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 Table of Contents  
Year : 2013  |  Volume : 2  |  Issue : 3  |  Page : 174-178

Performance of light-emitting diode fluorescence microscope for diagnosis of tuberculosis

1 Department of Microbiology, Lala Ram Sarup Institute of Tuberculosis and Respiratory Diseases, Sri Aurobindo Marg, New Delhi 110030, India
2 Department of Biostatistics, Lala Ram Sarup Institute of Tuberculosis and Respiratory Diseases, Sri Aurobindo Marg, New Delhi 110030, India
3 Department of TB and Chest, Lala Ram Sarup Institute of Tuberculosis and Respiratory Diseases, Sri Aurobindo Marg, New Delhi 110030, India

Date of Web Publication28-Feb-2017

Correspondence Address:
V P Myneedu
Department of Microbiology, Lala Ram Sarup Institute of Tuberculosis and Respiratory Diseases, Sri Aurobindo Marg, New Delhi 110030
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Source of Support: None, Conflict of Interest: None

DOI: 10.1016/j.ijmyco.2013.05.001

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Background: Fluorescence microscopy (FM) over the years has shown the potential for increasing the performance of microscopy. The present study was aimed to access the performance of the LED microscope for the detection of acid fast bacilli in a tuberculosis (TB) endemic country.
Methods: The study was conducted at a National Reference Laboratory (NRL) in New Delhi, India. Sputum samples were collected from suspected TB patients. Each sample was processed with Auramine O and ZN methods. Auramine O stained smears were evaluated using two different excitatory light sources (MVP and LED); and ZN stained smears were examined under light microscope. The mean time required to read the smears with different modalities was recorded. Bacterial cultures provided the reference standard.
Results: A total of 200 patients were included in this study. Sensitivity and specificity for the LED assessment, MVP assessment and light microscopy were 83.1% and 82.4%, 78.5% and 87.5% and 81.6% and 83.5%, respectively. Mean reading time was approximately three times faster than ZN microscopy. The mean time to read a negative smear was 2 min with fluorescence microscopy and 5 min with light microscopy with time savings of 60%.
Conclusion: Although the use of LED-FM only marginally increased sensitivity, the considerable time saving ability combined with very good acceptance and ease of use makes it a reliable alternative to other conventional methods available.

Keywords: Mycobacterium tuberculosis, Ziehl-Neelsen, Sensitivity, Negative predictive value, Time saving

How to cite this article:
Bhalla M, Sidiq Z, Sharma P P, Singhal R, Myneedu V P, Sarin R. Performance of light-emitting diode fluorescence microscope for diagnosis of tuberculosis. Int J Mycobacteriol 2013;2:174-8

How to cite this URL:
Bhalla M, Sidiq Z, Sharma P P, Singhal R, Myneedu V P, Sarin R. Performance of light-emitting diode fluorescence microscope for diagnosis of tuberculosis. Int J Mycobacteriol [serial online] 2013 [cited 2022 Jan 25];2:174-8. Available from: https://www.ijmyco.org/text.asp?2013/2/3/174/201226

  Introduction Top

Tuberculosis (TB) remains a major global health problem and ranks as the second leading cause of death from an infectious disease worldwide, after the human immunodeficiency virus (HIV). This disease disproportionately affects the poorer countries of the world where sputum smear microscopy mostly is the only diagnostic test available for the evaluation of patients with symptoms suggestive of pulmonary TB. Sputum smear microscopy, however, is associated with low and variable sensitivity [1]. Sensitivity is largely determined by the duration of microscopic examination. Where workloads are high and the amount of time spent examining the smears is low, sensitivity is correspondingly low [2]. Simple yet rapid approaches to reduce the laboratory workload and to increase the sensitivity of direct smear microscopy need to be explored urgently. A systematic review published in 2006 reported that fluorescence microscopy (FM) could improve sensitivity of smear microscopy by 10% over the conventional Ziehl–Neelsen (ZN) microscopy mostly used in poorer countries, and that specificities of the two techniques were comparable [3]. In addition to increased sensitivity, fluorescence microscopy also allows for more rapid screening of sputum smear specimens which is highly advantageous, particularly when high numbers of samples are screened per day, because the majority of laboratory time is spent confirming negative smear results. According to the International Union against Tuberculosis and Lung Disease technical guidelines for sputum microscopy, at least 5 min of screening is required to correctly identify a negative smear result when conventional light microscopy is used [4]. However, under routine conditions, the time spent per slide is often far less than the minimum required. A comparative study reported that a mean time of 1 min to examine a sputum smear with fluorescence microscopy achieved higher sensitivity and equivalent specificity than did conventional light microscopy with an examination time of 4 min [5]. Despite all the advantages, implementation of FM is problematic owing to the complexity of the equipment which involves the use of ultra-violet light generated by expensive mercury vapor lamps (MVP) with short life-spans and dependence upon a steady electrical supply [3]. Simple FM systems based on light-emitting diodes (LED-FM) have been identified as an alternative to conventional FM. Theses LED-FMs have long life-spans, do not produce UV light, and have minimal power requirements which makes their use at low-resource settings extremely feasible [6]. In 2010 the World Health Organization (WHO) recommended that conventional FM (CFM) be replaced by LED-FM in all settings where CFM is used, and that LED microscopy be phased-in as an alternative to conventional ZN microscopy [7].

The present study was undertaken to assess the diagnostic performance of LED microscopy compared with conventional FM and ZN light microscopy using mycobacterial culture as a reference standard.

  Methods Top

The study was conducted at the Department of Microbiology, Lala Ram Sarup (LRS) Institute of Tuberculosis and Respiratory Diseases, New Delhi, India – a 520-bedded tertiary care hospital dedicated to TB and respiratory diseases and was approved by the Institute's ethical committee. The Institute is also a National Reference Laboratory under the Revised National Tuberculosis Control Program (RNTCP), and a drug-resistant TB (DR-TB) site for the treatment of DR-TB, including MDR-TB patients. The laboratory is efficiently going through the regular rounds of proficiency testing, for both first- and second-line drug sensitivity by the Supra National Reference Laboratory, Institute of Tropical Medicine; Antwerp, Belgium.

Consecutive sputum specimens were collected from adult patients suspected of having pulmonary TB attending the out-patient department (OPD) of this Institute willing to participate in the study over a period of 9 months, i.e., from April 2011 to December 2011. Patients received instructions on the production of a good-quality specimen [8]. Each sample was divided into two parts. One part was sent for culture on Lowenstein Jensen (LJ) medium and the other part was used for making three direct smears. The smears were labeled as A (ZN), B (MVP) and C (LED). A separate random number for the purpose of coding was given to each of the three slides of each specimen by the supervisor of the group who did not screen the smears himself.

Smear staining and examination

Smears labeled as A were stained by Ziehl Neelsen staining and smears labeled as B and C were stained by Auramine O staining as per the standard protocol [9]. All the smears were read independently by the three microscopists trained in fluorescence and ZN microscopy and all discrepant slides were read by the umpire microbiologist. The ZN-stained slide was evaluated using a conventional light microscope. As recommended by the International Union against Tuberculosis and Lung Disease technical guide, 100 fields were covered with the use of an eyepiece with 10 × magnification and an oil immersion objective with 100 × magnification (total magnification, 1000 × ) [4]. The slides stained with Auramine O were read with the MVP and LED-FM with the use of an eyepiece with 10 × magnifications and an objective with 40 × magnification (total magnification, 400 × ). The microscopists were blinded to all previous results. Slides were randomly resorted after each evaluation, and separate data capture forms were used for each modality to eliminate the possibility of the first reading influencing the second. The time required for reading both ZN and Auramine-stained was estimated for every batch of slides examined.

Mycobacterial culture

Briefly, a loopful of decontaminated/concentrated sediment was inoculated onto Löwenstein–Jensen (LJ) media. Inoculated LJ slants were incubated at 37 °C in a CO2 incubator. Slants were inspected daily for growth for the first week and then weekly for the next 10 weeks. Positive growth was confirmed for Mycobacterium tuberculosis complex by the p-nitrobenzoic acid (PNBA) assay.

Statistical analysis

The data generated was entered in a binary format into an Excel spreadsheet (Microsoft). Sensitivity, specificity and positive and negative prediction values were calculated for all the three modalities using mycobacterial culture as a reference standard. The average time required to read the slides was compared between LED-FM, MVP-FM and ZN. The inter-reader reliability and reproducibility of reading between the three modalities were assessed by the calculation of Kappa coefficients.

  Results Top

A total of 200 sputum samples were evaluated during this 9-month study. Of these, 69 (34.5%) were culture positive, 123 (61.5%) were culture negative and eight (4%) got contaminated during incubation. After the exclusion of the contaminated samples, final analysis was performed with 192 samples. The results obtained from the three modalities were evaluated against the mycobacterial culture. The mean culture positives out of the actual 69 culture positives picked by ZN, MVP-FM and LED-FM were 57 (82.6%), 56 (81.1%) and 58 (84%), respectively. The overall combined sensitivity of LED-FM was 83.1%, and the overall combined specificity was 82.4% with an overall agreement of 82.6%. Details of the results obtained using the ZN microscope, MVP-FM and LED-FM are depicted in [Table 1],[Table 2],[Table 3], respectively.
Table 1: Comparison of the results obtained by the three microscopists using ZN microscope with culture.

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Table 2: Comparison of the results obtained by the three microscopists using MVP-FM with culture.

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Table 3: Comparison of the results obtained by the three microscopists using LED-FM with Culture.

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[Table 4] presents the overall combined positive predictive value, negative predictive value and Kappa stats in comparison with the culture obtained from the three different modalities used. The negative predictive value was found to be highest (89.7) in LED-FM with a Kappa value of 0.636.
Table 4: Overall combined specificity, sensitivity PPV, NPV and Kappa stats of the three microscopes.

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The mean time spent to read a negative smear with ZN microscope was 5 min and with fluorescence microscopy was 2 min. Thereby, demonstrating a time saving of 60% (approximately three times faster than ZN microscope) by fluorescence microscopy. However no difference was noted between LED and MVP.

  Discussion Top

In the light of recent WHO recommendations that LED microscopy be phased in as an alternative to conventional ZN microscopy, the present study was aimed at evaluating the performance of LED microscopy for the direct detection of AFB in a high-incidence setting [8]. In this study, although differences were not statistically significant, highest sensitivity was achieved with LED fluorescence microscopy with a good overall agreement of 82.6%. This is an important finding and supports previous studies that demonstrated the superior diagnostic performance of fluorescence microscopy, compared with conventional light microscopy [3],[10],[11],[12],[13],[14].

Smear reading was three times faster with LED-FM (mean time = 2 min) compared with data on conventional ZN microscopy (mean time = 5 min) with a time saving of 60%. This is consistent with 25–66% time saving when using FM compared with ZN microscopy reported in previous studies [15],[16]. Time saving with FM can be ascribed to quicker scanning of each field because of increased visibility of mycobacteria. The decreased magnification used with FM compared with light microscopy (400 × vs. 1000 × ) may have contributed towards the slight sensitivity differences noted as has already been mentioned in other studies [8],[17],[18]. The introduction of LED-FM in a high incidence setting would thus significantly reduce laboratory workloads and possibly allow better quality microscopy to be accomplished with the same human resources compared with ZN microscopy.

Sputum culture, which is regarded to be the gold standard for the detection of pulmonary TB, is hampered by excessive cost, infrastructure requirement and slow turnaround times. Although there is a definitive need for the improved access to sputum culture in areas with high rates of drug-resistant TB, the need for rapid smear results for the diagnosis of most infectious TB cases remains paramount. Good sensitivity and specificity and short evaluation time required makes LED-FM potentially useful, especially in resource-limited settings with a high burden of TB.

To date the major constraints to the implementation of fluorescence microscopy are the high price for fluorescence microscopes, maintenance and lack of sustainability. The mercury vapor lamps (MVP) that have been traditionally used as the excitatory light source in conventional fluorescence microscopes are energy inefficient, require an extensive power supply, and have limited lifespan (typically 200–300 h). A higher sensitivity of LED-FM in comparison with MVP-FM achieved in this study confirms previous observations that the LED provides a reliable alternative light source for fluorescence microscopy [19],[20].

This study had some limitations and failed to demonstrate statistically significant increases in sensitivity of FM. This might be explained by the great experience of the microscopists with the ZN method and the fact that they never used FM before being trained for the study. Nevertheless, this study's results are sufficient to demonstrate that LED-FM with almost similar sensitivity offers a highly feasible alternative to conventional ZN microscopy.

  Conclusion Top

This study shows that although LED-FM might not increase sensitivity compared with ZN microscopy, the faster reading of LED-FM smears combined with very good acceptability and ease of use, would support its introduction in peripheral laboratories in resource-poor settings. However, large scale field trials are required to assess the advantages and feasibility of replacing conventional ZN light microscopy with LED fluorescence microscopy as a first-line diagnostic test.

  Conflict of interest Top


  Acknowledgements Top

Author Dr. Manpreet Bhalla acknowledges the financial support provided by M/s Olympus India (P) Ltd. and the Fraen Micro LED Fluorescence module with Olympus/Magnus Microscope. Mr. Grish Chandra and Mrs. Shashi Kala are acknowledged for their technical support.

  References Top

A.M. Elliott et al, The impact of human immunodeficiency virus on presentation and diagnosis of tuberculosis in a cohort study in Zambia, J. Trop. Med. Hyg. 96 (1993) 1–11.  Back to cited text no. 1
A. Cambanis, A. Ramsay, V. Wirkom, E. Tata, L.E. Cuevas, Investing time in microscopy: An opportunity to optimize smear-based case detection of tuberculosis, Int. J. Tuberc. Lung Dis. 11 (2007) 40–45.  Back to cited text no. 2
K.R. Steingart et al, Fluorescence versus conventional sputum smear microscopy for tuberculosis: a systematic review, Lancet Inf. Dis. 6 (2006) 570–581.  Back to cited text no. 3
International Union against Tuberculosis and Lung Disease (IUATLD) technical guide, Sputum Examination for Tuberculosis by Direct Microscopy in Low-Income Countries, fifth ed., IUATLD, Paris, 2000.  Back to cited text no. 4
J. Bennedsen, S.O. Larson, Examination for tubercle bacilli by fluorescence microscopy, Scand. J. Respir. Dis. 47 (1966) 114– 120.  Back to cited text no. 5
Stop TB Partnership and World Health Organization, New Laboratory Diagnostic Tools for Tuberculosis Control, Retooling Task Force, WHO, Geneva, Switzerland, 2008.  Back to cited text no. 6
World Health Organization. <http://www.who.int/tb/ advisory_bodies/stag_tb_report_(2009)pdf>, 2011 (accessed 26.01.11).  Back to cited text no. 7
M.S. Khan, O. Dar, C. Sismanidis, K. Shah, P. Godfrey-Faussett, Improvement of tuberculosis case detection and reduction of discrepancies between men and women by simple sputumsubmission instructions: a pragmatic randomised controlled, trial, Lancet 369 (2007) 1955–1960.  Back to cited text no. 8
Laboratory services in tuberculosis control: Part II. Microscopy. WHO/TB/98.258, 1998.  Back to cited text no. 9
K. Prasanthi, A.R. Kumari, Efficacy of fluorochrome stain in the diagnosis of pulmonary tuberculosis co-infected with HIV, Indian J. Med. Microbiol. 23 (2005) 179–185.  Back to cited text no. 10
L.E. Kivihya-Ndugga et al, A comprehensive comparison of Ziehl–Neelsen and fluorescence microscopy for the diagnosis of tuberculosis in a resource poor urban setting, Int. J. Tuberc. Lung Dis. 7 (2003) 1163–1171.  Back to cited text no. 11
N.P. Singh, S.C. Parija, The value of fluorescence microscopy of Auramine stained sputum smears for the diagnosis of pulmonary tuberculosis, Southeast Asian J. Trop. Med. Public Health 29 (1998) 860–863.  Back to cited text no. 12
W. Githui et al, A comparative study on the reliability of fluorescence microscopy and Ziehl–Neelsen method in the diagnosis of pulmonary tuberculosis, East Afr. Med. J. 70 (1993) 263–266.  Back to cited text no. 13
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B.J. Marais et al, Use of light-emitting diode fluorescence microscopy to detect acid-fast bacilli in sputum, Clin. Infect. Dis. 47 (2008) 203–207.  Back to cited text no. 15
M. Bonnet et al, Bleach sedimentation: An opportunity to optimize tuberculosis smear microscopy in high HIV prevalence settings, Clin. Infect. Dis. 46 (2008) 1710–1716.  Back to cited text no. 16
J. Bennedsen, S.O. Larsen, Examination for tubercle bacilli by fluorescence microscopy, Scand. J. Respir. Dis. 47 (1996) 114– 120.  Back to cited text no. 17
A. Ramsay et al, New policies, new technologies: modeling the potential for improved TB microscopy services in Malawi, PLoS ONE 4 (2009) e7760.  Back to cited text no. 18
R.M. Anthony, A.H. Kolk, S. Kuijper, P.R. Klatser, Light emitting diodes for Auramine O fluorescence microscopic screening of Mycobacterium tuberculosis, Int. J. Tuberc. Lung Dis. 10 (2006) 1060–1062.  Back to cited text no. 19
N.V. Hung, D.H. Sy, R.M. Anthony, F.J.G. Cobelens, D. van Soolingen, Fluorescence microscopy for tuberculosis diagnosis, Lancet Infect. Dis. 7 (2007) 238–239.  Back to cited text no. 20


  [Table 1], [Table 2], [Table 3], [Table 4]

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