International Journal of Infectious Diseases 16 (2012) e663–e668

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International Journal of Infectious Diseases

journal homepage: www.elsevier.com/locate/ijid

Combination of adenosine deaminase activity and polymerase chain reaction in

bronchoalveolar lavage fluid in the diagnosis of smear-negative active pulmonary
tuberculosis
Viboon Boonsarngsuk *, Suwanna Suwannaphong, Chariya Laohavich
Division of Pulmonary and Critical Care Medicine, Department of Medicine, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok 10400, Thailand

A R T I C L E I N F O S U M M A R Y

Article history: Background: Some studies have assessed the diagnostic value of adenosine deaminase (ADA) activity in
Received 28 November 2011 bronchoalveolar lavage fluid (BALF) in the diagnosis of pulmonary tuberculosis (TB). However, a
Received in revised form 12 April 2012 conclusion has not been reached due to the limited number of patients with various pulmonary diseases
Accepted 16 May 2012
used as comparators. The objective of this study was to evaluate the efficacy of BALF ADA activity and TB
Corresponding Editor: Sheldon Brown, New PCR assay for diagnosing pulmonary TB.
York, USA
Methods: BAL samples from 424 patients with acid-fast bacillus-negative sputum smears who
underwent bronchoscopy for diagnostic evaluations of pulmonary diseases, were prospectively
Keywords: analyzed for ADA activity and TB PCR.
Tuberculosis Results: The median ADA activity of TB cases was significantly different from that of patients with solid
Bronchoalveolar lavage fluid tumor without endobronchial obstruction (p < 0.001), inactive TB (p = 0.04), and other (p = 0.038), while
Adenosine deaminase
this was not the case for the other pulmonary diseases. A cutoff BALF ADA activity of 3 U/l provided a
Polymerase chain reaction
sensitivity of 58.7% and specificity of 81.8% to differentiate TB from solid tumor without endobronchial
obstruction. The sensitivity of TB PCR in BALF was 28.1% with a specificity of 99.0%. The area under the
receiver operating characteristic (ROC) curve to differentiate TB from solid tumor without endobronchial
obstruction was significantly higher for the combination of ADA activity 3 U/l and TB PCR (0.77) than
for ADA activity 3 U/l alone (0.70, p < 0.001) or for TB PCR alone (0.64, p < 0.001). The sensitivity of the
combination of ADA activity 3 U/l and TB PCR was 72.7% and the specificity was 81.8%. In TB cases, a
greater radiographic extent of disease was associated with a higher median ADA activity (p = 0.017).
Conclusions: BALF ADA had limited value in differentiating pulmonary TB from some other pulmonary
diseases. To differentiate TB from solid tumor without endobronchial obstruction, a combination of BALF
ADA and TB PCR had marked additive diagnostic value.
ß 2012 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

1. Introduction Adenosine deaminase (ADA) has been proposed as a useful

Tuberculosis (TB) is a major global health problem. The clinical
manifestations of TB are highly variable, depending on the affected
organ, which is usually the lungs. Symptoms range from asymp-
tomatic, to non-specific complaints, to mild respiratory symptoms,
to respiratory failure. Radiographically, TB can present with varied
patterns of abnormality on chest radiographs, mimicking other
pulmonary diseases. The gold standard TB diagnosis requires the
growth of Mycobacterium in specimen cultures and confirmation
with biochemical tests or DNA probes. However, sensitivity is low.1,2
Thus, other diagnostic methods have been developed and investi-
gated in order to improve the diagnostic rate of TB.
* Corresponding author. Tel.: +66 2 201 1619.
E-mail address: bss-vb@hotmail.com (V. Boonsarngsuk).
surrogate marker of TB pleuritis, pericarditis, and peritonitis.
However, its diagnostic value in bronchoalveolar lavage fluid
(BALF) in the diagnosis of pulmonary TB remains ambiguous.3–7
The main reasons for this ambiguity are the limitations of
previous studies, which have lacked a properly designed control
population.
The PCR for TB is a rapid and reliable method for the diagnosis of
pulmonary TB. Although TB PCR provides high specificity, the
sensitivity has a wide range, from 36% to 97%, depending on the
results of smears and cultures.8–10
To date, there has been no study investigating the diagnostic
value of a combination of ADA and TB PCR in BALF in the diagnosis
of smear-negative pulmonary TB. In addition, data on BALF ADA
activity among various pulmonary diseases are limited. The aim of
the present study was to evaluate the efficacy of BALF ADA activity
and the TB PCR assay for diagnosing pulmonary TB and
distinguishing it from other pulmonary diseases.

1201-9712/$36.00 – see front matter ß 2012 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.ijid.2012.05.006
e664 V. Boonsarngsuk et al. / International Journal of Infectious Diseases 16 (2012) e663–e668
2. Materials and methods
2.1. Patients
We performed a prospective study that included adult patients
with acid-fast bacillus (AFB)-negative sputum smears who were
subjected to bronchoscopy for diagnostic evaluations of pulmonary
diseases between July 2010 and June 2011 at Ramathibodi Hospital.
Written informed consent was obtained from all patients before
starting the bronchoscopy procedure. This study protocol was
approved by the Ethics Committee on Human Experimentation of
Ramathibodi Hospital, Faculty of Medicine, Mahidol University.
The following clinical data were collected: general demograph-
ic information, underlying immunocompromised conditions (in-
cluding HIV disease, diabetes mellitus, alcoholism, cirrhosis,
chronic renal failure, malnutrition, connective tissue diseases,
hematologic and solid malignancies, organ transplant, systemic
steroid or other immunosuppressive drugs use, and receiving
chemotherapeutic agents), history of previous TB disease and
treatment, and chest radiographic findings. The radiographic
extent of disease was classified according to the criteria
established by the National Tuberculosis and Respiratory Disease
Association.11 Subjects were classified as having minimal disease if
lesions were non-cavitary, of slight to moderate density, and
involved a small part of one or both lungs. The total extent was
required to be less than the volume of one lung above the second
chondrosternal junction and the spine of the fourth or body of the
fifth thoracic vertebra. Subjects were classified as having
moderately advanced disease if they had more than minimal
disease but had a total extent of slight or moderately dense lesions
limited to the total volume of one lung, and that of dense lesions
limited to one-third the volume of one lung. Cavitary lesions were
required to be <4 cm in diameter. Subjects were classified as
having greatly advanced disease if lesions were more extensive
than the moderately advanced disease.
2.2. Bronchoalveolar lavage and sample processing
After examination of the entire bronchial trees, in cases where
there was an absence of a bronchoscopically visible lesion, BAL was
performed in the target bronchus leading to the lesion according to
imaging studies; 50 ml of normal saline was instilled and aspirated
into a trap. Repeated instillations of 50 ml normal saline up to a total
of 100–150 ml, or collection of 50 ml of retrieved fluid, was
considered adequate lavage. When a bronchoscopically visible
lesion was identified, bronchial washing was obtained by instillation
of 20 ml of normal saline over the lesion and by aspiration back until
50 ml of retrieved fluid was achieved. The choice of other sampling
techniques such as bronchial brushing, endobronchial biopsy, and
transbronchial biopsy were left to the examiner’s discretion.
The lavage fluid or washing fluid was submitted to AFB stain,
Mycobacterium culture, ADA determination, and TB PCR. Other
diagnostic investigations of BALF and other specimens were sent
for evaluation according to clinical suspicion.
BALF was centrifuged at 6000 rpm for 10 min and the ADA
activity of the supernatant was measured using the Diazyme ADA
assay kit (Diazyme Laboratories, CA, USA).12 The pellet was then
processed for TB PCR analysis. PCR for TB was performed using the
Multiplex TBC PCR module (BIORON Diagnostics GmbH, Ludwig-
shafen, Germany) in accordance with the manufacturer’s instruc-
tions.13
The physicians were not aware of the ADA or TB PCR results in
the diagnosis and decision making for disease management. A final
diagnosis was made upon agreement of clinical manifestations,
radiographic findings, microbiology results, cytopathology results,
and improvement or progression on follow-up.
2.3. Diagnosis of active pulmonary TB
A diagnosis of active pulmonary TB required one of the
following: (1) a positive Mycobacterium tuberculosis culture from
BALF; (2) the presence of granuloma in the respiratory tissue
histology without positive culture or evidence of any organisms or
causes other than TB; (3) a response to antituberculous medica-
tions revealed by improvement of clinical symptoms and chest
radiograph.
2.4. Inactive pulmonary TB
Inactive pulmonary TB was defined in the case of an
asymptomatic patient who had abnormal radiographic findings
consistent with pulmonary TB but no evidence of active disease,
and stable chest radiographs for 1 year on follow-up, without
treatment with antituberculous medication.
2.5. Statistical analysis
All data were analyzed with a statistical software package
(SPSS, version 16.0 for windows; SPSS Inc., Chicago, IL, USA). Data
for continuous variables were expressed as mean  standard
deviation (SD), and those of categorical variables were presented as
percentages. When data were non-normally distributed, median
values and ranges were reported instead.
Study patients were classified into 10 groups according to the
diagnosis. Groups were compared with the Mann–Whitney U-test,
Kruskal–Wallis one-way analysis of variance (ANOVA) test, and the
Chi-square test.
A receiver operating characteristic (ROC) curve was constructed
and the area under the ROC curve with 95% confidence intervals
(CI) was determined to find the cutoff BALF ADA activity for
differentiating TB from other pulmonary diseases that have
significantly different ADA activity from TB. ROC curves were
then compared for the cutoff BALF ADA activity, TB PCR, and the
combination of the cutoff BALF ADA activity and TB PCR.
3. Results
Four hundred and twenty-four patients were enrolled in the
study, comprising 216 males and 208 females. Their average age
was 57.6  15.2 years and they had various clinical symptoms and
radiographic findings. Box plots of ADA activity among the various
pulmonary diseases are shown in Figure 1. The median ADA activity of
TB cases was significantly different from that of patients with solid
tumor without endobronchial obstruction (p < 0.001), inactive TB
(p = 0.04), and other (p = 0.038) while this was not the case for the
other disease groups.
The ROC curve for ADA in differentiating TB and solid tumor
without endobronchial obstruction is shown in Figure 2. The area
under the ROC curve (AUC) was 0.71 (95% CI 0.65–0.78). A cutoff
BALF ADA activity of 3 U/l provided a sensitivity of 58.7% and
specificity of 81.8% to differentiate TB from solid tumor without
endobronchial obstruction.
PCR for TB was positive in 37 cases. Of these, three were defined
as false-positive, one in a solid tumor, one in a bacteria-infected
bronchiectasis, and one in an Aspergillus infection. All of them had
no evidence of pulmonary TB during 1 year of follow-up. The
sensitivity of TB PCR in BALF was 28.1% and the specificity 99.0%.
To differentiate TB from solid tumor without endobronchial
obstruction, the AUC was significantly higher for the combination
of ADA activity 3 U/l and TB PCR (0.77) than for ADA activity 3
U/l alone (0.70, p < 0.001) or for TB PCR alone (0.64, p < 0.001)
(Figure 3). The sensitivity of the combination of ADA activity 3 U/l
and TB PCR was 72.7% and the specificity 81.8%.
 V. Boonsarngsuk et al. / International Journal of Infectious Diseases 16 (2012) e663–e668
Figure 1. Distribution of BALF ADA activity among various pulmonary diseases (abbreviations: BALF, bronchoalveolar lavage fluid; ADA, adenosine deaminase; TB,
tuberculosis; CA, cancer; NTM, non-tuberculous mycobacteria).
Of 121 TB cases, there were 58 immunocompromised patients
with: HIV disease (n = 2), diabetes mellitus (n = 21), alcoholism
(n = 10), cirrhosis (n = 6), chronic renal failure (n = 8), malnutrition
(n = 9), connective tissue diseases (n = 11), hematologic malignan-
cies (n = 8), solid malignancies (n = 20), organ transplant (n = 4),
systemic steroid (n = 17), and other immunosuppressive drugs or
chemotherapeutic agents (n = 20). Immunocompromised status
did not impact on the median ADA activity and BALF TB PCR results
(Table 1).
A greater extent of radiographic disease (as defined by the
radiographic criteria established by the National Tuberculosis and
Respiratory Disease Association) was associated with higher
median ADA activity. In contrast, PCR TB positivity was not
significantly different between the three disease extent groups.
Figure 2. Receiver operating characteristic curve for bronchoalveolar lavage fluid
adenosine deaminase (ADA) activity, differentiating between tuberculosis and solid
tumor without endobronchial obstruction; asterisks represent the ADA activity.
e665
Positive BALF AFB smear and culture were associated with
positivity of TB PCR, resulting in higher positive TB PCR in definite
TB cases than probable cases, whereas these were not associated
with ADA activity.
4. Discussion
ADA activity has been used as a valuable marker for
differentiating tuberculous pleural effusion from other causes
of exudative pleural effusions.14,15 Recently, Porcel et al.
emphasized the diagnostic utility of pleural fluid ADA activity
in 2193 patients with different etiologies of pleural effusions.16
Patients with TB pleuritis had significantly higher ADA activities
than patients with non-TB effusions. However, differential
diagnoses of exudative pleural effusions are narrow when
comparing with parenchymal lung diseases, as in our patients.
Although many studies have reported that ADA activity in BALF of
patients with pulmonary TB is higher than in comparators,3–6
three major concerns have been debated. First, all non-pulmonary
TB patients were combined as comparators in the analyses, and
those with lung cancers formed the majority of these populations.
Second, with regard to the TB cases, they enrolled only those with
positive staining or culture of BALF, which cannot be applied to the
majority of TB patients who are smear- and culture-negative.
Finally, these studies have included relatively small numbers of
both TB cases and comparators.
Reechaipichitkul et al. separated the comparators into those
with malignancies and a miscellaneous group, which included
various causes of interstitial lung diseases, non-TB infectious
disease, and hematologic malignancy.7 In addition, they did not
enroll only smear- or culture-positive BALF cases, but included
smear- and culture-negative cases who responded to antitubercu-
lous medication, as in our study. They found BALF ADA of
pulmonary TB patients was not significantly different from the
other groups.
In the diagnosis of TB pleural effusion using ADA activity, there
is also false-positivity that is caused by both solid and
hematologic malignancies, bacterial infection, and connective
tissue diseases.16–18 In our study, these etiologies also caused
e666 V. Boonsarngsuk et al. / International Journal of Infectious Diseases 16 (2012) e663–e668

Figure 3. Receiver operating characteristic curve comparing the combination of a cutoff BALF ADA activity 3 U/l and TB PCR, BALF ADA activity 3 U/l alone, and TB PCR
alone in differentiating between TB and solid tumor without endobronchial obstruction (abbreviations: BALF, bronchoalveolar lavage fluid; ADA, adenosine deaminase; TB,
tuberculosis).

Table 1
Factors affecting BALF ADA activity and positivity of TB PCR in 121 TB cases

Variables Number BALF ADA, median (range) (U/l) p-Value Positive TB PCR, n (%) p-Value

Sex 0.18 0.49

Female 58 3.5 (0.0–44.0) 18 (31.0)
Male 63 3.0 (0.0–70.0) 16 (25.4)
Immunostatus 0.20 0.90
Immunocompetent 63 4.0 (0.0–57.0) 18 (28.6)
Immunocompromiseda 58 3.0 (0.0–70.0) 16 (27.6)
Radiographic extent 0.017 0.41
Minimal 42 3.0 (0.0–11.0) 9 (21.4)
Moderately advanced 45 3.0 (0.0–70.0) 13 (28.9)
Greatly advanced 34 5.0 (0.0–57.0) 12 (35.3)
BALF smear
Negative 105 3.0 (0.0–70.0) 26 (24.8)
Positive 16 6.5 (0.0–45.0) 0.14 8 (50.0) 0.036
PCR 0.84
Negative 87 4.0 (0.0–70.0) -
Positive 34 2.5 (0.0–45.0) -
Granuloma in histological samples 0.40 0.64
Negative 50 3.5 (0.0–70.0) 15 (30.0)
Positive 35 1.0 (0.0–30.0) 11 (31.4)
Not performed 36 4.5 (0.0–45.0) 8 (22.2)
Culture 0.78 0.001
Negative 57 5.0 (0.0–57.0) 8 (14.0)
Positive 64 3.0 (0.0–70.0) 26 (40.6)
TB diagnosisb 0.51 0.013
Probable 38 5.0 (0.0–57.0) 5 (13.2)
Definite 83 3.0 (0.0–70.0) 29 (34.9)

BALF, bronchoalveolar lavage fluid; ADA, adenosine deaminase; TB, tuberculosis.

a
Immunocompromised patients defined as those having HIV disease, diabetes mellitus, alcoholism, cirrhosis, chronic renal failure, malnutrition, connective tissue
diseases, hematologic and solid malignancies, organ transplant, systemic steroid or other immunosuppressive drugs, or chemotherapeutic agents.
b
A definite TB diagnosis was made in patients with either a positive M. tuberculosis culture from BALF, or the presence of granuloma in the respiratory tissue histology
without positive culture or evidence of any organisms or causes other than TB. Probable TB was diagnosed in patients who responded to antituberculous medications,
revealed by improvement of clinical symptoms and chest radiographs.
 V. Boonsarngsuk et al. / International Journal of Infectious Diseases 16 (2012) e663–e668
false-positive BALF ADA activities, giving ADA 3 U/l. ADA is
produced not only by mononuclear cells and lymphocytes, but
also by many different cell types, including neutrophils and red
blood cells.16,19 Therefore, we believe that in any organs, local ADA
would be high when affected not only by TB, but also by these
etiologies. Unfortunately, to date, ADA2, which is secreted only by
monocytes and macrophages and is believed to be a more efficient
diagnostic marker of TB pleuritis than total ADA activity,18,20 has
not been studied in BALF and this awaits further research.
Alternatively, an M. tuberculosis-specific BALF interferon-gamma
release assay has been reported recently with a high sensitivity
and specificity for the diagnosis of pulmonary TB.21 However,
more research is needed before any conclusions can be reached.
Thus a clinical approach currently remains necessary to narrow
down the differential diagnosis because ADA has limited value in
differentiating pulmonary TB from some other diseases.
In concordance with Orphanidou et al.,5 ADA activity was
significantly higher in patients with extensive disease than in those
with limited disease. In contrast, ADA activity did not correlate
with the presence of BALF smear, culture, and granuloma
formation. In pleural effusion, ADA expressed high activity in TB
pleuritis, which occasionally identified organisms or granulo-
mas.16 Thus, the extent of the lesions would have more influence
on ADA production than the organism loads and granulomatous
reactions.
The sensitivity of TB PCR in BALF in our study was quite low
compared with previous studies.8–10,22 However, in those studies,
only patients with smear- or culture-positive pulmonary TB were
enrolled for analysis. Thus, it is not surprising to obtain a high
positive rate for a PCR that detects an M. tuberculosis DNA segment
in patients who have high organism loads. In contrast, in smear-
and culture-negative pulmonary TB, the sensitivity of TB PCR has
been found to be relatively low, as in our study.9 However, the
specificity is still high regardless of the results of smear or
culture.8–10,22 Therefore, the BALF TB PCR has diagnostic utility
only when results are positive.
Due to there being no significant difference in BALF ADA activity
between TB and other pulmonary disease except for solid tumor
without endobronchial obstruction, we constructed a ROC curve
only for differentiating TB and solid tumor without endobronchial
obstruction, and demonstrated a cutoff activity of 3 U/l yielded
the greatest power distinction between TB and solid tumor.
Orphanidou et al.5 and Hassanein et al.6 demonstrated a cutoff
BALF ADA activity of 2.5 U/l, as determined by Giusti’s
colorimetric method. In pleural effusion, there was a strong
correlation between the Giusti method and the Diazyme assay for
the measurement of ADA.19 Pleural fluid with a cutoff value of 40
U/l in the Giusti method and 30 U/l in the Diazyme assay had a
concordance of 96.8%. Therefore, the cutoff ADA values determined
by the Giusti method in the previous studies are comparable to our
cutoff activity, which was measured by the Diazyme assay.
Although having a low sensitivity, TB PCR had a high specificity
and positive predictive value. Thus, when combined with ADA
activity, the diagnostic accuracy was higher than each alone.
There are some limitations to our study. We do not have BALF
differential cell count data. Therefore, we cannot explore the
correlation between ADA and different inflammatory cell types in
BALF and explain why some BALF had higher ADA than others. In
pleural fluid, the differential cell count is routinely recommended
because of its value in the differential diagnosis. In contrast, it has
limited value in BALF and is not recommended in general.
Nevertheless, Porcel et al. demonstrated a correlation between
ADA and leukocyte count, both neutrophils and lymphocytes, and
also erythrocyte count in pleural fluid.16 Thus, we believe that ADA
activity in BALF could be high in any inflammatory pulmonary
diseases, as shown in our results.
e667
In our study, we did not correct values for BALF urea or total
protein concentration. Although some authors have suggested the
use of local ADA activity, which be calculated from BALF ADA
corrected by serum ADA, serum urea (or albumin), and BALF urea
(or albumin),5 Kayacan et al. have shown BALF ADA activity to have
higher diagnostic accuracy than local ADA.3
In conclusion, BALF ADA has limited value in differentiating
pulmonary TB from some pulmonary diseases. A clinical approach
is necessary to narrow the differential diagnosis. When the
differential diagnosis leaves only TB and solid tumor, BALF ADA
is a useful tool. In addition, the combination of BALF ADA 3 U/l
and TB PCR had marked additive diagnostic value.
Conflict of interest: All authors declare that they do not have a
conflict of interest and do not have any financial relationships with
commercial entities that may have an interest in the subject of this
manuscript.
Ethical approval: This study protocol was approved by the Ethics
Committee on Human Experimentation of Ramathibodi Hospital,
Faculty of Medicine, Mahidol University. Written informed consent
was obtained from all patients.
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