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 Table of Contents  
Year : 2019  |  Volume : 3  |  Issue : 2  |  Page : 77-79

Sputum smear microscopy in tuberculosis: It is still relevant in the era of molecular diagnosis when seen from the public health perspective

State TB Demonstration Cum Training Centre, Intermediate Reference Laboratory, Kolkata, West Bengal, India

Date of Submission23-Mar-2019
Date of Decision28-Apr-2019
Date of Acceptance30-Apr-2019
Date of Web Publication17-Jun-2019

Correspondence Address:
Dr. Prasanta Kumar Das
2nd Floor, State TB Demonstration Cum Training Centre, Intermediate Reference Laboratory, DR BC Roy Post Graduate Institute of Paediatric Sciences, 38 Badan Roy Lane, Kolkata - 700 010, West Bengal
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/bbrj.bbrj_54_19

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Smear Microscopy has been the sole diagnostic support since the inception of the Revised national Tuberculosis Control Program. Treatment initiation and evaluation of prognosis both were dependant on the Smear Microscopy results. The recent commissioning of the CBNAAT machines raised questions on relevance of smear microscopy. The review explores the merits and demerits of the smear microscopy and other molecular technologies and debated on the relevance of microscopy when seen from the perspective of public health.

Keywords: Public health, sputum smear microscopy, tuberculosis control program

How to cite this article:
Das PK, Ganguly SB, Mandal B. Sputum smear microscopy in tuberculosis: It is still relevant in the era of molecular diagnosis when seen from the public health perspective. Biomed Biotechnol Res J 2019;3:77-9

How to cite this URL:
Das PK, Ganguly SB, Mandal B. Sputum smear microscopy in tuberculosis: It is still relevant in the era of molecular diagnosis when seen from the public health perspective. Biomed Biotechnol Res J [serial online] 2019 [cited 2022 May 19];3:77-9. Available from: https://www.bmbtrj.org/text.asp?2019/3/2/77/260486

  Background Top

Tuberculosis (TB) is one of the leading causes of morbidity and mortality today. Two billion people get infected and approximately two million death occurs annually.[1]

In 2015, there were 1315 deaths a day from TB in India. Number of new TB cases: 2,840,000; deaths from TB: 480,000; incidence: 217/100,000 population; percentage of people whose TB is not diagnosed: 49%; percentage of cases with HIV: 4%; estimated new TB cases with multidrug resistance: 2.5%.[2]

The WHO guidelines specify that microscopic examination of sputum samples should be the essential step in the investigation of patients who are suspected of having pulmonary TB. Incremental diagnostic yield of examination of three sputum specimens among smear-positive cases stood as the basis of this guideline.[3]

The Diagnostic procedure of Sputum Smear Microscopy was at question due to its low specificity and sensitivity after WHO had endorsed the new and rapid automated nucleic Acid amplification test (NAAT, Xpert MTB/RIF).

The review points out the advantage of sputum smear microscopy in the resourcelimited settings even after the commissioning of the NAATs for diagnosis of Tuberculosis.

  Methods Top

A comparative analysis of the technologies was done, evaluating the merits and demerits from the public health perspective as per the published studies available.

Enzyme-linked immunosorbent assay for TB testing is available. This assay is quite efficient and feasible in the public health system, but due to high sensitivity, it generates lot of false positives.

Both conventional and liquid culture by mycobacterial growth indicator tube (MGIT) techniques are efficient, but it is difficult to establish at the peripheral level. Although the selected cases can be tested in reference laboratory, it is very difficult to be used for universal drug sensitivity testing at the peripheral level as it requires Biosafety level III laboratories, skilled workforce, and, to some extent, labor intensive. However, the samples can be transported to the reference laboratories. From a public health point of view for extensive countrywide coverage and initial screening, it would not be a right choice.

Gas chromatography detects tuberculostearic acid in clinical samples, but it requires high-performance liquid chromatography with trained and skilled workforce. Moreover, the entire process is labor intensive. The test is not feasible when seen from a public health perspective.

Latex particle agglutination assays using monoclonal antibodies were used against the lipoarabinomannan; however, these tests were not specific, resulting in false positive cases.

Owing to the higher sensitivity and less turnaround time, DNA and RNA amplification techniques are increasingly used. However, sustainable logistic supplies from the support system of program and prompt maintenance from their end at the remote areas remains a question many a times, which makes this sputum microscopy more dependable tool for diagnostic and prognostic support.

  Discussion Top

Bright-field microscopy and tuberculosis

The primary method for the diagnosis of pulmonary TB is sputum smear microscopy in the resource-limited settings, amounting to an average of 95% of TB case detection.[4]

Attempts in recent years showed the importance of smear examinations with only two consecutive specimens. As there was no significant difference in the outcome of the patients between three or two sputum samples.

The overall clinical sensitivity is 22%–80% depending on the burden of mycobacteria, the type of AFB stain used, and the experience of laboratory technicians, whereas the positive predictive value of mycobacteria is more than 95%.

Fluorescent microscopy and tuberculosis

Fluorescence microscopy increases the sensitivity of sputum smear microscopy; additional data on specificity and on the clinical consequences associated with false-positive results are needed to guide implementation of this technology in high HIV-prevalence settings.[5] Cost constraints are major issues with fluorescent microscopy. This may be circumvented by the use of light-emitting diodes which costs <10% of a mercury vapor lamp. With a lifetime of >50,000 h, it can run on batteries and thus has been used in peripheral areas with definite operational advantages.[6]

Microscopy and culture

Although microscopy has an analytical sensitivity less than TB culture, establishing culture laboratories is a huge constraint while seen from the public health point of view.[7],[8] Moreover, the transport and packaging of samples from the remote, hard-to-reach areas, maintaining cold chain to ensure the viability of the organism, is a great challenging affair. Microscopy centers, on the other hand, are easy to be commissioned in those areas. The turnaround time even in the layer agar rapid cultures is not <13 days whereas smear microscopy results are available within 30 min. Although microscopy is nonspecific, for moribund or patients with severe illness, the technology will be an immense help. It has been seen that owing to improper transport and inadequate maintenance of cold chain, smear-positive culture contamination/negative has increased. If the number of samples from the remote, hard-to-reach areas getting contaminated increases, it would lead to substantial waste of resources.

Nucleic acid amplification tests and microscopy

NAATs have different analytical sensitivity based on different technical interventions. The line probe assay technology using the oligonucleotide probes has comparable sensitivity with the smear microscopy whereas the cartridge-based NAAT has more analytical sensitivity than smear microscopy.[9],[10] Setting up CBNAAT laboratories is a challenge in the remote areas. Moreover, sustainable maintenance of instrument and supply of logistics are the other challenges from the public health point of view.

Recent dissemination of CBNAAT machines at the block level has been very inspiring, but catering to that quantum of samples as in smear microscopy is yet to reach. For instance, in West Bengal, India, the 72 CBNAAT machines are being supplied with a daily output of 16 while the 865 microscopy centers have a capacity to undertake 40 examinations per day. Moreover, sustained supply of logistics and prompt maintenance at the periphery remains a constraint.

Here, microscopy has a substantial sensitivity with varied specificity [Table 1]. The test remains the most suitable for initial screening reaching out to millions and quite feasible when seen from public health perspective.
Table 1: Tuberculosis diagnostic methodology - sensitivity and specificity

Click here to view

This points out the need, relevance, and importance of sputum smear microscopy when seen from a public health perspective.

Advantages and relevance at the era of molecular diagnostics

Sputum microscopy is inexpensive, rapid, and substantially specific in the highly prevalent areas. It has been the global strategy of TB control. In the resource-limited settings, sputum smear microscopy is likely to remain the only sustainable technology. Sputum smear microscopy is the only cost-effective tool for diagnosing patients with infectious TB and to monitor their progress in treatment.


Sputum smear microscopy accounts for a limited sensitivity of 5000–10,000 AFB/ml. Around 45% below the CFU count is missed. However culture techniques are comparatively better and scores over smear microscopy.[11]

Smear Microscopy is having limitation in identification of species as it limits in the presence and absence of acid fast bacilli. The prevalence of mycobacterium TB (MTB) and non tubercular mycobacterium(NTM) determines the predictive values of a positive smear for Acid Fast Bacilli though a substantial amount of specificity is depicted in the highly prevalent areas.[12]

The other limitation for smear Microscopy is that It does not distinguish between viable and dead organisms, so patients who are on follow up after initiation of treatment are to be considered simultaneously for Liquid Culture to substantiate positivity for those who are smear positive. Further to note that for the patients who are HIV reactive and /or paucibacillary and the CFU is lower than the analytical sensitivity, simultaneous support of Liquid Culture system is needed.

  Conclusion Top

Ideally, the best diagnostic test for TB will be a test not influenced by the HIV status with high sensitivity, specificity, and positive and negative predictive values. It is a test that requires no electricity, refrigeration, or access to clean water, where sustainability is easy requiring minimum regular maintenance. A turnaround time is one hour.[12]

As of now there is no diagnostic tests available for tuberculosis specially in resource limited settings. However Sputum Smear Microscopy covers almost every aspect except for the sensitivity and specificity.

The overall clinical sensitivity of sputum AFB smear is 22%–80% depending on the burden of mycobacteria, the type of AFB stain used, and the experience of laboratory technicians, whereas the positive predictive value for mycobacteria is >95%.[13]

Till an ideal test evolves, it appears sputum smear microscopy is the only practically sustainable test to be considered before undertaking any other diagnostic procedure to TB.[12]

At this point, it is pertinent to remember with the reverence that the TB bacilli was discovered for the first time by Dr. Robert Koch in 1882 and the technology involved in its discovery was none other than sputum smear microscopy. The technology remains the foundation even in the era of molecular diagnostic technology for TB.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

  References Top

Global Tuberculosis Control: WHO Report 2011. Available from: http://www.who.int/tb/publications/global_report/2011/gtbr11_main.pdf. [Last accessed on 2012 Dec 22].  Back to cited text no. 1
TB Statistics India – National, Treatment Outcome, State. National TB Statistics India. Available from: https://www.tbfacts.org/tb-statistics-india/LAD. [Last accessed on 2019 Jan 28].  Back to cited text no. 2
Reider HL, Van Deun A, Kam KM, Kim SJ, Chonde TM, Trebucq A, et al., editors. Priorities for Tuberculosis Bacteriology Services in Low-Income Countries. Paris, France: International Union against Tuberculosis and Lung Disease; 2007.  Back to cited text no. 3
International Standards for Tuberculosis Care. Available from: http://www.who.int/tb/publications/2006/istc_report.pdf. [Last accessed on 2013 Mar 06].  Back to cited text no. 4
Steingart KR, Henry M, Ng V, Hopewell PC, Ramsay A, Cunningham J, et al. Fluorescence versus conventional sputum smear microscopy for tuberculosis: A systematic review. Lancet Infect Dis 2006;6:570-81.  Back to cited text no. 5
Hung NV, Sy DN, Anthony RM, Cobelens FG, van Soolingen D. Fluorescence microscopy for tuberculosis diagnosis. Lancet Infect Dis 2007;7:238-9.  Back to cited text no. 6
Luelmo F. What is the role of sputum microscopy in patients attending health facilities? In: Frieden T, editor. Toman's Tuberculosis: Case Detection, Treatment, and Monitoring – Questions and Answers. 2nd ed. Geneva: World Health Organization; 2004. p. 7-13.  Back to cited text no. 7
George G, Mony P, Kenneth J. Comparison of the efficacies of loop-mediated isothermal amplification, fluorescence smear microscopy and culture for the diagnosis of tuberculosis. PLoS One 2011;6:e21007.  Back to cited text no. 8
Perkins MD. New diagnostic tools for tuberculosis. Int J Tuberc Lung Dis 2000;4:S182-8.  Back to cited text no. 9
Automated Real Time Nucleic Acid Amplification Technology for Rapid and Simultaneous Detection of Tuberculosis and Rifamicin Resistance: Xpert MTB/RIF System. WHO Policy Statement; 2011. Available from: https://www.who.int/tb/publications/tb-amplificationtechnology-statement/en/. [Last accessed on 2019 May 16].  Back to cited text no. 10
Leung E, Minion J, Benedetti A, Pai M, Menzies D. Microcolony culture techniques for tuberculosis diagnosis: A systematic review. Int J Tuberc Lung Dis 2012;16:16-23, i-iii.  Back to cited text no. 11
Desikan P. Sputum smear microscopy in tuberculosis: Is it still relevant? Indian J Med Res 2013;137:442-4.  Back to cited text no. 12
[PUBMED]  [Full text]  
Lipsky BA, Gates J, Tenover FC, Plorde JJ. Factors affecting the clinical value of microscopy for acid-fast bacilli. Rev Infect Dis 1984;6:214-22.  Back to cited text no. 13


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