This study analyzed the diagnostic yield of sputum microbiological analysis for pulmonary tuberculosis (TB) over 10 years in Portugal. Three sputum samples were collected from 694 patients suspected of having pulmonary TB and analyzed with smear microscopy, culture, and nucleic acid testing. Sensitivity increased with additional sputum samples but was still low overall. While smear microscopy provided rapid results, culture remained the gold standard for diagnosis. The study highlights the need for improved diagnostic tools to help control the global TB burden.

1. JOURNAL CLUB
PREPARED BY
ARPITA CHANDRA

2. DIAGNOSTIC YEILD OF SPUTUM MICROBIOLOGICAL
ANALYSIS IN THE DIAGNOSIS OF PULMONARY
TUBERCULOSIS IN A PERIOD OF 10 YEARS.
PORTUGUESE JOURNAL
OF PULMONOLOGY
AUTHORS- Ana Tavares e Castro,
Mariana Mendes,Sara Freitas,
Paulo Cravo Roxo.
DEPARTMENT- Pulmonology Diagnostic center of
Coimbra,Portugal.
PUBLSHED IN- September-5,2014
vol-,2173-5115/© 2014 Sociedade Portuguesa de
Pneumologia

3. CONTENT
 Introduction
 Aim
 Material & Method
 Results
 Discussion
 Conclusion
 Acknowledgement
 References
KEYWORDS
Tuberculosis,
Sputum,
Microscopy,
Mycobacterium
tuberculosis

4. INTRODUCTION
Tuberculosis still associated with a high global
burden,estimated 8.6 million new cases and 1.3
million deaths in 2012 (most of poor countries).
Early diagnosis and immediate treatment are
essential to cure this airborne infectious disease
and prevent transmission in the community.

5. When pulmonary Tb is suspected-
 Smear microscopy
 Culture
 Nucleic acid amplification
testing should be done.
 laboratory analysis should be performed on
at least three sputum specimens.
 Culture identification is still the gold
standard.
 Slow growing organism.
 Solid media-(4-8 weeks)

6. In developing countries-
Smear microscopy
 Most commonly use
 Simple technique
 Identifies acid fast bacilli (low cost)
 High positive predictive value.
IN 2009 WHO recomended –
 Bright field microscopy should be replaced by
sensitive fluorescent light-emitting diode
(LED)microscopy.
 Nowadays, many countries, including Portugal,
use fluorescent microscopy on a routine basis.

7. Molecular testing-
Recent diagnostic instruments,
that can be used to simultaneously test for pulmonary
TB with higher sensitivity than sputum smear
microscopy and which could replace conventional
culture-based drug susceptibility testing.
At the Present time most widely used-
-ZN smear microscopy,
-solid or liquid medium culture.

8. AIM-
To analyze the sensitivities of these
conventional methods in a Portuguese
public health department.

9. MATERIAL AND METHODS
Patient selection-
A total of 694 patients were enrolled in this study.
Sample collection-3 sputum samples from each
patient collected in consecutive mornings.
Included- Suspected pulmonay TB.
Excluded- Non pulmonary or pleural TB, Latent
TB infection were excluded.

10. ●Patients were diagnosed with LTBI-
no symptoms or signs of the disease
except for a TST positive or a positive
interferon gamma release assays (IGRA)
test.
Tuberculin skin test-

11. Microbiology technique-
Sputum collection
 It was done at home immediately after
waking and washing the mouth with water,
avoiding toothpaste or antiseptic solution.
 Samples were collected to a sterile
container three days in a row, placed
inside a thermal bag at low temperature in
the fridge, avoiding overgrowth of other
bacteria, and delivered to the laboratory in
the fourth day.

12. Decontamination procedure-
 The N-acetyl-l-cysteine-sodium hydroxide(NALC-
NaOH) method (BBL® MycoPrep Specimen
Digestion and Decontamination Kit) prior to culture
analysis.
 prepared contiontrated or Indirect smears.
Microscopic staining technique-
 Ziehl-nehlson stain (TB Stain kit ZN).
 Two smears(Direct,Indirect) from each sample.
 Smears observed on optic microscope with oil
immersion lens.

13. Culture -
 sputum samples were inoculated on modified
Middlebrook 7H9 broth (BBL-MGIT).
 For the detection and recovery of mycobacteria
using the BACTEC-MGIT960 System.
 This incubator executes continuous monitoring
until there is a positive end to the testing
protocol.
 An instrument positive tube contains
approximately 105-106 colony-forming units per
milliliter (CFU/mL), detected by fluorescence
analysis.
 Positive culture were confirmed by smear
microscopy.

14. Middlebrook 7H9 broth BACTEC MGIT 960 System

15. LJ media-
 Solid egg based medium.
 Used for isolation & cultivation of MTB.
 Using a Jouan Incubator at 35°C upto 8 week.

16. Species Identification-
 A DNA probe test for in vitro routine (INNO-
LiPA MYCOBACTERYA v2 assay) was used
for the detection and identification of the genus
Mycobacterium and 16 different mycobacterial
species from solid culture or, if negative, from
liquid culture.
 Based on neucleotide differences in the
16S-23S rRNA.

17. Antibiotic senstivity testing
 Streptomycin, isoniazid, rifampicin and
ethambutol susceptibility testing were performed
according to the Method of Proportion (MOP)
procedure used with a semi-automated system
(BACTEC MGIT960 System).
Statistical analysis-
 Data analysis by SPSS Statistics.
 Sensitivity, specificity, positive and negative pre-
dictive measures were calculated for each and all
sputum sample analysis

18. RESULT-
Table-1 Demographic and clinical characterstics of patients
suspected of pulmonary tuberculosis.
Variable
Age
GENDER
Male
Female
BIRTH ORIGIN
Pourtugal
African
East Europe
South America
CRITERIA FOR TB SUSPICION
Passive screening
symptoms
Contact screening
Protocolized sceening
Other
PULMONARY TB
No
Yes
SPUTUM MYCOBACTERIA
MTB
M.avium
M.fortuitum
M. gordonae
M. kansasii
M. genus
No.
48.5 ± 18.6
452
242
672
10
9
3
631
29
27
7
409
285
180
9
2
2
1
1
%
65
35
97
1
1
0
91%
4
4
1
59
41
91
5
1
1
1
1

19. TABLE-2 Clinical characterstics of patients with pulmonary tuberculosis.
DIAGNOSIS
CONFIRMATION TECH.
Sputum analysis
Bronchial aspirate
Bronchoalveolar
lavage
Lung biopsy
RADIOLOGICAL PATTERN
Non cavitated
Cavitation
Normal
Unknown
COMORBIDITIES
No Comorbidities
Alcoholism
Diabetes mellitus
Neoplastic disease
IV drug abuse
HIV
Chronic liver disease
Chronic renal disease
HOUSING STATUS
Proper house
Social com. Resident
Prison inmate
Homeless
NO.
180
61
28
16
155
99
21
10
205
24
18
14
12
10
7
2
263
18
6
2
%
63
21
10
6
35
35
7
4
72
8
6
5
4
4
2
1
92
6
2
1%

20. TABLE-3 Diagnostic yield of sputum analysis submitted to the PDC
Laboratory from pateints suspected of pulmonary TB.
ONE SPECIMEN
Direct smear
Con. Smear
LJ medium
MGIT
Cumulative ana
TWO SPECIMEN
Direct smear
Con. Smear
LJ medium
MGIT
Cumulative ana
THREE SPECIMEN
Direct smear
Con. Smear
LJ medium
MGIT
Cumulative ana.
True
positive
result
55
70
95
125
146
58
79
108
147
170
64
82
119
158
180
PPV,%
100
97.2
96.3
93.3
93.6
98.3
95.3
95.6
90.7
91.4
98.5
95.3
95.2
90.8
91.4
NPV,%
64
65.4
68.1
71.4
74.2
64.3
66.3
69.5
74.1
77.4
64.9
66.6
70.8
75.6
78.9
Sensitivity%
19.3
24.6
33.3
43.9
51.2
24.4
27.7
37.9
51.6
59.6
22.5
28.8
41.8
55.4
63.2
Specificity%
100%
99.5
99.3
97.8
97.6
99.8
99%
98.8
96.3
96.1
99.8
99
98.5
96.1
95.8%

21. Discussion
 The microscopic staining technique-
standard procedure,
because it is swift, cheap and has a high
positive predictive value.
 On the other-hand,
culture remains the gold-standard .

22. In this study,
●Two-specimen examination senstivity in 8.4% but
the third-specimen added only 3.5% to sensitivity
rates.
---As this has been correspondingly included,
WHO recommendation- Same day 2 specimen.
●Sputum microscopy was performed with traditional
ZN microscopy but fluorescent LED microscopy
could have provided higher sensitivity rates.
●The fact that the WHO recommends a switch to
this more sensitive procedure, this technique was not
available at the PDC.
Conflicts of interest- NO

23. CONCLUSION-
 This study showed an incremental yeild
with more than one sputum sample.
 However overall sensitivity low,
suggesting a need for new diagnostic
strategies and novel and better diagnostic
tools.

24. Acknowledgement-
 This work was done by Pulmonology
Unit, Centro Hospitalar Cova da
Beira,Covilha, portugal.
 This research did not receive any specific
grant from funding agencies.
Ethical disclosures-
No experiments were performed on humans
or animals for this study.

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