Home | About us | Editorial Board | Search | Ahead of print | Current Issue | Archives | Instructions | Online submissionContact Us   |  Subscribe   |  Advertise   |  Login  Page layout
Wide layoutNarrow layoutFull screen layout
Lung India Official publication of Indian Chest Society  
  Users Online: 800   Home Print this page  Email this page Small font size Default font size Increase font size

  Table of Contents    
Year : 2018  |  Volume : 35  |  Issue : 4  |  Page : 307-311  

Impact of introducing light-emitting diode fluorescence microscopy services for diagnosis of pulmonary tuberculosis under Revised National Tuberculosis Control Program India

1 Department of Community Medicine and School of Public Health, PGIMER, Chandigarh, India
2 Revised National Tuberculosis Control Program, Chandigarh, India

Date of Web Publication26-Jun-2018

Correspondence Address:
Dr. Rahul Pandey
School of Public Health, PGIMER, Chandigarh- 160 012
Login to access the Email id

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/lungindia.lungindia_475_17

Rights and Permissions

Context: Light-emitting diode fluorescence microscopy (LED-FM) has been recommended by the WHO and the Government of India over the conventional bright-field microscopy using Ziehl–Neelsen (ZN) staining for the diagnosis of sputum smear-positive tuberculosis (TB) suspects. Aim: The aim of this study was to assess the effectiveness of LED-FM in detecting TB cases. Setting and Design: This retrospective cohort study was carried out in December 2016 using secondary data of the years 2011–2012 from designated microscopy centers (DMCs) at Chandigarh, India. Methodology: Two peripheral DMCs where conventional ZN microscopy was used were taken as controls comparable to two peripheral DMCs which used LED-FM services in programmatic settings. The record of all suspected TB patients who underwent sputum smear examination pre- and post-LED-FM introduction was compared for measuring smear-positive cases, conformational grading, and time taken to read per slide examined. Chi-square was applied to access the statistical significance. Results: Out of total 8850 cases registered after the inception of LED-FM microscopy services, case detection rate was 13.3%, with significant decrease in case detection rate at tertiary level DMCs, there was significant increase in case detection rate at peripheral DMCs operating with LED-FM in contrast to ZN microscopy which was used previously. Scanty grade smear increased significantly by 9.0%. The time taken per slide examination decreased by 57.1% (from 7 min on an average to 3 min on an average) after LED-FM was used. Conclusion: LED-FM is easy to use, takes lesser time to examine slides, and has longer self-life. It also eases laboatory technician's task in reporting scanty grade positives which use to be challenging previously. The results of this study provide enough evidences to scale up the installation process and usage of LED-FM at DMCs for TB diagnosis in Indian settings.

Keywords: Light-emitting diode fluorescence microscopy, Revised National Tuberculosis Control Program, tuberculosis, Ziehl–Neelsen microscopy

How to cite this article:
Goel S, Pandey R, Kumar M, Kankaria A, Khaneja R. Impact of introducing light-emitting diode fluorescence microscopy services for diagnosis of pulmonary tuberculosis under Revised National Tuberculosis Control Program India. Lung India 2018;35:307-11

How to cite this URL:
Goel S, Pandey R, Kumar M, Kankaria A, Khaneja R. Impact of introducing light-emitting diode fluorescence microscopy services for diagnosis of pulmonary tuberculosis under Revised National Tuberculosis Control Program India. Lung India [serial online] 2018 [cited 2020 Jan 21];35:307-11. Available from: http://www.lungindia.com/text.asp?2018/35/4/307/235052

   Introduction Top

Tuberculosis (TB) is a major public health threat and is responsible for 1.5 million deaths globally.[1] As per the WHO Global report, the estimated global TB incidence cases in 2015 were 10.9 million with 25% of them reported from India.[1] Globally, India has the highest burden of multidrug-resistant-TB cases and second highest burden of HIV-TB cases. It is estimated that about 170 million workdays are lost annually in India due to this disease which is equivalent to an annual economic cost of 130 billion dollars.[2] Besides, this an undiagnosed TB positive case can infect 10–15 people in his/her entire life.[3] Such consequences can be catastrophic for our national ambition of eliminating TB and Government of India's pledge of health for all.

Sustainable development goals (2015–2030) and End TB strategy (2017–2025) aims to culminate TB epidemic and reduce the incidence as well as absolute number of TB deaths (baseline 2015) to 90%. Revised National TB Control Program (RNTCP) under its strategic plan for 2012–2017 also envisage for detection of 90% of all incident TB cases as well as to treat them. Among the enlisted strategies of RNTCP, use of sputum microscopy is the primary method of diagnosis for symptomatic cases.[4] Use of Ziehl–Neelsen (ZN) staining under the direct microscopy was a landmark intervention that increased case detection rate from 40% to 70% in the program settings.[5] Although ZN staining has high sensitivity and specificity for acid-fast bacilli (AFB), the ambitious goals set by the Government of India to eliminate TB by 2030 needs more technological advancement for diagnosis purposes under RNTCP.[6]

LED FM has been recommended by the WHO and Government of India to be introduced for diagnostic purposes in place of conventional ZN staining. Studies conducted globally have documented a similar or higher sensitivity (∼8%–10%) and specificity for LED FM as compared to ZN staining-based conventional microscopy for sputum smear-positive TB case detection.[7],[8],[9],[10],[11],[12] In addition, LED FM was reported to consume less time per slide examination because slides are examined at lower magnification thus covering much larger area. It is also reported that, as compared to conventional microscopy, LED FM needs lower maintenance and can be operated even on batteries. These features make it suitable for Indian settings where high TB burden is present, and hence, the workload on laboratory staff is excessive.[9],[11] Few infrastructural issues such as irregular electric supply, sluggish equipment maintenance procedures may add on for suboptimal performance which necessitates need of technological advancement-like light-emitting diode fluorescence microscopy (LED-FM) in programmatic settings.[7],[9],[11] The RNTCP had a target of replacing the existing conventional microscopy technique by LED-FM with priority at health facilities with higher workload.[4]

Merely, a solitary study in India by Reza et al. in 2013 at 15 randomly selected medical colleges has been conducted which documented that LED-FM has increased effectiveness in diagnosis of TB positives. However, evidence for the same at the peripheral microscopy centers in Indian setting is lacking [9] which bleaks the advocacy needed for its rapid installation at all the designated microscopy centers (DMCs) in the country.

The present study was done with an overall aim of assessing the effectiveness of LED-FM in detecting TB cases. Specifically, the objectives are to document difference in case detection rate, to identify any change in conformational grades and to examine time take to read per slide before and after introduction of LED-FM at different levels of healthcare facility.

   Methodology Top

Study settings

This retrospective cohort study was conducted in Chandigarh, India, with a population of 1.1 million. The organizational structure of RNTCP in India consists of a Central TB division, State TB cell, District TB center, TB unit (TU), DMC, and directly observed treatment short course (DOTS) center. Chandigarh has 3 TU, 17 DMC, and around 171 DOTS centers.[13] Chandigarh started the revitalization of diagnostic technique in the year 2012 with the introduction of Auramine O based LED-FM at DMCs. Five DMCs were equipped with LED-FM during the period of study, out of which only four were operational.

Each presumptive TB case has to undergo two sputum smear examination with one on spot and another with early morning sputum specimen. A presumptive TB case with one or both sample positives is confirmed as a pulmonary TB case.[4] LED-FM is now recommended to be used instead of conventional ZN staining microscopy to examine these sputum smears of suspected TB cases.

Study population, sample size, and sampling technique

For the study purpose, four DMCs where LED-FM was operational were selected. Among these, two were in tertiary level medical institutes and two at peripheral health centers. The case detection rates for these DMCs were compared before (2011) and after (2012) introduction of LED FM. In addition, two peripheral DMCs were chosen as controls which were using conventional method (ZN Staining) for the detection of TB cases. These cases and controls were located in same type of localities and cover almost similar populations.

The study population included population with presumptive TB referred to the above-mentioned DMCs for sputum smear examination. The results of sputum examination were compared before (2011) and after the introduction of LED-FM (2012). Assuming 90% of case detection rate, 80% power of the study and confidence interval of 95%, an appropriate sample size of 1200 was calculated.

Study variables and data analytics

The dependent variables were change in case detection rate, AFB grading for cases, and time consumed in examining each slide after the introduction of LED FM whereas, the independent variables were age, gender, types of DMCs, and number of sputum smears examined. These details were collected from the designated RNTCP microscopy laboratory registers. Data were tabulated into the Microsoft Excel and cross-tabulated with pre- and post-LED-FM introduction. Statistical significance of the result was analyzed by Pearson's Chi-square test, P ≤ 0.05 was taken statistically significant.

Ethical approval

Ethical and administrative approval for the study was taken from the State TB Program Officer, Chandigarh, to access the record from the DMCs.

   Results Top

A total of 7631 TB symptomatics were examined in the preintervention period, out of which 1049 (13.7%) were found positive. Similarly, 8850 symptomatic were examined postintervention (Introduction of LED-FM) in same setup, out of which 1173 were found positives (13.2%). The patient population characteristics before and after the introduction of LED FM was almost similar [Table 1].
Table 1: Sociodemographic profile of the patients in the study

Click here to view

Thus as compared with ZN microscopy, there was a significant decrease in case detection rate after the introduction of LED FM at tertiary DMCs (P = 0.03) whereas, a significant increase in case detection rate (percentage change + 13.7%) with LED FM at peripheral DMCs was noticed (P = 0.04) [Table 1].

Two similar peripheral DMCs were selected as control where no such intervention was done during the period (2011–2012), and they showed a decrease in case detection rate over the period (percentage change − 7.12%) [Table 2].
Table 2: Comparison of case detection rate between Designated Microscopy Centre before and after introduction of light-emitting diode fluorescence microscopy

Click here to view

There was a significant increase in scanty grading by 9% and 1+ grade by 0.1%, although the 2+ and 3+ grades showed a decrease of 3.1% and 4.2%, respectively, after the introduction of LED-FM for case detection [Table 3].
Table 3: Sputum smear examination results, by scanty grading in selected Designated Microscopy Centers before and after introduction of light-emitting diode fluorescence microscopy

Click here to view

On observation and interview, it was found that average time taken by laboratory technician (LT) to examine the slide under LED-FM was 3 min (2–4 min) post-LED-FM introduction as compared to an average of 7 min (8–9 min) for ZN microscopy.

   Discussion Top

This study assessed and compared the case detection rate before and after the introduction of LED FM in operational settings. We found that there was a decrease in case detection rate when LED-FM was introduced at tertiary level healthcare institutes. Yet at peripheral health facilities, after the usage of LED-FM, there was a significant increase in case detection rate. Further, the study observed that the number of scanty grade smears increased significantly and time required per slide examination decreased to almost half after using LED-FM at microscopy centres.

Contrary to our finding, study done by Reza et al.[9] Chaidir et al.[10] Xia et al.[11] and Ba et al[12] found increase in case detection rate after LED-FM usage irrespective of healthcare settings. These studies have credited technical advancement associated with LED-FM for the increase in sensitivity and case detection rate in addition to other factors such as training of staff, better work capacity, and better infrastructure. Similar to our finding, Bonnet et al. at healthcare clinic in Kenya,[14] Minion et al.[15] at tertiary care centers in Canada have documented that there was no rise in case detection rate when LED-FM was used.

Similar to earlier literature,[9] we found a percentage increase in scanty grade smears (P < 0.05) after introducing LED-FM.[9],[11] There are attributes of LED FM which makes it more sensitive than ZN. These are better absorbability of mycolic acid for carbol auramine (used in LED-FM) as compared to carbol fuschin (used in ZN microscopy) resulting in staining larger number of bacilli as well as high-power fields for examining larger field area with easier identification with sharper contrast between background and bacilli.[16] Moreover, studies have questioned the quality of carbol fuschin powder used for ZN stain.[8] LTs have showed confidence in reporting scanty grade case after the intervention and this will endorse the RNTCP objective of improving case detection rate.

The LTs in our study reported that ZN microscopy took around 7 min (6–8 min) on an average for confirmatory diagnosis whereas LED-FM takes merely 3 min (2–4 min). Moreover, LED-FM working does not require dark room, or warm up time and their diodes work for longer time. Besides it has lesser negative health consequence as no chance of dissemination of mercury vapors as is the case for ZN microscopy.[15],[16] These facts have been documented in earlier literature too.[8],[9] The reduction of time after introducing LED-FM under RNTCP will lessen the laboratory workload which will improve the quality of work. Another Indian study by Bhalla et al. has reported a marginal increase in sensitivity with use of LED FM; considerable time saving and convenience.[17] Contrary, Marzouk et al. in Tunisia concluded similar time taken by LED FM as compared to conventional microscopy.[18]


In this study, we covered all the types of healthcare facilities under the domain of RNTCP which provided a comprehensive picture of utility of LED-FM in different settings. This effort was lacking in Indian [9] as well as global scenarios.[8],[10],[11],[12] Further, the study covered all the domains suggested under RNTCP for good microscopy such as case detection rate, specificity, time per slide, and ease of working.


Since it was a secondary data analysis of aggregated data, we had no check on the data quality, although RNTCP maintains a high standard in data recording procedures. Further nonavailability of patient, wise disaggregated data limited the presentation of results and scope for regression analysis.

   Conclusion and Recommendation Top

We conclude that there is an increase in scanty sputum cases with a decrease in the time taken by LED microscopy compared to ZN microscopy, which underscores the need for its installation in diagnostic laboratories at all levels of healthcare facilities of developing nations. We suggest future research on disaggregated data for detailed analysis measuring effectiveness of LED FM in diverse healthcare settings.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

   References Top

World Health Organization. Global Tuberculosis Report. World Health Organization; 2016. Available from: http://www.who.int/tb/publications/global_report/en. [Last accessed on 2017 May 25].  Back to cited text no. 1
Moitra S, Sen S, Mukherjee S, Das P, Sinha S, Bose M. Study of prevalence and outcome of standardized treatment on category I pulmonary tuberculosis cases in North India: A single center experience. Community Acquir Infect 2015;2:83-92.  Back to cited text no. 2
  [Full text]  
Tuberculosis (TB). Fact Sheet. World Health Organization. Available from: http://www.who.int/mediacentre/factsheets/fs104/en/. [Last accessed on 2017 May 25].  Back to cited text no. 3
Revised National Tuberculosis Control Program. National Strategic Plan for Tuberculsois Control. India: Central TB Division, Directorate General of Health Services. Ministry of Health and Family Welfare; 2012-2017.  Back to cited text no. 4
Das D, Selvakumar N. Can LED fluorescence microscopy replace Ziehl-Neelsen microscopy in tuberculosis detection? Int J Tuberc Lung Dis 2012;16:1558.  Back to cited text no. 5
Central TB Division, Directorate of Health Services, Ministry of Health and Family Welfare. I RNTCP Annual Status Report 2012. Central TB Division, Directorate of Health Services, Ministry of Health & Family Welfare; 2012. Available from: http://www.tbcindia.nic.in/pdfs/TB India 2012- Annual Report.pdf. [Last accessed on 2017 May 24].  Back to cited text no. 6
World Health Organization. Fluorescent Light Emitting Diode (LED) Microscopy for Diagnosis of Tuberculosis: Policy Statement. Available from: http://www.who.int/tb/publications/2011/led_microscopy_diagnosis_9789241501613/en/index.html. [Last accessed on 2017 May 24].  Back to cited text no. 7
Cuevas LE, Al-Sonboli N, Lawson L, Yassin MA, Arbide I, Al-Aghbari N, et al. LED fluorescence microscopy for the diagnosis of pulmonary tuberculosis: A multi-country cross-sectional evaluation. PLoS Med 2011;8:e1001057.  Back to cited text no. 8
Reza LW, Satyanarayna S, Enarson DA, Kumar AM, Sagili K, Kumar S, et al. LED-fluorescence microscopy for diagnosis of pulmonary tuberculosis under programmatic conditions in India. PLoS One 2013;8:e75566.  Back to cited text no. 9
Chaidir L, Parwati I, Annisa J, Muhsinin S, Meilana I, Alisjahbana B, et al. Implementation of LED fluorescence microscopy for diagnosis of pulmonary and HIV-associated tuberculosis in a hospital setting in Indonesia. PLoS One 2013;8:e61727.  Back to cited text no. 10
Xia H, Song YY, Zhao B, Kam KM, O'Brien RJ, Zhang ZY, et al. Multicentre evaluation of Ziehl-Neelsen and light-emitting diode fluorescence microscopy in China. Int J Tuberc Lung Dis 2013;17:107-12.  Back to cited text no. 11
Ba F, Rieder HL. A comparison of fluorescence microscopy with the Ziehl-Neelsen technique in the examination of sputum for acid-fast bacilli. Int J Tuberc Lung Dis 1999;3:1101-5.  Back to cited text no. 12
National Health Mission U.T. Chandigarh; 2016. Available from: http://www.nrhmchd.gov.in. [Last accessed on 2016 Sep 18].  Back to cited text no. 13
Bonnet M, Gagnidze L, Githui W, Guérin PJ, Bonte L, Varaine F, et al. Performance of LED-based fluorescence microscopy to diagnose tuberculosis in a peripheral health centre in Nairobi. PLoS One 2011;6:e17214.  Back to cited text no. 14
Minion J, Pai M, Ramsay A, Menzies D, Greenaway C. Comparison of LED and conventional fluorescence microscopy for detection of acid fast bacilli in a low-incidence setting. PLoS One 2011;6:e22495.  Back to cited text no. 15
Richards OW. The staining of acid-fast tubercle bacteria. Science 1941;93:190.  Back to cited text no. 16
Bhalla M, Sidiq Z, Sharma PP, Singhal R, Myneedu VP, Sarin R, et al. Performance of light-emitting diode fluorescence microscope for diagnosis of tuberculosis. Int J Mycobacteriol 2013;2:174-8.  Back to cited text no. 17
  [Full text]  
Marzouk M, Ferjani A, Dhaou M, Ali MH, Hannachi N, Boukadida J, et al. Comparison of LED and conventional fluorescence microscopy for detection of acid-fast bacilli in an area with high tuberculosis incidence. Diagn Microbiol Infect Dis 2013;76:306-8.  Back to cited text no. 18


  [Table 1], [Table 2], [Table 3]


    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Access Statistics
    Email Alert *
    Add to My List *
* Registration required (free)  

  In this article
    Conclusion and R...
    Article Tables

 Article Access Statistics
    PDF Downloaded180    
    Comments [Add]    

Recommend this journal