PLENO DK CRP 1 & 2

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ALICIA
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SAMUEL
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CRP DK 1
KELOMPOK 4
 Dzce Tp Fakltesi Dergisi 2008; 3:39-42

The Validity of the Rapidly Diagnostic Tests for Early Detection of Urinary
Tract Infection
Mustafa YILDIRIM1, dris AHN2, Abdlkadir KKBAYRAK1, kr KSZ3,
Selda ACAR2, M. Tevfik YAVUZ2
1
Department of Clinical Microbiology and Infectious Diseases, University of Duzce, Faculty of Medicine,
Duzce, Turkey, 2Department of Microbiology and Clinical Microbiology, University of Duzce, Faculty of
Medicine, Duzce, Turkey, 3Department of Microbiology and Clinical Microbiology, Duzce Ataturk State
Hospital, Duzce, Turkey.

SUMMARY
Urinary tract infectious (UTI) is the most common of all bacterial infections; the purpose of the
present study was to determine the validity of rapidly diagnostic tests for the early detection of UTIs in patients.
128 patients who had UTIs and control group consisted of 128 subjects who had not UTIs were included to the
study. Urine specimens obtained from the patients were evaluated for possible UTI by Gram staining, microscopic
pyuria, dipstick (nitrite and leukocyte esterase), and quantitative urine culture. Using the quantitative urine culture
as the gold standard (reference test), the sensitivity, specificity, and positive (PPV) and negative predictive values
(NPV) of all the screening tests were determined and compared. The sensitivity, specificity, PPV, and NPV for
the four screening methods were calculated against the urine culture (reference method) for the diagnosis of UTI.
In conclusion, validity of Gram stain was found higher compared to other rapid diagnostic tests.
Key words: Urinary tract infection, Gram stain, screening tests

riner Sistem Enfeksiyonlarnn Erken Saptanmasnda Hzl Tan

Testlerinin Deeri
ZET
riner sistem enfeksiyonlar (S) tm bakteriyel enfeksiyonlarn en sk nedenidir; sunulan bu
almann amac riner sistem enfeksiyonlarnn erken saptanmasnda hzl tan testlerinin deerinin
belirlenmesidir. S olan 128 hasta ve kontrol grubu olarak 128 salkl kii almaya dahil edildi. Hastalardan
elde edilen idrar rnekleri Gram boyama, mikroskopik piyri, dipstik (nitrit ve lkosit esteraz) ve kantitatif idrar
kltr ile olas S ynnden deerlendirildi. Tm tarama testlerinin duyarll, zgll, pozitif (PPV) ve
negatif kestirim deerleri (NPV) altn standart olarak idrar kltrleri kullanlarak belirlendi ve karlatrmalar
yapld. Bu drt tarama metodu iin duyarllk, zgllk, PPV ve NPV deerleri idrar kltr sonularna (referans
yntem) gre hesapland. Sonu olarak, Gram boyama dier hzl tan yntemleri ile karlatrldnda daha
deerli bulundu.
Anahtar kelimeler: riner system enfeksiyonu, Gram boyama, tarama testleri

INTRODUCTION contamination from fecal bacteria that colonize

Urinary tract infectious (UTI) is
defined by the presence of organisms in the
urinary tract, which is usually sterile. However,
since asymptomatic colonization of the urinary
tract can occur, other features such as the
presence of inflammatory markers or
follow-up cultures may be needed to
definitively diagnose a UTI. Clinically
important infections usually occur due to
bacteria, although viruses, fungi, and parasites
can also cause infection (1).
The gold standard for diagnosis of UTI
is growth of pathogenic bacteria in a urine
culture. However, diagnosis is complicated by
the perineal area and distal urethra. In the
1950s, Kass studied adult women and
established a threshold of 100,000 CFU per ml
in a voided specimen as the standard to define
a positive urine culture (2). Although urine
culture is the gold standard for diagnosis of
UTI, results are not available for 24 to 48 h.
Rapid techniques to predict UTI include urine
dipstick tests for leukocyte esterase and nitrites
and various forms of urinalysis, including
Standard microscopy on a centrifuged
specimen, high-powered microscopy with a
hemacytometer, and Gram stain of unspun
urine for organisms (1).

Mustafa YILDIRIM ve ark. 39

 Dzce Tp Fakltesi Dergisi 2008; 3:39-42
The purpose of the present study was
to determine the validity of rapidly diagnostic
tests for the early detection of UTIs in patients.
MATERIALS AND METHODS
This was a cross-sectional study
conducted prospectively in Duzce Medical
Faculty Hospital and its clinical laboratories.
One hundred and twenty eight patients who
had UTIs and control group consisted of 128
subjects who had not UTIs were included to
the study. The urine specimens were sent to the
hospital laboratories in sterile containers, and
trained laboratory technologists performed the
tests using standard laboratory procedures.
Urine for culture was refrigerated if not plated
within 10 minutes of receipt. The urine
dipstick, urine white blood cell count/mm3 and
Gram stain tests were performed immediately
on fresh urine. Extra urine was saved by each
laboratory and stored at 2C to 6C, and the
two nonstandard tests (cell count and Gram
stain) were performed daily.
Results of the dipstick test (Multistix
10SG 228, Bayer Diagnostics, Elkhart, IN)
were interpreted visually according to standard
color charts. The leukocyte esterase measu-
rement was read after 2 minutes and recorded
as negative, trace, small (+1), moderate (+2),
or large (+3). The nitrite measurement was
read at 60 seconds and recorded as negative or
positive.
For the WBC count, uncentrifuged
urine was drawn into a Neubauer (Reichert,
Buffalo, NY) hemocytometer by capillary
action. Leukocytes were counted on one side of
the chamber and multiplied by 1.1 to obtain a
total cell count per cubic millimeter.
Quantitative urine cultures and Gram
stain were performed in the hospital
microbiology laboratory. Urine received in
sterile containers was inoculated onto blood
and MacConkey agar plates with a 0.01 mL
calibrated loop, incubated at 35C, and
examined daily for growth for 2 days. Smears
were prepared using 2 drops of uncentrifuged
urine on a slide within the standardized marked
area (1.5 cm in diameter), air-dried, fixed, and
Gram-stained. The average number of bacteria
per 10 oil immersion fields, morphology, and
Gram stain were recorded. A positive urine
culture was defined as growth of a urinary tract
pathogen at 104 colony-forming units per
milliliter (CFU/mL) (3).
Mustafa YILDIRIM ve ark.
Using the quantitative urine culture as
the gold standard (reference test), the
sensitivity, specificity, and positive and
negative predictive values of all the screening
tests were determined and compared. The
sensitivity, specificity, positive predictive
value (PPV), and negative predictive value
(NPV) for the four screening methods were
calculated against the urine culture (reference
method) for the diagnosis of UTI.
RESULTS
Gram staining, microscopic pyuria,
nitrite, and leukocyte esterase test and culture
methods were evaluated for the diagnosis of
the UTI and shown in Table 1. Comparison of
Sensitivity, Specificity, PPV, and NPV values
are shown in Table 2.
DISCUSSION
Several rapid screening tests are used
commonly to make a presumptive diagnosis of
UTI, including dipstick biochemical analysis of
urine for nitrites or leukocyte esterase, as well
as microscopic examination of urine for
formed elements including white blood cells or
bacteria. Numerous studies have been pub-
lished concerning the usefulness of these tests
in diagnosing UTI (4-6).
The urine Gram stain, has been
proposed both as a more sensitive and specific
method for identifying patients with UTI and
as a means of screening for when to have a
urine culture performed (7, 8). A study by
Lockhart and colleagues (9) of 207 patients
with fever found the Gram stain to have a
sensitivity of 94% and specificity of 92%.
Hoberman et al. (8) found 96% sensitivity for
the enhanced urinalysis (urine white blood cell
count/mm3 plus Gram stain) in a similar
sample of 4253 children < 24 months of age,
with a positive urine culture defined as 50
000 CFU/mL. According to the present study,
for predicting a positive urine culture, the
presence of any bacteria on a Gram-stained
urine specimen offers the better combination of
sensitivity and specificity than other tests
evaluated. However, the dipstick test performs
nearly as well, with a slightly lower sensitivity
for the presence of any nitrite or LE (Table 2).
Many bacteria (mainly gram negative)
that cause UTI can metabolize dietary nitrate in
the urine to nitrite. Detection of nitrite in urine
is therefore a valuable indicator of bacterial
invasion of the bladder (10). In a
40
 Dzce Tp Fakltesi Dergisi 2008; 3:39-42

Table1. Comparison of urine culture (reference test) and screening tests for diagnosis of UTI (n =128)
Urine Gram staining Micros. pyuria Nitrite Leukocyte esterase
Culture
Pos Neg Pos Neg Pos Neg Pos Neg
Pos 111 17 41 87 79 49 61 67
(n=128) (86.7%) (13.3%) (32%) (68%) (61.7%) (38.3) (47.7%) (52.3)
Neg 0 128 8 120 4 124 6 122
(n=128) (0.0%) (100%) (6.2%) (93.8%) (3.1%) (96.9%) (4.7%) (95.3%)
resulted in a lower accuracy, compared to the
meta-analyse (11), overall, the sensitivity of test for nitrites, lower predictive values of
the urine dipstick test for nitrites was low (45- positive test results and similar predictive
60% in most situations) with higher levels of values of negative test results (11). In present
specificity (85-98%). The typically low pre- study, the urine dipstick test for leukocyte
test probabilities resulted in high predictie esterase had 47.7% sensitivity, 95.3%
values of negative test resuts. The test for specificity, 91.0 PPV and 64.5% NPV. These
nitrites had its highest accuracy in specific results were similar to literature.
populations such as pregnant women, urology Because Gram stainings cost and
patients and elderly people. Only in the elderly difficulty of performance are much greater
did the test for nitrites reach a high sensitivity, than the for the dipstick test, it is not as
while in pregnant women sensitivity was the attractive as the dipstick test in clinical
lowest. Although statistically not significant, practice. In combination with the cell count, it
the test for nitrites might perform better in comprises the enhanced urinalysis. The cell
asymptomatic patients and in patients who are count alone shows no advantage over dipstick
not on antibiotics. In this study, the urine tests or Gram stain because it had comparable
dipstick test for nitrites had 61.7% sensitivity, sensitivity and is less specific. As a less
96.9% specificity, 95.2 PPV and 71.7% NPV. expensive screen for when to send a urine
Results of the present study were similar to culture and perform a Gram stain (12). The
other studies. urine dipstick test for leukocyte esterase and
nitrite continues to be a low-cost excellent
Table2.Comparison the sensitivity, specificity, screening test for UTI (13). Of all the tests
PPV, and NPV to Gram staining, microscopic studied, it is the only one not requiring Clinical
pyuria, nitrite, and leukocyte esterase in the Laboratory Improvement Amendments
early diagnosis of UTI. certification and can be performed by the
bedside nurse or physician. The strategy of
Test Sensitivity Specificity PPV NPV
urine dipstick and culture tests for patients for
(%) (%) (%) (%)
Gram 86.7 100 100 88.3
whom a UTI is considered is less costly,
staining identifies patients with UTI, and allows one to
Microscopic 32.0 93.7 83.7 58.0 screen which patients should begin
pyuria presumptive treatment.
Nitrite 61.7 96.9 95.2 71.7 In conclusion, according to the present
Leuk. 47.7 95.3 91.0 64.5 study, of the four screening tests, Gram
esterase staining had the highest sensitivity, specificity,
PPV and NPV. Accordingly, Gram stain seems
White cells are normally found in to be useful according to dipstick tests. Gram
urine, but an increase (pyuria) is an indication stain can be recommended highly as a rapid
of inflammatory change. Leukocyte esterase tool to rule out the diagnosis of UTI in both the
measurement has been shown to give an clinical and the laboratory setting.
accurate estimate of the number of leucocytes
present (10). Sensitivity of the urine dipstick Correspond author: Mustafa YILDIRIM
test for leukocyte-esterase was, in general, Faculty of Medicine, Duzce Department of Clinical
Microbiology and Infe. Dis.81620, Duzce, Turkey
slightly higher than for the dipstick test for
E-mail: mustafayildirim4@yahoo.com
nitrites (48-86%), while the specificity was
slightly lower (17-93%). Generally, this

Mustafa YILDIRIM ve ark. 41

 Dzce Tp Fakltesi Dergisi 2008; 3:39-42
REFERENCES
1. Zorc J.J., Kiddoo D.A., Shaw K.N. Diagnosis
and Management of Pediatric Urinary Tract
Infections. Clinical Microbiology Reviews,
Apr., p. 417422, 2005.
2. Kass, E.H. Asymptomatic infections of the
urinary tract. Trans. Assoc. Am. Physicians;
69:5664, 1956.
3. Hellerstein S. Recurrent UTI in children.
Pediatr Infect Dis J. 1: 271281, 1992.
4. Hurlbut T.A., Littenberg B, and the Diagnostic
Technology Assessment Consortium. The
diagnostic accuracy of rapid dipstick tests to
predict urinary tract infection. Am J Clin
Pathol. 96:582588, 1991.
5. Lohr J.A. Use of routine urinalysis in making a
presumptive diagnosis of urinary tract
infection in children. Pediatr Infect Dis J.
10:646650, 1991.
6. Todd J.K. Diagnosis of urinary tract infections.
Pediatr Infect Dis J.1:126131, 1982.
7. Hoberman A., Wald E.R., Penchansky L.,
Reynolds E.A., Young S. Enhanced urinalysis
as a screening test for urinary tract infection.
Pediatrics. 91:11961199, 1993.
8. Hoberman A, Wald E.R., Reynolds E.A.,
Penchansky L., Charron M. Is urine culture
Mustafa YILDIRIM ve ark.
necessary to rule out urinary tract infection in
young febrile children? Pediatr Infect Dis J.
15:304309, 1996.
9. Lockhart G.R., Lewander W.J., Cimini D., et al.
Use of urinary Gram stain for detection of
urinary tract infection in infants. Ann Emerg
Med. 25:3135, 1995.
10. Oreson R., Groschel D.H.M. Leucocyte
esterase activity and nitrite test as a rapid
screen for significant bacteriuria. J Clin
Microbiol 21:840-842, 1985.
11. Deville W., Yzermans J.C., Duijn N.P.,
Bezemer P.D., Windt D. and Bouter L.M. The
urine dipstick test useful to rule out infections.
A meta-analysis of the accuracy. BMC
Urology4:4.2004URLhttp://www.biomedcentr
al.com/1471-2490/4/4.
12. Shaw K.N., McGowan K.L., Gorelick M.H.,
Schwartz J.S. Screening for urinary tract
infection in infants in the emergency
department: which test is best? Pediatrics 101,
1998.URL:http://www.pediatrics.org/cgi/conte
nt/full/101/6/e1
13. Shaw K.N., Hexter D., McGowan K.L.,
Schwartz J.S. Clinical evaluation of a rapid
screening test for urinary tract infections in
children. J Pediatr. 118:733-736, 1991.
42
Background: Urinary tract
infectious (UTI) is the
most common of all
bacterial infections; the
purpose of the present
study was to determine
the validity of rapidly
diagnostic tests for the
early detection of UTIs in
patients. 128 patients
who had UTIs and control
group consisted of 128
subjects who had not UTIs
were included to the
study.
Findings: Urine specimens
obtained from the patients
were evaluated for possible
UTI by Gram staining,
microscopic pyuria, dipstick
(nitrite and leukocyte
esterase), and quantitative
urine culture. Using the
quantitative urine culture as
the gold standard (reference
test), the sensitivity,
specificity, and positive
(PPV) and negative
predictive values (NPV) of
all the screening tests were
determined and compared.
The sensitivity, specificity,
PPV, and NPV for the four
screening methods were
calculated against the urine
culture (reference method)
for the diagnosis of UTI.
Results

Gram staining,
microscopic pyuria,
nitrite, and leukocyte
esterase test and culture
methods were evaluated
for the diagnosis of the
UTI and shown in Table 1.
Comparison of Sensitivity,
Specificity, PPV, and NPV
values are shown in Table
2.
In conclusion, validity
of Gram stain was
found higher
compared to other
rapid diagnostic tests.
 Diagnosis Worksheet
VIABILITY
Was there an independent,
blind comparison with a
reference (gold) standard
of diagnosis?
Was the diagnostic test
evaluated in an appropriate
spectrum of patients (like
those whom it would be used in
practice)?
Was the reference standard
applied regardless of the
diagnostic test result?
Was the test (cluster of tests) Yes. Disebutkan telah ada
validated in a second,
independent group of patients ?
Yes. Gram
staining,microscopic
pyuria,dipstick nitrite and
leucosyite eritrase .
Cant tell. Sampel terdiri dari
128 pasien UTI dan 128 pasien
no UTI . Namun tidak
dijelaskan lebih jauh detail
mengenai subjek penelitian .
No. Tidak disebutkan bahwa
pemeriksaan 4 jenis test
tersebut dilakukan tanpa
terdahulu tahu apakah hasil
kultur urinnya sebagai gold
standard + atau -
penelitian sebelumnya .
IMPORTANT

Sensitivity : 32% probability :

Specificity : 0,835%
93,7 % Post-test Prob
LR+ : 5,08% Pre-test Prob :
50%
LR - : 0,725
PPV :83,67%
NPV : 57,97
Prevalence :
Pretest Odds : 1
Post-test Odds :
5,08%
Post-test
 APPLICABILITY
Is the diagnostic test
available, accurate and
precise in your setting ?
Can you generate a clinically
sensible estimate of your
patients pre-test probability ?
Are the study patients similar
to your own?
Is it unlikely that the disease
possibilities or probabilities
have changed since the
evidence was gathered?
Will the resulting post-test
probabilities affect your
management and help your
patient?
Could it move you across a
test-treatment threshold?
Would your patient be a
willing partner in carrying it
out?
Yes,pewarnaan gram juga
pyuria mikroscopic,tes
dipstick,pemeriksan nitre dan
leukosit esterase merupakan
pemeriksaan yang sudah
tersedia,dapat dilakukan
mudah dan hasilnya dapat
diperoleh dengan cepat
cannot tell,karena
karakteristik penelitian subjek
penelitian tidak diterangkan
dengan jelas .
Yes,dengan pemeriksaan gram
penyakit UTI menjadi lebih
banyak diketahui,sehingga
prevalensi dan probabilitasnya
meningkat .
Yes, test ini dapat membantu
dokter untuk mendiagnosa
sedini mungkin dan dilakukan
tatalaksana sesegara mungkin .
Yes,pewarnaan gram juga
pyuria mikroscopic,tes
dipstick,pemeriksan nitre dan
leukosit esterase merupakan
pemeriksaan noninvasive dan
biayanya tidak mahan
CRP DK 2
KELOMPOK 4

PRIDE PRESENT
 EVALUATION OF REAL-TIME PCR OF
PATIENT PLEURAL EFFUSION FOR DIAGNOSIS
OF TUBERCULOSIS
Rosso et al. BMC Research Notes 2011, 4:279
http://www.biomedcentral.com/1756-0500/4/279

SHORT REPORT Open Access

Evaluation of real-time PCR of patient pleural

effusion for diagnosis of tuberculosis
Franciele Rosso1, Candice T Michelon2, Rosa D Sperhacke3, Mirela Verza2, Liliane Olival4, Marcus B Conde4,
Renata L Guerra4, Arnaldo Zaha1 and Maria LR Rossetti2,5*

Abstract
Background: Pleural tuberculosis (TB) diagnosis often requires invasive procedures such as pleural biopsy. The aim of
this study was to evaluate the role of real-time polymerase chain reaction (PCR) for the IS6110 sequence of M.
tuberculosis in pleural fluid specimens as a rapid and non-invasive test for pleural TB diagnosis.
Findings: For this cross-sectional study, 150 consecutive patients with pleural effusion diagnosed by chest
radiography, who were referred for diagnostic thoracocentesis and pleural biopsy and met eligibility criteria, had a
pleural fluid specimen submitted for real-time PCR testing. Overall, 98 patients had pleural TB and 52 had pleural
effusion secondary to other disease. TB diagnosis was obtained using acid-fast bacilli (AFB) smear or culture for
mycobacteria and/or histopathologic examination in 94 cases and by clinical findings in 4 cases. Sensitivity, specificity,
positive and negative predictive values of PCR testing for pleural TB diagnosis were 42.8% (95% CI 38.4 - 44.8), 94.2%
(95% CI 85.8 - 98.0), 93.3% (95% CI 83.6 - 97.7), and 48.5% (95% CI 44.2 - 50.4), respectively. The real-
time PCR test improved TB detection from 30.6% to 42.9% when compared to AFB smear and culture methods
performed on pleural fluid specimens, although the best sensitivity was achieved by combining the results of
culture and histopathology of pleural tissue specimens.
Conclusion: The real-time PCR test of pleural fluid specimens is a useful and non-invasive additional assay for fast
diagnosis of pleural TB.

Introduction possible complications requiring specialized

Diagnosis of pleural tuberculosis (TB) remains a medical care that is not widely available [6].
chal- lenge due to its nonspecific clinical Nucleic acid ampli- fication assays allow the
presentation and direct detection of M. tubercu- losis in clinical
paucibacillary nature. Conventional methods, specimens within hours of their receipt in the
such as laboratory, providing a potential tool for diagnos-
direct testing for acid-fast bacilli (AFB) and ing paucibacillary forms of TB [7]. Although the
culture of pleural fluid, lack sensitivity (less role of nucleic acid amplification tests has been
t h a n 5% and 40%, respectively) [1-3]. Despite reasonably well defined in p ul mo n a ry
improved detection rates with new methods, tuberculosis, their place in pleurisy evaluation
high pleural fluid levels of adenosine deaminase is not clear [7,8]. Real-time polymer-
(ADA) may be found in other diseases, espe- ase chain reaction (PCR) is a technology that has
cially empyema and parapneumonic effusions, advan- tages over conventional PCR testing,
and there is no established cut-off value for such as the fast availability of results, decreased
measurement of inter- feron-gamma in pleural risk of contamination and quantification of
fluid [4,5]. bacterial load [9].
Therefore, a definitive diagnosis of pleural TB In Brazil, TB has an initial presentation as a
* Correspondence: mrossett@terra.com.br
2still depends
Centro de on demonstration
Desenvolvimento Cientfico e Tecnolgico,of M. tuberculosis
Fundao Estadual de pleural disease in about 9% of adults and is the
or caseous
Produo granulomas
e Pesquisa em Sade - Av. Ipiranga,in5400,
pleural biopsy,Porto
ZIP code: 90610-000, an main cause of pleural effusion, corresponding
Alegre,
invasiveBrazil method with to over 50% of cases [10,11]. In this study, we
Full list of author information is available at the end of the article
evaluated the role of a real- time PCR assay for
This isIS6110
2011 Rosso et al.; licensee BioMed Central Ltd.the an open access article distributed
sequence M. tubercu-
of under the terms of thelosis
Creative
inCommons
pleural
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
fluid
any medium, provided the original work is properly cited. specimens to evaluate the efficiency and
practicality of the test for diagnosing pleural TB.
Rosso et al. BMC Research Notes 2011, 4:279 Page 2 of 6
http://www.biomedcentral.com/1756-0500/4/279

Materials and methods DNA extraction

Study population DNA was extracted from clinical specimens
From January 2003 to July 2005, all patients according to previously described methodology
presenting with pleural effusion on chest with modifications [13]. Briefly, pleural fluid
radiography referred by their clinicians to the samples were concentrated by centrifugation,
Pulmonary Service for Diagnostic Methods, and 100 L of lysis buffer (8 M guanidine
Thoracic Diseases Institute/Clementino Fraga hydrochloride, 0.08 M Tris HCl, 0.04 M EDTA and
Filho University Hospital Federal University of Rio 2% Tri t o n X-100) was added to the pellet and
de Janeiro for diagnostic thoracocentesis were vortexed. Lysed samples were boiled for 10 min
eligible for the study. Inclusion criteria were age and centrifuged, and the resultant
18 years old, absence of known history or supernata nts were transferred to a tube
clinical or radiographic evi- dence of renal, containing 2.5 L of silica suspension. After centri-
cardiac or liver failure, and no known diagnosis fugation, the supernatant was removed, and the
of cancer or TB at the moment of enrollment. All pellet was washed twice with washing buffer (8
included patients signed a consent form and M guanidine hydrochloride and 0.08 M Tris HCl)
were submitted to an interview, physical and once with 70% ethanol. The tubes were
examination, a n d thoracocentesis followed by dried with closed lids at 56C for 10 min and
pleural biopsy with Copes needle. Patients with open lids at room temperature for 5 min.
were excluded f rom the study if t h e diagnostic Elution buffer (1 TE) was added, and the tubes
p ro ce d u re s were n o t c o mp l et e d or if t h e were homogenized and incubated for 10 min at
pleural effusion etiology remained undetermined 56C. After centrifugation, the supernatant was
at the end of the diagnostic investigation. Pleural collected for further experimentation. An
effusion was deemed limited if it affected less extraction negative control (tube containing no
than one third of the hemithorax, moderate if it organisms) and an extraction posi- tive control
affected between one third and two thirds of (dilution of a commercial M. tuberculosis H37Rv
the hemithorax, and extensive if it affected reference
Real-time PCRstrain
assay ATCC27294 containing 50 col-
more than two thirds of the hemithorax. This Amplification
ony forming units)reactions were performed
were processed in each setwith
of
study was approved by the Research Ethics primers INS-1/INS-2 as previously described,
DNA extractions.
Committees
Clinical specimens of the Federal University of Rio de which generated a 245-base pair (bp)
Two
Janeiro pleural
and biopsy
of the fragments and an aliquot
State Foundation of Healthof amplification product [14]. PCR was performed
pleural
Research and fluid Production
were stained using
of Porto the Brazil.
Alegre, Ziehl- using the ABI Prism 7500 system (Applied
Neelsen method and cultured in Lwenstein Biosystems, Foster City, California), and the
Jensen and Sabouraud medium following amplified product was detected with SYBR Green
standard protocols [12]. All specimens with I dye. The reac- tion was op ti mi zed to ob tai n
positive mycobacteria culture results were tested the best amplification kinetics. The final
using biochemical me t h o d s to distinguish M. reaction volume (20 L) contained a mixture of 5
tuberculosis f ro m o t h e r nontuberculous L of extracted target DNA, 10 L (0.8 ) of SYBR
myc obact eri a . T h r e e pleural biopsy fragments Green PCR Master Mix (Applied Biosystems),
were submitted to histopatho- logical primers INS-1/INS-2 (5 pmol) and water. After 2
examination after hematoxylin and eosin stain- min of incubati on at 50C a n d 10 min at 95C,
ing, and an aliquot of pleural fluid was stained 40 cycles of amplification were performed with a
using the Papanicolaou me t h o d . Immediately thermal profile of 95C for 15 sec and 68 C for 1
after pleural fluid collection, an aliquot of 500 min. A final cycle (95C for 15 sec, 60C for 1
L was f rozen a t min, 95C for 15 sec) was added for the
-20 C a n d se n t to t h e State F o u n d a t i o n of dissociation curve. The amplification and disso-
H e a l t hTB definition
Pleural Research and Production of Porto ciation curves were analyzed with 7500 System
A diagnosis
Alegre of pleural
for real-time PCRTBtesting.
was made using the Software (Applied Biosystems) with threshold and
following tests: AFB smear, the growth of M. baseline analy- sis a ut o def i ne d by t he software.
tuberculosis (MTB) in pleural fluid or pleural biopsy A negative c o n t ro l (master mix with water
tissue, the presence of granulomatous instead of template DNA) and a positive control
inflammation in histopathologic study of the (pAMP-1 plasmid containing an IS6110 245-bp
pleural biopsy tissue, or the resolution of clinical inserted fragment) were included for each set of
and radiograph abnormalities after six months of PCRs. The sample was defined as positive when it
stan- dard antituberculosis treatment (clinical had a detectable cycle threshold (Ct) and the
diagnosis). melting tempera- t u re (Tm) was the sa me as
for th e positive c o n t ro l (88.8C). O u r g ro u p
has o b t ai n ed successful results using the
IS6110 region to detect M. tuberculosis
Rosso et al. BMC Research Notes 2011, 4:279 Page 3 of 6
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complex DNA, and thus this region was chosen Table 1 Demographic characteristics of 150 patients with
for the study [15,16]. pleural effusion.
TB non-TB p value
Presence of inhibitors (n = 98) (n = 52)
For all specimens with negative results, an internal Mean age (years) 36 60 <0.01
con-
trol (pAMP-1 plasmid containing an exogenous Gender [n (%)] 0.40
600-bp
f ragment obtained f rom the pUC13 plasmid Male 67 (68.4) 32 (61.5)
digested
with EcoRI enzyme) was added to the PCR (10 Female 31 (31.6) 20 (38.5)
fg/L,
the detection limit concentration) to evaluate the Race [n (%)] 0.12
pre-
sence of inhibitors. This sequence was amplified White 45 (46.4) 31 (59.6)
as
described by Cortez-Herrera et al [17] using the Non-white 52 (53.6) 21 (40.4)
same
primers INS-1/INS-2 to generate a 664 bp Unknown 1 0
amplicon. Pleural effusion extension*&[n (%)] 0.03
PCR products were analyzed using electrophoresis
on a
1.5% agarose gel stained with ethidium bromide Limited 25 (25.8) 18 (35.3)
and
visualized with an ultra-violet illuminator to allow Moderate 59 (60.8) 20 (39.2)
the
identification of the amplicon size [14]. Extensive 13 (13.4) 13 (25.5)
Unknown
* Chest radiographic aspects 1 1
Results
Statistical analysis
& Limited: pleural effusion affecting less than one third of the hemithorax
Pleural effusion location* [n (%)] 0.63
Moderate: pleural effusion affecting between one third and two thirds of the
A total of 158 patients were enrolled in the study. Unilateral
The Chi-squared test was used to compare hemithorax Extensive: pleural effusion affecting93more
(95.9)
than two thirds of48
the(94.1)
Eight subjects were excluded because they did
frequencies hemithorax.
not complete
between groups, and Fishers exact test was used Bilateral 4 (4.1) 3 (5.9)
all
when diagnostic procedures . A mo n g the
remaining
the absolute150 number patients,
compared98 (65.3%) had
was less thanpleural
5. Unknown
value (NPV) were 42.8% (42/98) 1 1
and 48.5%
Sensitiv-
TB, and 52 (34.7%) had (49/101), respectively, while specificity and
ity, specificity,
pleural effusion positive predictive
secondary value disease
to other (PPV), (33 Parenchyma predictive
positive alteration* [n (%)]value (PPV) were 94.2% 0.22
negative
neoplasia, (49/52) a n d 93.3% (42/45), respectively.
predictive
8 unspecifiedvalue (NPV) and 95%
inflammatory confidence
disease, interval
7 pneumonia, No 65 (67) 29 (56.9)
Pleural fluid o b ta i ne d fro m f our TB pa tien ts
(CI)
3 ede- migenic syndrome, and 1 systemic lupus repeatedly d e m o n s t r a t e d inhibition in t h e
were calculated. Analysis Yes 32 (33) 22 (43.1)
erythematosus). Among was the perf ormedwith
patients using TB amplification reaction; therefore, inhibition results
SPSS
diagnosis, all had a nega- tive HIV test. A cough were only included in the denominator
Unknown 1 1 for test
12.0 software (SPSS Ins. Chicago, IL, USA).
was described in 69.4% (68/98), thoracic pain in accuracy cal- culations. Three of the four TB
75.5% (74/98), fever in 82.6% (81/98) and patients with inhibited PCR results in pleural fluid
dyspnea in 20.4% (20/98) of the TB patients. (75%) were male, white and
Demo- graphic characteristics of the patients presented with unilateral pleural effusion without
are presented in Table 1. asso- ciated pulmonary parenchyma alteration
Among the pleural TB cases, 4 (4%) were on chest radio- graphy. All of the m had positive
diagnosed based o n the resol utio n of clinical cultures from pleural fluid specimens and
a n d ra d i o g ra p h abnorma liti es after six d e mo n s t ra t i o n of granuloma in pleural biopsy.
m o n t h s of s t a n d a rd a n t i-TB treat ment , a nd 94 Among the 98 TB patients, pleural fluid analysis
(96%) had diagnosis confirmed by laboratory pro- vided positive results in a total of 60
methods. The most frequent finding was the patients (61.2%), with 18 specimens positive by
demonstration of granuloma in pleural biopsy culture only, 30 speci- mens positive by PCR
(88.8%; 87/98 patients). AFB smear in pleural only, and 12 specimens positive in both
fluid and pleura tissue specimens contributed methods. Therefore, TB was detected in pleural
one case each (1%). Both also had cultures fluid by culture in 30.6% (30/98) of the patients
positive for MTB. Th e culture was positive in and by PCR testing in 42.9% (42/98) of the
pleural fluid and pleural tissue specimens in patients. Pleural biopsy analysis provided
30.6% (30/98) a n d 64.3% (63/98) of the positive results in a total of 90 patients (91.8%),
patients, respectively. with 3 specimens positive by culture
The accuracy of the PCR test for pleural TB
Rosso et al. BMC Research Notes 2011, 4:279 Page 4 of 6
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Table 2 Accuracy of real-time PCR testing in pleural fluid for TB diagnosis

PCR TB non-TB Sens (%) Spec (%) PPV (%) NPV (%)
(95% CI) (95% CI) (95% CI) (95% CI)
Positive (n = 45) 42 3 42.8 93.3
Negative (n = 101) 52 49 (38.4 - 44.8) 94.2 (83.6 - 97.7) 48.5
Inhibited (n = 4) 4 0 (85.8 - 98.0) (44.2 - 50.4)
Total (n = 150) 98 52
PCR test accuracy was calculated using the inhibition results only in the denominator.
PCR: Polymerase chain-reaction; TB: tuberculosis; Sens: sensitivity; Spec: specificity; PPV: positive predictive value; NPV: negative predictive value; CI: confidence
interval

only, 27 positive by histopathology only O u r study evaluated the real-time PCR

(presence of granuloma), and 60 specimens testing in pleural fluid, which does not require
positive in both methods. Therefore, TB in pleural an invasive proce- dure for specimen collection,
tissue was detected by the cul- ture in 64.3% a fact that should be con- sidered in a setting
(63/98) of the patients and by histopatho- logic where pleural biopsy is not widely available. Our
e x a mi na t io n in 88.8% (87/98) of the patients . results demonstrated real-time PCR sensi- tivity
Table 3 shows the results of real-time PCR, of 42.8% when TB cases are defined based on
culture for mycobacteria, and histopathology clin- ical criteria (with or without laboratory
performed in pleural fluid and pleura tissue confirmation). Although we have f ound higher
specimens of the 98 TB patients, including those PCR sensitivity t h a n other studies evaluating
Discussion
with solely clinical diagnoses. similar clinical specimens and target nucleic acid
Molecular methods have been shown to be sequences for amplification (IS6110) [21-24], our
importan t tools for confirming pleural TB results corroborate the suboptimal sensitiv- ity as
diagnosis because of the main limitation of PCR testing for replacing
their high specificity (>90%), but they are non- conventional diagnostic tests for diagnosis of
invasive pleural TB. Pleural fluid specimens in 4 patients
and low risk, unlike the gold-standard method of with positive cul- ture for TB and demonstration
pleural biopsy [8]. Nevertheless, the sensitivity of of granuloma in pleural biopsy repeatedly
PCR testing has been varied, ranging from 37% - demonstrated inhibition of the nucleic acid
77% across studies, mainly due to different amplification reaction. Nonetheless, even
target nucleic acid sequences and the without these substances and the 4 false
amplification methods used [8]. Low sensitivity negative results, test sensitivity would not be
values can be explained by the low bacillary load much improved, from 42.8% to 46.9%. Three false
and the presence of substances that inhibit positive samples were observed. This could be
amplification in pleural fluid [18]. Some studies potentially due to cross-contamination th a t
comparing conventional PCR methods with real- could have o cc u rred during sp eci men
time PCR testing have demon- strated higher processing, although the guidelines for
sensitivities for the latter (difference of about diagnostic methods were fol- lowed. The
30% with real-time PCR sensitivity up to 68%) guidelines include unidirectional work flow
when both tests are performed in the same comprising the physical separation of specimen
specimens, especially in pleural biopsies [19,20]. proces- sing and reagent preparation and the
In addition, real- time PCR has the advantages use of positive and negative con trols du ring
of short turn-around time, lower risk of sample D N A extraction a n d PCR amplification [16,25].
Table 3 Results of real-time
contamination, PCR to
and ability testing, culture
quan- tify for MTB (pleural/tissue specimen), and histopathologic study
bacterial Real-time PCR of pleural fluid was able to
(presence of granulomatous inflammation in pleural tissue) among 98* patients with pleural TB diagnosis
load, raising interest for its use in pleural detect TB in 30 patients with negative AFB
Culture for MTB in pleural Culture for MTB in pleural Histopathologic study of pleural
effusion tests [9]. fluid smears
tissue and culture in pleural
tissue fluid a n d in 3 of 4
positive negative positive patients
negative with pleural
positive TB negative Total
Real-time PCR
positive 12 30 27 15 36 6 42 (42.9%)
negative/inhibition 18 38 36 20 51 5
Total 30 (30.6%) 63 (64.3%) 87 (88.8%) 98 (100%)
PCR: Polymerase chain-reaction. MTB: Mycobacterium tuberculosis. *Four patients with pleural TB had a diagnosis based only on clinical criteria, and three of them had
positive PCR results.
Rosso et al. BMC Research Notes 2011, 4:279 Page 5 of 6
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diagnosed based on clinical criteria alone. study and drafted the manuscript. LO: participated in the design of the study
Therefore, PCR testing was responsible for an and statistical analysis. MBC: helped in the acquisition of funding, participated
in the analysis and interpretation of the data and revised the manuscript. RLG:
improvement in TB diagnosis from 30.6% to participated in statistical analysis, interpretation of data and drafted the
42.9% when compared to AFB smear and manuscript. AZ: drafted and revised the manuscript. MLRR: helped in the
culture methods performed on pleural fluid acquisition of funding, participated in analysis and interpretation of data and
revised the manuscript. Participated in the general supervision of the research
specimens. In addition, the combined results of group. All authors read and approved the final manuscript.
culture and PCR in pleural fluid specimens
provided an even bet ter rate of pleural TB
Competing interests
detection of 61.2% (60/98). Although the best The authors declare that they have no competing interests.
sensitivity was achieved by combining
Received: 8 March 2011 Accepted: 5 August 2011
the results of pleura tissue culture and
Published: 5 August 2011
histopathology (91.8%, 90/98), the prompt
confirmation of tuberculous pleuritis in about 43% References
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doi:10.1186/1756-0500-4-279
Cite this article as: Rosso et al.: Evaluation of real-time PCR of patient
pleural effusion for diagnosis of tuberculosis. BMC Research Notes 2011
4:279.

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 Background
Pleural tuberculosis diagnosis
often requires invasive
procedures such as pleural
biopsy.
Objective
To evaluate the role of real-
time polymerase chain reaction
(PCR) for the IS6110 sequence
of M. tuberculosis in pleural
fluid specimens as a rapid and
non-invasive test for pleural TB
diagnosis.
 Method
Cross-sectional study
Sample : 150 consecutive
patients with pleural effusion.
Overall, 98 patients had pleural
TB and 52 had pleural effusion
secondary to other disease.
Criteria
Age > 18 years old
Absence of known history or
clinical or radiographic
evidence of renal, cardiac or
liver failure and no known
diagnosis of cancer or TB at the
moment of enrollment.
Results
PCR TB NON SEN SPE PPV NPV
-TB S C
+ (n=45) 42 3 42.8 93.3
- (n=101) 52 49 94.2 48.
5
Inhibited (n=4) 4 0
Total (n=150) 98 52

PCR test accuracy was calculated using

the inhibition results
only in the denominator.
 Conclusion
The real-time PCR test of pleural fluid
specimens is an useful and non-invasive
additional assay for fast diagnosis of
pleural TB.
 Diagnosis Worksheet
VIABILITY
Was there an independent,
blind comparison with a
reference (gold) standard
of diagnosis?
Yes. The absence of known
history of clinical or
radiographic evidence of
renal, cardiac, or liver failure
and no known diagnosis of
cancer or TB at the moment
of enrollment.

Was the diagnostic test Yes. Because all patients

evaluated in an appropriate presenting with pleural
spectrum of patients (like effusion on chest radiography.
those whom it would be used in
practice)?

Was the reference standard Yes. Because there are

applied regardless of the negative controls and positive
diagnostic test result? controls that were included for
each sets of PCRs.

Was the test (cluster of tests) Yes.

validated in a second,
independent group of patients ?
Are the results of this diagnostic study
important?

PCR Totals
Present Absent
C. Positive 42 3 45
Diagnos
Negativ 56 49 105
is of
e
Culture
s and
HP
98 52 150
IMPORTANT

Sensitivity : 13,912
42,85% Post-test
Specificity : probability :
94,23% 93,3%
LR+ : 7,4 Post-test Prob
LR - : 0,6 Pre-test Prob :
28%
PPV : 93,33%
NPV : 48,5%
Prevalence :
65,3%
Pretest Odds :
1,88
Post-test Odds :
 APPLICABILITY
Is the diagnostic test
available, accurate and
precise in your setting ?
Can you generate a clinically
sensible estimate of your
patients pre-test probability ?
Are the study patients similar
to your own?
Is it unlikely that the disease
possibilities or probabilities
have changed since the
evidence was gathered?
Will the resulting post-test
probabilities affect your
management and help your
patient?
Could it move you across a
test-treatment threshold?
Would your patient be a
willing partner in carrying it
out?
Would the consequences of the Yes.
No. Because pleural fluid
specimens in 4 patients with
positive culture for TB and
demonstration of granuloma
in pleural biopsy repeatedly
demonstrated inhibition of
the nucleic acid amplification.
Yes
No. It is likely to be changed
since the difference between
post-test probability and pre-
test probability more than 20%
Yes, because rapid diagnosis
and initiation of chemotherapy
are essential to prevent
secondary fibrothorax and to
avoid subsequent pulmonary or
extrapulmonary TB
development.
Yes, since its non-invasive
and not really different with
biopsy procedure.
Thank you