MYCOBACTERIOLOGY AND

AEROBIC ACTINOMYCETES

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Diagnostic Performance of Pleural Fluid Adenosine Deaminase

for Tuberculous Pleural Effusion in a Low-Incidence Setting
Matthew Blakiston,a,d Weldon Chiu,b Conroy Wong,c Susan Morpeth,a Susan Taylora

a Microbiology Department, Counties Manukau Health, Auckland, New Zealand

b Biochemistry Department, Counties Manukau Health, Auckland, New Zealand
c
Respiratory Department, Counties Manukau Health, Auckland, New Zealand
d Microbiology Department, Auckland District Health Board, Auckland, New Zealand

ABSTRACT The challenges associated with diagnosing tuberculous pleural effusion

have led to the use of pleural fluid adenosine deaminase (pfADA) as a biomarker for
Mycobacterium tuberculosis infection. This study retrospectively reviewed the diag-
nostic performance of pfADA, the pleural fluid lactate dehydrogenase (LD)/ADA ra-
tio, and combinations of these two parameters in 1,637 episodes of pleural effusion
in the low-tuberculosis (TB)-incidence setting of Auckland, Aotearoa New Zealand,
from between March 2008 and November 2014. The median pfADA in 57 TB pleural
effusion episodes (58.1 U/liter) was significantly higher (P ⬍ 0.001) than in 1,580
non-TB pleural effusions (11.4 U/liter). The median LD/ADA ratio in TB pleural effu-
sion (8.2) was significantly lower (P ⬍ 0.001) than in non-TB pleural effusions (30.5).
The pfADA and pleural fluid LD/ADA ratio AUCROC values (that is, receiver operating
characteristic [ROC] curve analysis with determination of the ROC area under the
curve) were 0.93 and 0.94, respectively. The pfADA thresholds of ⱖ15 and ⱖ30 U/li-
ter demonstrated sensitivities of 100% (95% confidence internal ⫽ 93.7 to 100) and
93.0% (83.3 to 97.2), specificities of 62.7% (60.3 to 65.0) and 87.3% (85.6 to 88.9),
positive predictive values (PPVs) of 8.8% (6.9 to 11.2) and 20.9% (16.4 to 26.4), and
negative predictive values (NPVs) of 100% (99.6 to 100) and 99.7% (99.3 to 99.9), re-
spectively. LD/ADA ratio thresholds of ⬍25 and ⬍15 demonstrated sensitivities of
100% (93.5 to 100) and 89.1% (78.2 to 94.9), specificities of 61.6% (59.1 to 64.0) and
84.8% (82.9 to 86.5), PPVs of 8.5% (6.6 to 10.9) and 17.3% (13.3 to 22.0), and NPVs of
100% (99.6 to 100) and 99.5% (99.0 to 99.8), respectively. A combination of pfADA ⱖ 30
U/liter and an LD/ADA ratio ⬍ 15 increased the specificity and PPV to 97.8% (96.9 to
98.4) and 57.3% (46.5 to 67.5) but decreased the sensitivity to 85.5% (73.8 to 92.4).
The primary value of pfADA in a low-TB-incidence setting, such as Auckland, is in
utilization of its high NPV. Received 28 February 2018 Returned for
modification 11 April 2018 Accepted 16 May
2018
KEYWORDS ADA, pleural TB
Accepted manuscript posted online 23 May
2018
Citation Blakiston M, Chiu W, Wong C,

P leural effusion is a common condition with a broad etiology (1). In practice the
assessment of clinical, demographic, and pleural fluid cellular and chemical char-
acteristics enables the differential diagnosis to be narrowed (1). Tuberculosis (TB) is a
Morpeth S, Taylor S. 2018. Diagnostic
performance of pleural fluid adenosine
deaminase for tuberculous pleural effusion in a
low-incidence setting. J Clin Microbiol
leading cause of pleural effusion worldwide and is important to consider even in a low
56:e00258-18. https://doi.org/10.1128/JCM
incidence area such as Aotearoa New Zealand (NZ) that has an annual incidence of new .00258-18.
TB cases of 6.4 per 100,000 population (2). TB pleural effusion is a paucibacillary Editor Betty A. Forbes, Virginia
infection that follows the rupture of a subpleural focus (3). It typically presents with an Commonwealth University Medical Center

exudative lymphocytic effusion and can be definitively diagnosed by the identification Copyright © 2018 American Society for
Microbiology. All Rights Reserved.
of Mycobacterium tuberculosis in pleural tissue or pleural fluid (3, 4). Culture and
Address correspondence to Matthew Blakiston,
histological examination of pleural tissue specimens can identify M. tuberculosis or mattbl@adhb.govt.nz.
demonstrate necrotizing granulomas in approximately 90% of cases (3, 4). However,

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Blakiston et al. Journal of Clinical Microbiology

pleural biopsy is a relatively invasive procedure with associated risks and costs. Pleural
fluid collection and culture is less invasive but is severely limited by a sensitivity of
approximately 30% (3, 4). M. tuberculosis nucleic acid amplification tests on pleural fluid
are similarly limited by low sensitivity (4, 5). M. tuberculosis may be isolated from
respiratory samples in up to 50% of TB pleural effusion cases (3, 4).
The limitations of traditional diagnostic methods can make it challenging to confirm
or exclude TB pleural effusion. This has led to the investigation and use of biomarkers
such as pleural fluid adenosine deaminase (pfADA) to supplement standard investiga-
tions (5). ADA is an enzyme involved in purine degradation and mononuclear leukocyte
maturation and is most abundantly found in T lymphocytes and macrophages (4). Two
meta-analyses utilizing summary receiver operating characteristic (ROC) curve analysis
have reported pfADA Q-point values (a summary measure of test performance corre-
sponding to the highest common value of sensitivity and specificity) for TB pleural
effusion of 91 and 93%, respectively, suggesting reasonable discriminatory ability (6, 7).
However, pfADA studies have been predominantly performed in locations with an
intermediate or high TB incidence and thus have limited generalizability to low-
incidence settings, where the nonspecific nature of pfADA elevation results in a low
positive predictive value (PPV). Nonetheless, with a high sensitivity and negative
predictive value (NPV), it may still be useful in low incidence settings (1, 5). A recent
study from southern England reported a sensitivity and NPV (with a threshold of ⱖ35
IU/liter) of 85.7 and 99.9%, respectively, for TB pleural effusion (8). Interpretation in
conjunction with other pleural fluid variables, such as cellular differential, protein level,
and gamma interferon, has been reported to increase test specificity (5). A pleural fluid
lactate dehydrogenase (LD)/serum LD ratio has also been described within multipa-
rameter scoring systems (5). Local anecdotal observations have suggested that pleural
LD is generally lower in TB pleural effusion than in other inflammatory pleural effusions
(that have an elevated ADA) and that assessment of this relationship may have
diagnostic utility.
The appropriate use and interpretation of pfADA depend on the local TB prevalence,
the aim of testing, the ADA assay method used, and the associated ADA threshold
selected (5, 6, 9). To facilitate optimal use and interpretation in a low-TB-incidence
setting, this study retrospectively investigated the diagnostic performance of pfADA
and the pleural fluid LD/ADA ratio (individually and in combination) for TB pleural
effusion in Auckland, NZ.

MATERIALS AND METHODS

This was a retrospective observational study of individuals ⱖ15 years old who had pfADA testing
performed at Counties Manukau Health (CMH) hospital laboratory in Auckland, NZ, between March 2008
(when pfADA testing was introduced) and November 2014. The study population had two subgroups.
The first subgroup was a population of CMH patients who had primarily undergone routine laboratory
initiated pfADA testing if their effusion was exudative (as per Lights criteria [1]) or unknown nature (if
Lights criteria could not be calculated). CMH patients with nonroutine pfADA testing on transudative
effusions were also included in the study population. The second subgroup population included
individuals referred from other Auckland health providers for pfADA testing. The study population was
identified from the electronic laboratory records, and associated demographic, laboratory, and clinical
information was extracted from electronic clinical records. Data collection began in November 2015,
giving a minimum of 12 months between an episode of pleural effusion occurring and review of
electronic records taking place. During the study time frame, pfADA testing was performed using the
Diazyme ADA assay (Diazyme Laboratories, Poway, CA) on the Abbot Architect CI8000 autoanalyzer. The
LD measurement method was a lactate-to-pyruvate forward reaction using NADH absorbance at 340 nm
on the Abbott Architect CI8000. The practice at CMH from March 2008 to March 2010 was for all
exudative pleural effusions to undergo pfADA testing and mycobacterial culture utilizing the automated
BacT/Alert broth culture system (bioMérieux, Marcy-Etoile, France) and Lowenstein-Jensen slopes. Since
March 2010, only effusions with pfADA ⱖ 15 U/liter underwent routine (laboratory-initiated) mycobac-
terial culture. During the study time frame, cytological examination was routinely performed on
exudative effusions.
Pleural effusions were classified by etiology as follows. “TB pleural effusion” was divided into
confirmed and probable categories. “Confirmed TB-pleural effusion” indicated M. tuberculosis identified
by culture in pleural fluid or pleural tissue or a nonpleural site with an otherwise unexplained pleural
effusion. “Probable TB-pleural effusion” indicated subsequent (nonconcurrent) microbiological diagnosis
of TB within 18 months or clinical diagnosis and treatment for TB with a good clinical response. All TB

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Performance of pfADA in a Low-TB-Incidence Setting Journal of Clinical Microbiology

pleural effusions were grouped together for analysis. “Non-TB pleural effusion” represented negative
laboratory investigations for M. tuberculosis and/or the absence of a clinical or microbiological diagnosis
of TB over the subsequent clinical course. “Malignant effusions” were also divided into confirmed and
probable categories. “Confimed malignant effusion” indicated positive pleural fluid cytology or positive
pleural tissue histology or confirmed malignancy at another site with radiological evidence of metastatic
thoracic involvement. “Probable malignant effusion” indicated a documented clinical diagnosis based on
a suggestive radiological and clinical course and the absence of an alternative cause. All malignant
effusions were grouped together for analysis. “Parapneumonic effusions” (PPEs) were classified by a
documented clinical diagnosis and the absence of an alternative cause identified over the clinical course
and were subdivided into complicated and simple categories. Superinfected malignant effusions were
included in this group. “Complicated PPE” (CPPE) indicated a positive pleural fluid culture (excluding skin
contaminants) or positive direct microscopy (Gram stain) or pH ⬍ 7.2 (glucose ⬍ 3.4 mmol/liter if the pH
was unavailable) or a pleural LD greater than 1,000 U/liter or a radiologically loculated effusion. A “simple
PPE” (SPPE) indicated a PPE that did not meet the criteria for CPPE. Other etiologies were determined by
the documented clinical diagnosis. Effusion etiology was classified as unknown (non-TB) when no clinical
or pathological diagnosis could be determined from records.
Data were analyzed on a per-episode basis. Repeated pleural fluid samples from the same patient
(with the same given etiology) collected within an 8-week time frame were considered to be the same
episode. The sample with the highest ADA value was included for per episode analysis. Microsoft Excel
(Microsoft Corporation, Redmond, WA) and Prism v7 (GraphPad Software, La Jolla, CA) were utilized for
data management and analysis. Categorical demographic data were summarized using frequency counts
and proportions. Non-normally distributed continuous variables were summarized using the median, the
interquartile range, and the range. A two-tailed Mann-Whitney test was used to evaluate for significant
differences in the median pfADA, LD, and LD/ADA between the different etiologies. The overall
discriminatory performance of pfADA and the pleural fluid LD/ADA ratio for TB pleural effusion were
assessed utilizing ROC curve analysis (with determination of the ROC area under the curve [AUCROC]). The
sensitivity, specificity, PPV, and NPV for TB pleural effusion were calculated for preselected pfADA
thresholds of ⱖ15 and ⱖ30 U/liter. These values were selected based on current CMH laboratory usage
for assessing likelihood of TB pleural effusion. A threshold of ⬍30 U/liter suggesting a low likelihood of
TB has been utilized since 2008, based on audit of first 120 samples tested (9). A threshold of ⬍15 U/liter,
suggesting a very low likelihood of TB, was used as a cutoff for routine M. tuberculosis culture since 2010.
The LD/ADA ratio thresholds were selected after data collection. Multiparameter analysis, including
pfADA and the pleural fluid LD/ADA ratio, was also performed. Institutional research office approval was
obtained from CMH and Auckland District Health Board.

RESULTS
We identified pfADA testing on 2,209 pleural effusion samples between March 2008
and November 2014. After exclusion of 572 duplicate samples, 1,637 separate episodes
of pleural effusion from 1,576 individuals were included in the analysis. This included
1,458 (89.1%) from CMH and 179 (10.9%) referred samples. The majority were exudative
effusions (1,338, 81.7%), followed by unknown nature (177, 10.8%) and transudates
(122, 7.5%). Of the 1,392 effusions with an available cellular differential, 970 (69.7%)
were mononuclear leukocyte predominant (⬎50%). Pleural fluid mycobacterial culture
had been performed in 1,125 (68.7%) of 1,637 episodes.
TB pleural effusion accounted for 57 (3.5%) of 1,637 episodes (from 57 individuals).
Fifty (87.7%) were considered confirmed, as per the study definition, by concurrent
culture based identification of M. tuberculosis (in the pleural space or another site), and
seven were (12.3%) considered probable. One individual had multidrug-resistant TB.
Non-TB pleural effusion accounted for 1,580 (96.5%) of 1,637 episodes. The demo-
graphics (by episode) of the total population and the TB/non-TB populations are
presented in Table 1. The 55 TB-pleural effusions with available laboratory data were all
exudative. Of 52 TB pleural effusions with an available cellular differential, 48 (92.3%)
were mononuclear predominant. The median pleural fluid pH was 7.35, and the median
pleural fluid glucose was 4.8 mmol/liter. The pleural fluid mycobacterial culture was
positive for M. tuberculosis in only 22 (38.6%) of 57 episodes. All 57 TB pleural effusions
had negative acid-fast microscopy. Malignancy (490/1,580, 31.0%) was the most com-
mon etiology of non-TB pleural effusion (with 449 [91.6%] of 490 considered con-
firmed), followed by SPPE (270/1,580, 17.1%), CPPE (266/1,580, 16.8%), congestive heart
failure (CHF; 165/1,580, 10.4%), occurrence in association with intra-abdominal (IA)
infection or post-IA surgery (66/1,580, 4.2%), non-CHF fluid maldistributive states (renal
failure, hepatic failure, hypoalbuminemia, sepsis-associated capillary leak, and iatro-
genic fluid overload; 65/1,580, 4.1%), post-coronary artery bypass graft (CABG) and
other cardiac surgery (56/1,580, 3.5%), hemothorax (35/1,580, 2.2%), and nonpneu-

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TABLE 1 Population demographicsa

No. (%) of samplesb
Non-TB pleural effusion TB pleural effusion
Parameter Total (n ⴝ 1,637) (n ⴝ 1,580) (n ⴝ 57)
Median age in yrs (IQR) 67 (54–78) 67 (53–78) 31 (16–50)
Male 963 (58.8) 929 (58.8) 34 (59.6)

Ethnicity
Pa៮ keha៮ (NZ European) 660 (40.3) 658 (41.6) 2 (3.5)
Pacific peoples 395 (24.1) 381 (24.1) 14 (24.6)
European other 192 (11.7) 192 (12.2) 0 (0)
Ma៮ oric 185 (11.3) 178 (11.3) 7 (12.3)
Asian peoples 165 (10.1) 133 (8.4) 32 (56.1)
Other/unknown 40 (2.4) 38 (2.4) 2 (3.5)

Place of birth
Aotearoa New 820 (50.1) 811 (51.3) 9 (15.8)
Zealand
Overseas 685 (41.8) 649 (41.1) 36 (63.2)
Unknown 132 (8.1) 120 (7.6) 12 (21.1)

CMH samples 1,458 (89.1) 1,425 (90.2) 33 (57.9)

Referred samples 179 (10.9) 155 (9.8) 24 (42.1)
aCMH, Counties Manukau Health; IQR, interquartile range; NZ, New Zealand; TB, tuberculosis.
bExcept as noted otherwise in column 1.
cIndigenous peoples of NZ.

monic pleural infection (17/1,580, 1.1%). Miscellaneous causes (60/1,580, 3.8%) and
unknown (non-TB) etiology (90/1,580, 5.7%) accounted for the remaining non-TB
pleural effusions.
The median pfADA in TB pleural effusion (58.1 U/liter) was significantly (P ⬍ 0.001)
higher than in non-TB pleural effusion (11.4 U/liter) and in all categories of non-TB
pleural effusion except for culture or microscopy positive CPPE (Table 2 and Fig. 1). The
median pleural fluid LD in TB pleural effusion was significantly (P ⬍ 0.001) higher than
in pleural effusions associated with CHF, non-CHF fluid maldistribution states, SPPE,
CABG or other cardiac surgery, IA infection or IA surgery, miscellaneous causes, and

TABLE 2 Pleural fluid ADA, LD, and LD/ADA ratioa

U/liter (IQR)
LD/ADA ratio
Etiology No. ADA LD (IQR)
Tuberculous pleural effusion 57 58.1 (45.1–74.8) 453.0 (342.0–647.0) 8.2 (5.9–11.0)

Nontuberculous pleural effusion 1,580 11.4 (6.8–19.4) 317.0 (152.0–708.5) 30.5 (19.5–47.4)
Complicated parapneumonic effusion (CPPE) 266 31.9 (21.7–57.6) 1,438.0 (816.5–2,960.0) 45.8 (32.8–61.6)
Culture- or microscopy-positive CPPE (n ⫽ 72) 56.2 (29.0–115.0) 2,305.0 (1,330.0–5,000.0) 45.0 (35.0–60.3)
Culture- and microscopy-negative CPPE (n ⫽ 194) 27.7 (19.3–45.1) 1,222.0 (781.3–2,432.0) 46.7 (32.3–61.6)
Nonpneumonic pleural infection 17 22.6 (16.8–58.4) 1,189.0 (490.0–2,835.0) 47.3 (24.9–47.3)
Simple parapneumonic effusion 270 11.1 (6.9–16.7) 260.0 (156.0–478.0) 25.7 (17.3–36.6)
Malignancy 490 11.4 (8.0–16.6) 382.5 (214.5–618.8) 33.1 (20.3–53.5)
Hematological (n ⫽ 43) 16.5 (12.3–51.5) 389.5 (235.5–1,012.0) 18.5 (12.6–32.2)
Nonhematological (n ⫽ 447) 10.9 (7.7–16.0) 381.5 (214.0–612.3) 34.8 (21.2–54.7)
Congestive heart failure (CHF) 165 4.3 (3.2–6.7) 107.5 (79.8–136.3) 23.9 (17.9–32.1)
Non-CHF fluid maldistributive states 65 5.1 (3.5–8.0) 110.0 (76.5–194.5) 21.9 (15.6–32.9)
Post-CABG or other cardiac surgery 56 9.1 (6.1–10.8) 217.0 (136.8–337.8) 26.2 (19.9–37.1)
Hemothorax 35 12.5 (7.7–17.1) 431.0 (259.0–753.0) 38.7 (31.9–60.6)
Intra-abdominal (IA) infection/post-IA surgery 66 13.3 (7.5–18.8) 305.0 (204.3–522.8) 28.1 (18.5–47.3)
Miscellaneousb 60 11.0 (7.5–18.1) 276.0 (172.0–449.0) 26.9 (14.6–38.6)
Unknown (nontuberculous) 90 9.9 (5.1–15.4) 166.5 (113.0–308.0) 23.7 (14.6–32.3)
aADA, LD, and LD/ADA values are displayed as medians and interquartile ranges (IQRs). The numbers in parentheses in the etiology column are the numbers of
episodes in the respective subgroups of CPPE and malignant pleural effusions. LD (and thus LD/ADA) was only available for 1,596 of 1,637 samples, including 55 of
57 TB-PE. ADA, adenosine deaminase; CABG, coronary artery bypass graft; LD, lactate dehydrogenase.
bIncludes drug reactions, pulmonary emboli, asbestos-related “fibrinous pleuritis,” autoimmune conditions, systemic vasculitides, viral serositis, and esophageal rupture.

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Performance of pfADA in a Low-TB-Incidence Setting Journal of Clinical Microbiology

FIG 1 Pleural fluid ADA by etiology. A boxplot shows medians, interquartile ranges, and ranges. CPPE,
complicated parapneumonic effusion; CHF, congestive heart failure; IA infect/surg, intraabdominal
infection or post-IA surgery; NPPI, nonpneumonic pleural infection; non-CHF FMS, non-CHF fluid mal-
distribution state; SPPE, simple parapneumonic effusion.

unknown causes; it was significantly (P ⬍ 0.01) lower than in effusions associated with
CPPE and nonpneumonic pleural infection; and it was not significantly (P ⬎ 0.05)
different than in effusions associated with malignancy and hemothorax (Table 2). The
median pleural fluid LD/ADA ratio in TB pleural effusion (8.2) was significantly (P ⬍
0.001) lower than in non-TB pleural effusion (30.5) and in all categories of non-TB
pleural effusion (Table 2 and Fig. 2). The median pfADA level in pleural fluid M.

FIG 2 Pleural fluid LD/ADA ratio by etiology. A boxplot shows medians, interquartile ranges, and ranges.
CPPE, complicated parapneumonic effusion; CHF, congestive heart failure; IA infect/surg, intraabdominal
infection or post-IA surgery; NPPI, nonpneumonic pleural infection; non-CHF FMS, non-CHF fluid mal-
distribution state; SPPE, simple parapneumonic effusion.

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FIG 3 ROC curve for pleural fluid ADA and LD/ADA ratio.

tuberculosis culture-positive TB pleural effusions (65.6 U/liter) did not differ significantly
(P ⫽ 0.12) from pleural fluid culture-negative TB pleural effusions (56.2 U/liter). The
median pleural LD/ADA ratio was higher in pleural fluid M. tuberculosis culture-positive
TB pleural effusions (9.9) than pleural fluid culture-negative TB pleural effusions (7.5)
with borderline significance (P ⫽ 0.05). The pfADA and the LD/ADA ratio had an AUCROC
of 0.93 (95% confidence interval [CI] ⫽ 0.91 to 0.95) and 0.94 (95% CI ⫽ 0.92 to 0.97),
respectively (Fig. 3). In analysis restricted to the consecutive CMH population the
AUCROC for pfADA and the LD/ADA ratio was 0.94 (CI ⫽ 0.92 to 0.95) and 0.95 (CI ⫽ 0.92
to 0.97), respectively. In analysis restricted to the referred episodes the AUCROC for
pfADA and the LD/ADA ratio was 0.92 (CI ⫽ 0.87 to 0.96) and 0.91 (CI ⫽ 0.86 to 0.96),
respectively. Similarly, in analysis restricted to confirmed exudative effusions the AU-
CROC for pfADA and the LD/ADA ratio was 0.91 (CI ⫽ 0.89 to 0.93) and 0.95 (CI ⫽ 0.93
to 0.97). The sensitivity, specificity, PPV, and NPV of pfADA (ⱖ15 and ⱖ30 U/liter), the
pleural fluid LD/ADA ratio (⬍25 and ⬍15), and the associated dual-parameter combi-
nations are shown in Table 3.

TABLE 3 Diagnostic performance of pleural fluid ADA and LD/ADA ratio for TB pleural
effusiona
% (CI)
Parameter(s) Sensitivity Specificity PPV NPV
ADA ⱖ 15 U/liter 100 (93.7–100) 62.7 (60.3–65.0) 8.8 (6.9–11.2) 100 (99.6–100)
ADA ⱖ 30 U/liter 93.0 (83.3–97.2) 87.3 (85.6–88.9) 20.9 (16.4–26.4) 99.7 (99.3–99.9)
LD/ADA ratio ⬍ 25 100 (93.5–100) 61.6 (59.1–64.0) 8.5 (6.6–10.9) 100 (99.6–100)
LD/ADA ratio ⬍ 15 89.1 (78.2–94.9) 84.8 (82.9–86.5) 17.3 (13.3–22.0) 99.5 (99.0–99.8)
ADA ⱖ 15 U/liter and 100 (93.5–100) 86.9 (85.2–88.5) 21.2 (16.7–26.6) 100 (99.7–100)
LD/ADA ⬍ 25
ADA ⱖ 15 U/liter and 89.1 (78.2–94.9) 92.7 (91.3–93.9) 30.3 (23.7–37.7) 99.6 (99.1–99.8)
LD/ADA ⬍ 15
ADA ⱖ 30 U/liter and 92.7 (82.7–97.1) 96.6 (95.6–97.4) 49.0 (39.6–58.5) 99.7 (99.3–99.9)
LD/ADA ⬍ 25
ADA ⱖ 30 U/liter and 85.5 (73.8–92.4) 97.8 (96.9–98.4) 57.3 (46.5–67.5) 99.5 (99.0–99.7)
LD/ADA ⬍ 15
aNotethat LD was available in only 1,596 of 1,637 episodes of pleural effusion, including 55 of 57 TB-PE.
ADA, adenosine deaminase; CI, 95% confidence interval; LD, lactate dehydrogenase; NPV, negative
predictive value; PPV, positive predictive value.

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DISCUSSION
Pleural fluid ADA has been investigated as a biomarker for TB pleural effusion
because of the limitations of classical diagnostic methods (5). While ADA and other
biomarker diagnostics fail to provide sufficient information for TB treatment in the era
of multidrug resistance, it may have a supplementary role in the investigation of pleural
effusion. To improve the use and interpretation of pfADA in a low-incidence setting,
this study describes its diagnostic performance for TB pleural effusion in Auckland, NZ.
Consistent with the epidemiology of TB in NZ, the TB pleural effusion population
evaluated was younger and contained a higher proportion of individuals of Asian
ethnicity and an overseas place of birth than the non-TB population (2). The median
pfADA level was significantly higher in TB pleural effusion (58.1 U/liter) than in non-TB
pleural effusions (11.4 U/liter). However, the range of pfADA levels in TB did overlap
with pleural effusions of non-TB etiology, in particular with other inflammatory etiol-
ogies, such as CPPE, nonpneumonic pleural infections, and hematological malignancy.
The median pleural fluid LD/ADA ratio was significantly lower in TB pleural effusion (8.2)
than in non-TB etiologies (30.5) with similar, if less extensive, overlap. The high AUCROC
for both parameters does, however, suggest a good intrinsic discriminatory ability, and
a similar performance was evident in the consecutive CMH and referred subpopula-
tions.
Pleural fluid ADA thresholds of ⱖ15 and ⱖ30 U/liter displayed a high sensitivity and
NPV. This suggests that a low pfADA level, in this setting, would make TB an unlikely
cause of pleural effusion. Similarly, a pfADA greater than 110 to 120 U/liter appears to
make TB pleural effusion unlikely; the highest pfADA level in TB pleural effusion was
110 U/liter, which is well below the upper levels seen in some CPPE and malignant
effusions. Not unexpectedly, pfADA had a low PPV in this setting due to its elevation
in other, more common, inflammatory pleural conditions. In isolation, the pleural fluid
LD/ADA ratio performed similarly to pfADA alone. However, the dual-parameter com-
bination of an elevated pfADA plus the LD/ADA ratio increased specificity and PPV.
Considering both the absolute pfADA level and its relative abundance compared to LD
may provide direction to further investigation in effusions of unclear etiology. Despite
a high NPV for TB pleural effusion, the interpretation of pfADA (and the associated
LD/ADA ratio) still requires consideration of the individual pretest probability of TB and
recognition of potential fallibilities such as nonrepresentative sampling from a possibly
nonhomogenous space and analytical error or variability.
Limitations of this study include its retrospective nature and the associated potential
for etiological attribution error and/or bias associated with investigators assigning
cause from clinical records. There is also potential for confirmation bias since clinical
investigations for TB may have been influenced by the pfADA result. The ADA levels
reported and assessed in this article are based on results from the non-Giusti Diazyme
assay. These results (in particular the absolute levels) should not be generalized to
results from other ADA measurement methods, like the Giusti method (which has
shown a good correlation but a positive bias compared to Diazyme assay ADA levels [9,
10]), or alternative assays, without local confirmation. The interpretation of pfADA levels
in children and the impact of biological or pharmacological immunosuppression was
not assessed in this study, and results cannot be directly generalized to these patient
groups. Similarly, the performance pfADA should not be generalized to ADA measure-
ment in other specimen types. The pfADA and LD results in CHF and other fluid
maldistributive states should not be interpreted as representative of these disease
states in general, since predominantly samples of exudative (or unknown) nature
underwent pfADA testing in this population.
The diagnosis of TB pleural effusion is challenging and frequently multifaceted in
nature. The interpretation of supplementary biomarkers such as pfADA depends on
local TB prevalence and the associated aim of testing. In a low-TB-incidence setting,
such as Auckland, New Zealand, this study suggests that the primary value of pfADA

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Blakiston et al.
testing is in the utilization of its high NPV. Consideration of pfADA levels relative to LD
can provide additional information.
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