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FLUORESCENCE MICROSCOPY (FM) for the detection of tuberculosis (TB) has been in use since the
mid-1940s and is the method of choice for large laboratories in industrialised nations.1,2 A systematic review has shown that FM is on average 10% more
sensitive than conventional light microscopy, has comparable specificity and takes significantly less time to
read smears.3,4 The main barrier to the widespread
implementation of FM has traditionally been the cost
of fluorescent microscopes and their maintenance.5
Light emitting diode (LED) fluorescent microscopes
have been identified as an alternative to conventional
FM. Compared to mercury vapour fluorescent microscopes, LED microscopes are less expensive, have lower
maintenance requirements, require less power and are
able to run on batteries.57 LED bulbs have a very long
half-life, there is no ultraviolet light production and
they are reported to perform equally well without a
darkroom. These qualities make them feasible for use
in low-resource settings, and recent World Health
Organization (WHO) policy has recommended that
Correspondence to: Camilla Rodrigues, P D Hinduja National Hospital and Medical Research Centre, Veer Savarkar Marg,
Mahim (West) Mumbai, India. Tel: (+91) 22 2444 7795. Fax: (+91) 22 2444 9151/2318. e-mail: dr_crodrigues@hinduja
hospital.com
Article submitted 30 April 2010. Final version accepted 29 September 2010.
484
RESULTS
During the 6-month study period, 3729 clinical specimens (2781 pulmonary and 948 extra-pulmonary)
from 2306 patients were received for smear microscopy. Of these, 2275 specimens (1358 pulmonary and
917 extra-pulmonary) were processed for mycobacterial culture and were included in our analysis.
Among the pulmonary specimens, 1050 (91%) were
sputum and the remaining 308 (9%) included bronchoalveolar lavage (BAL), bronchial washings, tracheal secretions and other non-sputum specimens from
the respiratory tract. Extra-pulmonary specimens included abscesses (n = 296), biopsy/aspirates (n =
247), lymph nodes (n = 140), bone marrow (n = 3),
specimens from the gastro-intestinal system (n = 43),
the genitourinary system (n = 7), urine (n = 70), adrenal (n = 2), CSF (n = 35), joint (n = 17), thoracic
Table 1
485
Pulmonary
ZN
Direct
Concentrated
LED1
Direct
Concentrated
LED2
Direct
Concentrated
Mean LED
Direct
Concentrated
Extra-pulmonary
ZN
Direct
Concentrated
LED1
Direct
Concentrated
LED2
Direct
Concentrated
Mean LED
Direct
Concentrated
Smear+/culture+
n/N
Sensitivity (95%CI)
Smear/culture
n/N
617/746
531/743
0.827 (0.7980.854)*
0.715 (0.6810.747)*
586/611
593/611
0.959 (0.9400.973)
0.971 (0.9550.983)
555/746
488/743
0.744 (0.7110.775)
0.657 (0.6210.775)
577/611
583/611
0.944 (0.9230.961)
0.954 (0.9340.969)
613/746
556/743
0.822 (0.7920.849)*
0.748 (0.7150.779)*
546/611
554/611
0.894 (0.8660.917)
0.907 (0.8810.928)
584/746
522/743
0.783 (0.7510.812)*
0.703 (0.6680.735)*
561.5/611
569/611
0.920 (0.8950.940)
0.931 (0.9080.950)
90/241
48/228
0.373 (0.3120.438)*
0.211 (0.1590.247)*
578/676
550/584
0.855 (0.8260.881)*
0.942 (0.9100.969)*
54/241
42/228
0.224 (0.1730.282)
0.184 (0.1350.242)
634/676
548/584
0.938 (0.9170.955)
0.938 (0.9030.969)
110/241
72/228
0.456 (0.3920.522)*
0.316 (0.2550.382)*
566/676
530/584
0.837 (0.8070.864)*
0.908 (0.8710.941)*
82/241
57/228
0.340 (0.2810.404)
0.250 (0.1950.314)
600/676
539/584
0.888 (0.8610.910)
0.923 (0.8870.955)
Specicity (95%CI)
* Indicates statistical signicance between direct and concentrated smear readings, based on non-overlapping 95%CIs.
ZN = Ziehl-Neelsen; LED = light emitting diode; FM = uorescent microscopy; + = positive; = negative; CI = condence interval.
ZN
Direct
Concentrated
LED1
Direct
Concentrated
LED2
Direct
Concentrated
Mean LED
Direct
Concentrated
Mean (95%CI)
Median [IQR]
2.48 (2.382.59)
1.11 (1.051.17)
2.40 [2.162.89]
1.05 [0.941.24]
1.08 (0.901.26)
0.42 (0.390.45)
0.90 [0.811.17]
0.41 [0.340.51]
1.73 (1.631.84)
0.74 (0.650.83)
1.71 [1.392.04]
0.66 [0.560.81]
1.41 (1.301.51)
0.58 (0.540.63)
1.39 [1.131.58]
0.53 [0.470.63]
* Estimates for direct smears measured over 115 days; estimates for concentrated smears measured over 72 days with ZN and 62 days with LED.
CI = condence interval; IQR = interquartile range; ZN = Ziehl-Neelsen;
LED = light emitting diode.
486
Table 3
Inter-rater agreement
Comparison
Direct
LED1LED2
LED1ZN
LED2ZN
Concentrated
LED1LED2
LED1ZN
LED2ZN
Agreement
%
Kappa*
77.7
75.8
75.6
0.92
0.87
0.90
82.6
74.5
70.1
0.91
0.75
0.77
DISCUSSION
The goal of this study was to implement and evaluate
LED microscopy for the direct detection of AFB in a
high-incidence setting. Similar to many high-incidence
laboratories, our technologists are highly skilled and
experienced with ZN microscopy, but are unfamiliar with fluorescent microscopy. Despite the wellestablished benefits of FM, it has not been adopted
widely in low- and middle-income countries, mainly
because of the high maintenance equipment and upfront costs associated with traditional mercury-vapour
fluorescent microscopes. Through the introduction of
LED technology, FM has now become a feasible modality for TB diagnosis.11 Combined with the recent
WHO recommendations for LED microscopy to replace conventional FM and be phased in as an alternative for conventional ZN microscopy, it is anticipated that many TB laboratories currently using ZN
microscopy will consider switching to LED FM in the
near future.8
Here we report our experience in introducing LED
microscopy, and our attempts to validate its performance in a real-world setting. Through this process,
many pitfalls were encountered and lessons were
learned that are often unappreciated or unreported
487
CONCLUSIONS
LED FM has significant benefits over ZN microscopy
and conventional FM. To realise these benefits, careful implementation is needed when introducing LED
FM into laboratories unfamiliar with the technique.
488
Ensuring adequate training, detailed standard operating procedures and quality assurance programmes
will maximise accuracy. While evidence from implementation studies is very valuable, internal validation
of diagnostic devices through operational research
can be difficult and presents challenges often not encountered in tightly controlled research environments.
Acknowledgements
The authors thank Parmanand Deepchand Hinduja National Hospital and Medical Research Centre for their encouragement and
support. This study was supported by grants from the Canadian
Institutes of Health Research (CIHR MOP-89918) and European
Commission (TBSusgent, EU-FP7). MP was supported by a CIHR
New Investigator Award, and JM was supported by a Quebec Respiratory Health Training Fellowship. None of these agencies were
involved in the conduct or review of this study.
References
1 Lempert H. Fluorescence microscopy in the detection of tubercle bacilli. Lancet 1944; 244: 818822.
2 Marais B J, Brittle W, Painczyk K, et al. Use of light-emitting
diode fluorescence microscopy to detect acid-fast bacilli in sputum. Clin Infect Dis 2008; 47: 203207.
3 Steingart K R, Henry M, Ng V, et al. Fluorescence versus conventional sputum smear microscopy for tuberculosis: a systematic review. Lancet Infect Dis 2006; 6: 570581.
4 Bennedsen J, Larsen S O. Examination for tubercle bacilli by
fluorescence microscopy. Scand J Respir Dis 1966; 47: 114
120.
5 Van Deun A, Chonde T M, Gumusboga M, Rienthong S. Performance and acceptability of the FluoLED Easy module for
tuberculosis fluorescence microscopy. Int J Tuberc Lung Dis
2008; 12: 10091014.
6 Affolabi D, Torrea G, Odoun M, et al. Comparison of two
LED fluorescence microscopy build-on modules for acid-fast
smear microscopy. Int J Tuberc Lung Dis 2010; 14: 160164.
7 Anthony R M, Kolk A H, Kuijper S, Klatser P R. Light emitting
diodes for auramine O fluorescence microscopic screening of
Mycobacterium tuberculosis. Int J Tuberc Lung Dis 2006; 10:
10601062.
8 World Health Organization. Fluorescent light emitting diode
(LED) microscopy for diagnosis of tuberculosis: policy statement. Geneva, Switzerland: WHO, 2010. http: //www.who.int/
tb/dots/laboratory/who_policy_led_microscopy_july10.pdf
Accessed July 2010.
9 Kent P T, Kubica G P. Public health mycobacteriology: a guide
for the level III laboratory. Atlanta, GA, USA: US Department
of Health and Human Services, Public Health Service, Centers
for Disease Control, 1985.
RSUM
C O N T E X T E : Laboratoire de mycobactriologie dun centre de rfrence de la tuberculose (TB) Mumbai, Inde.
O B J E C T I F S : Evaluer lexamen microscopique par fluorescence (FM) au moyen de la technologie des diodes
lectroluminescentes (LED) pour la dtection des bacilles
acido-rsistants dans un centre de rfrence tertiaire dans
un pays o la TB est endmique.
S C H M A : On a introduit la FM LED dans un laboratoire expriment en ce qui concerne lexamen microscopique au Ziehl-Neelsen (ZN), mais peu habitu FM.
On a valu FM LED paralllement avec les services de
routine dexamen microscopique ZN et on la compar
avec les cultures mycobactriennes servant de standard
de rfrence.
R S U LTAT S : Les chantillons provenant de 1357 cas
pulmonaires et de 917 cas extrapulmonaires ont t examins au cours de ltude. Nous avons trouv pour FM
LED une sensibilit de 78,3% ainsi quune spcificit de
92,0% par rapport la culture mycobactrienne pour
RESUMEN
M A R C O D E R E F E R E N C I A : El laboratorio de micobacteriologa en un centro de referencia de tuberculosis en
Mumbai, India.
O B J E T I V O S : Evaluar la microscopia de fluorescencia con
tcnica de diodos electroluminescentes (LED) en la deteccin de los bacilos acidorresistentes en un centro terciario de referencia de un pas con tuberculosis (TB)
endmica.
M T O D O S : Se introdujo la tcnica de microscopia de
fluorescencia (FM) con LED en un laboratorio con experiencia en el estudio sistemtico por microscopia con
coloracin de Ziehl-Neelsen (ZN) y sin competencias
en FM. Se compar la nueva tcnica con el servicio
sistemtico de microscopia con coloracin de ZN, tomando el cultivo de micobacterias como la tcnica de
referencia.
R E S U LTA D O S : En el estudio se examinaron 1357 muestras de origen pulmonar y 917 extrapulmonares. En promedio, la microscopia de fluorescencia con LED ofreci