Fluorescent light-emitting diode (LED) microscopy for

diagnosis of tuberculosis

Policy statement
WHO Library Cataloguing-in-Publication Data

Fluorescent light-emitting diode (LED) microscopy for diagnosis of tuberculosis: policy

statement.

1.Tuberculosis - diagnosis. 2.Microscopy, Fluorescence - methods. 3.Lighting -

instrumentation. 4.Developing countries. I.World Health Organization.

ISBN 978 92 4 150161 3 (NLM classification: WF 220)

World Health Organization 2011

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WHO/HTM/TB/2011.8
Contents

Abbreviations
Executive summary. 3
1. Background... 4
2. Evidence for policy formulation 4
2.1 Synthesis of evidence.. 4
2.2 Management of declarations of interest 5
3. Summary of results. 5
4. Policy recommendations 6
5. Intended audience.. 7
6. References.. 7
7. Annexes. 8
Annex 1: Expert Group members. 8
Annex 2: WHO staff members.. 10
Annex 3: STAG-TB members 11

1
Abbreviations

CI confidence interval
GRADE grades of recommendation assessment, development and evaluation
LED light-emitting diode
STAG-TB Strategic and Technical Advisory Group for Tuberculosis
TB tuberculosis
WHO World Health Organization

2
Executive summary

Conventional light microscopy of Ziehl-Neelsen-stained smears prepared directly from sputum

specimens is the most widely available test for diagnosis of tuberculosis (TB) in resource-limited
settings. Ziehl-Neelsen microscopy is highly specific, but its sensitivity is variable (2080%) and is
significantly reduced in patients with extrapulmonary TB and in HIV-infected TB patients.
Conventional fluorescence microscopy is more sensitive than Ziehl-Neelsen and takes less time,
but its use has been limited by the high cost of mercury vapour light sources, the need for regular
maintenance and the requirement for a dark room.
Light-emitting diodes (LED) have been developed to offer the benefits of fluorescence microscopy
without the associated costs. In 2009, the evidence for the efficacy of LED microscopy was
assessed by the World Health Organization (WHO), on the basis of standards appropriate for
evaluating both the accuracy and the effect of new TB diagnostics on patients and public health.
The results showed that the accuracy of LED microscopy was equivalent to that of international
reference standards, it was more sensitive than conventional Ziehl-Neelsen microscopy and it
had qualitative, operational and cost advantages over both conventional fluorescence and Ziehl-
Neelsen microscopy.
On the basis of these findings, WHO recommends that conventional fluorescence microscopy be
replaced by LED microscopy, and that LED microscopy be phased in as an alternative for
conventional Ziehl-Neelsen light microscopy. The switch to LED microscopy should be carefully
phased in at country level, with LED technology that meets WHO specifications. Countries using
LED microscopy should train laboratory staff, validate the technique, introduce appropriate quality
assurance and monitor the effect on TB case detection rates and treatment outcomes.

3
 Policy statement

Fluorescent light-emitting diode (LED) microscopy for diagnosis

of tuberculosis

1. Background
Direct sputum smear microscopy is the most widely used means for diagnosing pulmonary TB
and is available in most primary health-care laboratories at health-centre level. Most laboratories
use conventional light microscopy to examine Ziehl-Neelsen-stained direct smears; this has been
shown to be highly specific in areas with a high prevalence of TB but with varying sensitivity (20
80%).
Fluorescence microscopy is more sensitive (10%) than conventional Ziehl-Neelsen microscopy,
and examination of fluorochrome-stained smears takes less time. Uptake of fluorescence
microscopy has, however, been limited by its high cost, due to expensive mercury vapour light
sources, the need for regular maintenance and the requirement for a dark room.
LED microscopy was developed mainly to give resource-limited countries access to the benefits
of fluorescence microscopy. First, existing fluorescence microscopes were converted to LED light
sources. Considerable research and development subsequently resulted in inexpensive, robust
LED microscopes or LED attachments for routine use in resource-limited settings.
In comparison with conventional mercury vapour fluorescence microscopes, LED microscopes
are less expensive, require less power and can run on batteries; furthermore, the bulbs have a
long half-life and do not pose the risk of releasing potentially toxic products if broken, and LED
microscopes are reported to perform equally well in a light room. These qualities make LED
microscopy feasible for use in resource-limited settings, bringing the benefits of fluorescence
microscopy (improved sensitivity and efficiency) where they are needed most.

2. Evidence for policy formulation

2.1 Synthesis of evidence
In September 2009, WHO assessed the evidence for the efficacy of LED microscopy in a
systematic, structured way. The first step was a systematic review and meta-analysis of
published and unpublished data with standard methods appropriate for studies of diagnostic
accuracy. The second step was the convening of an expert group to evaluate the strength of the
evidence, recommend operational and logistical considerations for use of LED microscopy in
national TB control programmes and identify gaps to be addressed by future research. The third
step was presentation of draft recommendations to the WHO Strategic and Technical Advisory
Group for Tuberculosis (STAG-TB) for endorsement.
In accordance with current WHO standards for evidence assessment in the formulation of policy
recommendations, the grades of recommendation assessment, development and evaluation
(GRADE) system (1) was used by the Expert Group to assess the findings of the systematic
review. This approach provides a systematic, structured framework for evaluating both the
accuracy of new interventions and their impact on patients and public health.
The Expert Groups findings and the final GRADE evaluation (2) were presented to STAG-TB in
November 2009. STAG-TB recognized that the evidence was compelling and that there was a
large body of work on LED microscopy and advised WHO to proceed with policy guidance on its
use (3). STAG-TB also asked WHO to prepare an overarching policy framework to guide the use
of new TB diagnostics, methods and approaches at country level (3). This document provides a
pragmatic summary of the evidence and recommendations for LED microscopy. It should be read
4
in conjunction with the detailed findings of the Expert Group (which include the GRADE tables)
and the WHO framework for using TB diagnostics (2). The framework gives the context for use of
one or more of the currently approved WHO diagnostic tools and methods in relation to country
infrastructure, resources, TB epidemiology and TB policy reform.
The existing TB diagnostic tools are not mutually exclusive: they can be used in various
combinations in country screening and diagnostic algorithms, which are highly setting- and
resource-specific. Expert laboratory input is therefore needed to define the most cost-effective
and efficient algorithms for individual countries, guided by WHO standards (e.g. for laboratory
biosafety) and procedures and in the context of overall, integrated, laboratory strengthening.

2.2 Management of declarations of interest

Expert Group members were asked to submit completed declaration of interest forms, which were
reviewed by the WHO secretariat before the Expert Group meeting. None of the members
declared any conflict of interest. The declaration of interest statements were summarized by the
co-chair of the Expert Group meeting at the start of the meeting. No additional declarations were
made.
Selected individuals with intellectual or research involvement in LED microscopy were invited as
observers to provide technical input and answer technical questions on the methods. These
individuals did not participate in the GRADE evaluation and were asked to leave the meeting
during the final discussions, when the recommendations were developed. They were also not
involved in writing the final meeting report, nor in preparation of the STAG-TB documentation or
the final WHO policy statements.
The process for evidence synthesis and policy development was reviewed by the WHO
Guidelines Review Committee, and the policy recommendations were approved in June 2010.
The target date for review is 2015.

3. Summary of results
Accuracy of LED in comparison with reference standards: LED microscopy showed 84%
sensitivity (95% confidence interval [CI], 7689%) and 98% specificity (95% CI, 8597%)
against culture as the reference standard. When a microscopic reference standard was used,
the overall sensitivity was 93% (95% CI, 8597%), and the overall specificity was 99% (95%
CI, 9899%). A significant increase in sensitivity was reported when direct smears were used
rather than concentrated smears (89% and 73%, respectively).

Accuracy of LED in comparison with Ziehl-Neelsen microscopy: LED microscopy was

statistically significantly more sensitive by 6% (95% CI, 0.113%), with no appreciable loss in
specificity, when compared with direct Ziehl-Neelsen microscopy.

Accuracy of LED in comparison with conventional fluorescence microscopy: LED microscopy

was 5% (95% CI, 011%) more sensitive and 1% (95% CI, -0.7% - 3%) more specific than
conventional fluorescence microscopy.

In qualitative assessments of user characteristics and outcomes in relation to implementation,

such as time to reading, cost-effectiveness, training and smear fading, the main findings were:

o In comparison with Ziehl-Neelsen, LED showed similar gains in time for reading as
conventional fluorescence microscopy, with about half the time for smear examination.

o Cost assessments predict better cost-effectiveness with LED than with Ziehl-Neelsen
microscopy, with improved efficiency.

5
 o Qualitative assessments of LED microscopy confirmed many anticipated advantages,
including use of the devices without a dark room, durability and portability (in the case of
attachment devices); user acceptability in all field studies was reported as excellent.

LED may be useful for diagnosing other diseases, e.g. malaria and trypanosomiasis, reducing
the costs involved in providing integrated laboratory services.

Possible barriers to widescale use of LED include training of laboratory staff unfamiliar with
fluorescence microscopy and the fading of inherently unstable fluorochrome stains. Evidence
from standardized training suggests that full proficiency in LED microscopy can be achieved
within 1 month.

Adequate evidence is available to recommend the use of auramine stains for LED
microscopy. Other commercial and in-house fluorochrome stains are not recommended.

Evidence on the effect of fading of fluorochrome stains on the reproducibility of smear results
over time suggests that current external quality assurance programmes should be adapted.

The introduction of LED might affect the cost of other diagnostic modalities, e.g. light
microscopy for examining urine, stools and blood, which should be retained at peripheral
health laboratory level.

No studies were found on the direct effect of LED microscopy on outcomes important to
patients, such as cure and treatment completion.

Further research is required on the outcomes of LED microscopy that are important to
patients and on combinations of LED microscopy with novel approaches for early case
detection or sputum processing.

4. Policy recommendations
The GRADE process confirmed that there is sufficient generalizable evidence to recommend
strongly the use of LED microscopy. WHO therefore recommends that:
conventional fluorescence microscopy be replaced by LED microscopy with auramine
staining in all settings where fluorescence microscopy is currently used;
LED microscopy be phased in as an alternative to conventional Ziehl-Neelsen light
microscopy in both high- and low-volume laboratories;
the switch to LED microscopy be made according to a carefully phased implementation plan,
with LED technology that meets WHO specifications;
countries using LED microscopy address the following issues:
- training requirements, especially for laboratory staff unfamiliar with fluorescence
microscopy techniques;
- country validation, i.e. demonstrating equivalent performance of LED with Ziehl-Neelsen
or conventional fluorescence microscopy at country level during the introductory phase;
- introduction of WHO-endorsed programmes for internal quality control and external
quality assurance; and
- monitoring of trends in TB case detection and treatment outcomes after introduction of
LED microscopy.

6
WHO will assist countries in the use of LED microscopy by:
preparing and disseminating technical specifications for LED devices to guide countries,
technical and funding agencies in the purchase of high-quality equipment;
preparing and disseminating standard operating procedures for LED microscopy;
preparing and disseminating programmes for internal quality control and external quality
assurance for LED microscopy; and
facilitating, with partners and technical agencies, a coordinated approach to standardized
training on LED microscopy at country level.

5. Intended audience
This policy statement should be used to guide the use of LED microscopy for TB diagnosis in
national TB control programmes. It is intended for use by national TB control programme
managers and laboratory directors, in coordination with external laboratory consultants, donor
agencies, technical advisors, laboratory technicians, laboratory equipment procurement officers,
warehouse managers, other service providers, other relevant government officials and
implementing partners involved in country-level TB laboratory strengthening. People responsible
for programme planning, budgeting, resource mobilization and training for TB diagnostic services
may also benefit from reading this document.

6. References
1. http://www.gradeworkinggroup.org
2. http://www.who.int/tb/laboratory/policy_statements/en/index.html
3. http://www.who.int/tb/advisory_bodies/stag/en/index.html

7
7. Annexes
Annex 1: Expert Group Members

Dr Maryline Bonnet
Epicentre, c/o MSFCH Prof Paul R Klatser
78 Rue de Lausanne Co-Chair of Subgroup on Optimizing Smear
116 - CH, 1211 Geneve Microscopy, STP New Diagnostics Working
Switzerland Group
e-mail: [email protected] Head of Department
KIT Biomedical Research
Dr Saidi Egwaga Royal Tropical Institute
National TB/Leprosy Programme Meibergdreef 39
Ministry of Health, TB/Leprosy Unit 1105 AZ Amsterdam
PO Box 9083 The Netherlands
Dar Es Salaam e-mail: [email protected]
Tanzania
e-mail: [email protected] Dr Madhukar Pai
Co-Chair of Subgroup on Evidence
Dr Bernard Fourie Synthesis for TB Diagnostics, STP New
Chief Scientific Officer and Director of South Diagnostics Working Group
African Operations Assistant Professor
Medicine in Need Inc. USA and Medicine in Department of Epidemiology, Biostatistics &
Need South Africa (Pty) Ltd Occupational Health
PO Box 12660, Queenswood 0121 McGill University
Pretoria 1020 Pine Avenue West
South Africa Montreal, H3A 1A2
e-mail: [email protected] Canada
e-mail: [email protected]
Dr Christy Hanson
Chair of STP Retooling Task Force Dr John Ridderhof
US Agency for International Development Chair of STP Global Laboratory Initiative
(USAID), USAID/BGH/HIDN/ID Working Group
3.7.23, 3rd Floor, Ronald Reagan Bldg Associate Director for Laboratory Science
20523-5900 - Washington, DC National Center for Preparedness, Detection
USA and Control of Infectious Diseases, CCID
e-mail: [email protected] Centers for Disease Control and Prevention
1600 Clifton Rd NE,
Dr Moses Joloba MS-C12
Head of National TB Reference Laboratory Atlanta, Georgia 30333
Department of Medical Microbiology USA
Microbiology-Pathology Building e-mail: [email protected]
Uganda
e-mail: [email protected] Dr Bertie Squire
Co-Chair of Subgroup on TB Diagnostics
Dr Marija Joncevska and Poverty, STP New Diagnostics Working
Regional laboratory specialist Group
Project HOPE Central Asia Liverpool School of Tropical Medicine
162 Kunaeva st. Pembroke Place
050010 Almaty Liverpool
Kazakhstan UNITED KINGDOM
e-mail: [email protected] e-mail: [email protected]

8
 Dr Jessica Minion
Dr Karen Steingart Medical Microbiology
Francis J. Curry National Tuberculosis MSC Epidemiology
Center McGill University
University of California, San Francisco 1020 Pine Avenue West
3180 18th Street, Suite 101 Montreal, H3A 1A2
San Francisco, CA 94110-2028 Canada
USA Email: [email protected]
e-mail: [email protected]
Dr Catharina Boehme
Mr Javid Syed Foundation for Innovative New Diagnostics
TB/HIV Project Director (FIND)
Treatment Action Group 71 Avenue Louis-Casai
611 Broadway, Suite 308 1216 Geneva
New York, NY 10012 Switzerland
USA e-mail:
e-mail: [email protected]
[email protected]
Dr C N Paramasivan
Dr Armand van Deun Foundation for Innovative New Diagnostics
Bacteriology Consultant (FIND)
International Union Against Tuberculosis 71 Avenue Louis-Casai
and Lung Disease 1216 Geneva
Mycobacteriology Unit Switzerland
Institute of Tropical Medicine e-mail:
Nationalestraat 155 [email protected]
B-2000 - Antwerpen
Belgium Dr Eric Adam
e-mail: [email protected] Foundation for Innovative New Diagnostics
(FIND)
Dr Adithya Cattamanchi 71 Avenue Louis-Casai
Assistant Professor of Medicine 1216 Geneva
San Francisco General Hospital Switzerland
Division of Pulmonary and Critical Care email: [email protected]
Medicine
Room 5K1 Dr Richard Bumgarner
1001 Potrero Avenue Independent Consultant
San Francisco, California 94110 Health Economics and Finance
Email: [email protected] Program and Institutional Evaluations
Tuberculosis Control Adviser
1715 Abbey Oak Drive
Vienna, VA 22182
USA
e-mail: [email protected]

9
Annex 2 : WHO staff members

Leopold Blanc: [email protected]

Jean Iragena: [email protected]
Paul Nunn: [email protected]
Karin Weyer: [email protected]
Matteo Zignol: [email protected]

WHO-TDR

L Cuevas: [email protected]
F Moussy: [email protected]
Andrew Ramsay: [email protected]
S Swaminathan: [email protected]
Sanne Van Kampen: [email protected]

WHO-OTHER

WHO Guidelines Review Committee Secretariat:

grcsecretariat@who

10
Annex 3 : STAG-TB members

Mr Faruque Ahmed Prof Francis Drobniewski

Director, Health Program Director, Health Protection Agency
BRAC National Mycobacterium Reference Unit
Dhaka Institute for Cell and Molecular Sciences,
Bangladesh Barts and the London School of Medicine
London
Ms Olayide Akanni United Kingdom
Executive Director
Journalists Against AIDS (JAAIDS) in Nigeria Dr Wafaa El-Sadr
Abuja Director, CIDER
Nigeria Mailman School of Public Health
Columbia University
Dr Salah Al Awaidy New York, NY
Director USA
Department of Communicable Disease
Surveillance & Control Dr Paula I Fujiwara
DGHA, HQ, Ministry of Health Director, Dept of HIV and Senior Advisor
Muscat The Union (Paris, France)
Oman USA

Dr R V Asokan Mr Case Gordon

RNTCP Project Coordinator World Care Council
Indian Medical Association Viols en Laval
Punalur - Kerala France
India
Prof Vladimir Malakhov
Dr Kenneth Castro National Center for External Quality
Director, Division of TB Elimination Assessment in Laboratory Testing
Centers for Disease Control and Prevention of Russian Federation
Atlanta, GA Moscow
USA Russia

Dr Jeremiah Muhwa Chakaya Dr Mao Tan Eang

STAG-TB Chair Advisor to the Minister of Health
Technical Expert Director, National Center for Tuberculosis and
National Leprosy and TB Programme Leprosy Control
Ministry of Health Ministry of Health
Nairobi Phnom Penh
Kenya Cambodia

Dr Elizabeth Corbett Dr Megan Murray

Reader in Infectious and Tropical Diseases Associate Professor of Epidemiology
London School of Tropical Medicine & Hygiene Harvard University School of Public Health
and MLW Research Programme Department of Epidemiology
Blantyre Boston, MA
Malawi USA

11
Dr Yogan Pillay Dr Pedro Guillermo Suarez
Deputy Director General TB & TB-HIV/AIDS
Strategic Health Programmes Center for Health Services
Pretoria Management Sciences for Health
South Africa Arlington, VA USA

Dr Minghui Ren Dr Rosalind G Vianzon

Director-General National TB Programme Manager
Department of International Cooperation National Center for Disease Control and Prevention
Ministry of Health Department of Health
Beijing Manila
China Philippines

Dr Marieke van der Werf Prof Wang Longde

Head, Unit Research, Senior Epidemiologist Dean, School of Public Health
KNCV Tuberculosis Foundation Peking University
The Hague Beijing
The Netherlands China

Dr Rajendra Shukla Dr Yuthichai Kasetjaroen

Joint Secretary Director
Ministry of Health & Family Welfare Bureau of Tuberculosis
New Delhi Ministry of Health
India Bangkok
Thailand

12
For further information please contact:

Stop TB Department
World Health Organization
20 Avenue Appia
1211 Geneva 27
Switzerland

Telephone: + 41 22 791 21 11
Facsimile (fax): + 41 22 791 31 11

Website: http://www.who.int/topics/tuberculosis/en/
ISBN 978 92 4 150161 3