
patient. Screening is then offered to household and any
other close contacts, which usually involves a visit to the
TB screening clinic. After a TST, contacts will need a
further visit to have the result read and if testing is in-
conclusive, they should be referred to a TB specialist. [1]
All this equates to a significant amount of medical,
health visitor and nursing time and it may well be that
£30 per contact is a gross underestimate. Without a diary
analysis of this time spent screening contacts, it was
however, difficult to formally assess this. Overall, our
estimate of £9,550 saved in contact tracing costs shows
that PCR virtually pays for itself.
Microscopically, 5,000-10,000 AFB ml-1 must be pre-
sent for smear-positivity, compared to 10-100 AFB ml-1
that are required for culture positivity. Therefore, a
smear-positive respiratory sample signifies a high bacte-
rial load and an increased risk of infectivity. In a mini
review of available nucleic acid amplification tests
(NAAT) for the detection of MTB in clinical specimens
(21 studies), the mean sensitivity in smear-positive
specimens was 94.5%. [9] The positive-predictive value
and sensitivity of the PCR in our study was 98%, which
is in-keeping with previous data on this particular real-
time PCR assay (100% sensitivity). [4] In view of this,
and the fact that PCR is expensive, we feel that per-
forming PCR on only one of the three smear positive
specimens should be enough to aid diagnosis and decide
which patients’ contacts do and do not need to be
screened. In contrast, NAAT sensitivity on smear-nega-
tive specimens was much lower, at 69.3%. [10] However,
as smear-negativity signifies a low bacterial load, these
patients are likely to have a low risk of infectivity, so

B. J. Renton et al. / HEALTH 1 (2009) 63-66
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contact tracing becomes less of an urgent issue. As rapid
liquid culture takes approximately 8-10 days in our hos-
pital, we therefore felt that routine use of PCR in this
group of patients would be unlikely to be cost-effective.
In a recent article by Taegtmeyer et al., the authors
found that PCR had a direct impact on clinical manage-
ment in one third of smear-positive patients in whom it
was used. This included patients for whom treatment
was changed and situations in which contact tracing ex-
ercises were either commenced or stopped. PCR signifi-
cantly reduced the time to identification of the myco-
bacteria and detection or exclusion of rifampicin resis-
tance, which allowed prompt alterations in management
when needed. They concluded that there would be addi-
tional clinical benefit from PCR being applied to all
smear-positive specimens in low prevalence settings. [10]
We would concur with this, and believe that our study
illustrates that direct PCR testing of all smear-positive
patients can also be cost-effective.
In conclusion, although current national guidance on
direct PCR recommends its use principally to confirm
true MTB before a large contact tracing exercise is un-
dertaken, our results suggest that it may have a useful
and cost-effective role in routine clinical practice on
smear-positive respiratory samples, particularly in areas
with a high incidence of EMI. Whilst there are reports
suggesting that the incidence of EMI has increased over
the past few decades, this observation has not been con-
clusively established due to the lack of a comprehensive
surveillance system. [11] This data would be particularly
helpful in deciding which areas in the UK may benefit
from direct PCR testing on all smear-positive respiratory
specimens. Such an approach should be reflected in the
National Institute of Clinical Excellence (NICE) guide-
lines when they are next revised.
REFERENCES
[1] CG33 Tuberculosis – National Institute of Clinical Ex-
cellent (NICE) guidelines. March 2006.
[2] Corless, J.A., Stockton, P.A., Davies, P.D. (2000) Myco-
bacterial culture results of smear-positive patients with
suspected pulmonary tuberculosis in Britain. Eur Respir
J, 16: 976-9.
[3] National Collaborating Centre for Chronic Conditions:
clinical diagnosis and management of tuberculosis and
measures for its prevention and control. (2006). London,
Royal College of Physicians.
[4] Cramer, S.O., Matinmehr, F, Hillemann, D. (2004) Fast
and sensitive detection of the Mycobacterium tuberculo-
sis complex by real-time PCR, Clinical Microbiology
and Infection, 10(3), 418.
[5] Drobniewski, F.A., Watterson, S.A., Wilson, S.M., Harris,
G.A. (2000) A clinical, microbiological and economic
analysis of a national service for the rapid molecular di-
agnosis of tuberculosis and rifampicin resistance in My-
cobacterium tuberculosis. J Med Microbiol, 49:271-8.
[6] Dowdy, D.W., Maters, A., Parrish, N., Beyrer, C.,
Dorman, S.E. (2003) Cost-effectiveness analysis of the
Gen-Probe amplified Mycobacterium tuberculosis direct
test as used routinely on smear-positive respiratory
specimens. J Clin Microbiol, 41(3), 948-953.
[7] van Cleeff, M., Kivihya-Ndugga, L., Githui, W.,
Ng’ang’a L., Kibuga, D., Odhiambo, J., et al. (2005)
Cost-effectiveness of polymerase chain reaction versus
Ziehl-Neelsen smear microscopy for diagnosis of tuber-
culosis in Kenya. Int J Tuberc Lung Dis, 9:877-83.
[8] Zalekis, R. (2005) Side-effects of Tuberculosis treatment.
Breathe, 2(1), 69-73.
[9] Piersimoni, C. and Scarparo, C. (2003) Minre-
view–Relevance of commercial amplification methods
for direct detection of Mycobacterium tuberculosis com-
plex in clinical samples. J Clin Microbiol, 41(12), 5355-
5365.
[10] Taegtmeyer, M., Beeching, N.J., Scott, J., Seddon, K.,
Jamieson, S., Squire, S.B., Mwandumba, H.C., Miller,
A.R.O., Davies, P.D.O., Parry, C.M. ( 2007) The Clinical
impact of nucleic acid amplification tests on the diagno-
sis and management of tuberculosis in a British hospital.
Thorax Online.
[11] Griffith, D.E., Aksamit, T., Barbara, A., et al. (2007)
American thoracic society guidelines: Diagnosis, treat-
ment and prevention of nontuberculous mycobacterial
diseases. Am J Resp and Crit Care Med, 175, 367-417.
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