MANAGEMENT OF

TUBERCULOSIS

A Guide For
Low Income Countries

Fifth edition
2000

Donald A Enarson, Hans L Rieder, Thuridur Arnadottir, Arnaud Trébucq

International Union Against Tuberculosis

and Lung Disease
68 boulevard Saint-Michel, 75006 Paris, France

The publication of this Guide was made possible thanks to the support of
MISEREOR, Postfach 1450, Mozartstrasse 9, 5100 Aachen, Germany
Editor:
International Union Against Tuberculosis and Lung Disease (IUATLD)
68 boulevard Saint-Michel, 75006 Paris, France
Authors: D.A. Enarson, H.L. Rieder, T. Arnadottir, A. Trébucq

© International Union Against Tuberculosis

and Lung Disease (IUATLD)

June 2000

All rights reserved.

No part in this publication may be reproduced without the prior permission

of the authors, the publisher, and the publishing house.

ISBN: 2-914365-00-4

II
 PREFACE

While tuberculosis has declined considerably in industrialised countries,

the disease still poses a serious and even increasing problem in many low
income countries, affecting the health and social welfare of millions of
people. Fighting tuberculosis is a challenge to all who are concerned about
health and development. Thanks to modern antituberculosis medications,
it has become possible to cure practically all patients suffering from this
potentially fatal disease. Successful treatment, however, presupposes ade-
quate medication, close supervision of staff, direct observation of med-
ication swallowing and monitoring of treatment results by bacteriological
examination. Our project partners, including religious and other non-gov-
ernmental organisations, have repeatedly expressed their need for a con-
cise description of how to recognise, cure and combat tuberculosis today
in low income countries.
We have joined together with the International Union Against
Tuberculosis and Lung Disease in offering this Guide. The first edition of
10,000 copies, in English and French, was out of print in four years. The
second edition of 18,000 copies in English, French and Spanish, was out
of print in three years. It has also been translated into Chinese and
Mongolian. The third completely revised edition, in English, French and
Spanish, was published in 1994; 10,000 copies were out of print within
eighteen months. It was slightly edited in a fourth edition of which
10,000 copies were distributed, and which was out of print within two years.
The Guide contains a description of tuberculosis and its identification.
The treatment of tuberculosis and the organisation and management of tuber-
culosis services and the structure within which such services can be deliv-
ered, even under the most stringent socio-economic conditions, is outlined.
The tuberculosis situation is evaluated, and the interventions designed to
bring it under control are discussed. This fifth edition has been thoroughly
edited. The order of presentation has been revised to provide a more log-
ical flow of ideas. The new edition addresses issues that previously lacked
explanation, most notably the resistance of Mycobacterium tuberculosis to
medications and its impact on the management of tuberculosis.
We hope this will be a useful guide for tuberculosis control for those
who are valiantly shouldering the seemingly overwhelming task in remote
rural areas and in overcrowded urban slums. It will also be of interest to

III
health planners and co-ordinators as well as those in charge of training
health workers.
Professor Dr. Josef Sayer
Executive Director, Misereor

Acknowledgements
The following persons are gratefully acknowledged for their contribution
to the production of this Guide: Dr Raul Diaz for the organisation of
production and distribution, Ms Clare Pierard for reviewing the text. We
acknowledge Dr Magdalene Oberhoffer and Professor Heinrich Jentgens,
whose inspiration led to the production of the earliest versions of this Guide.
We would like to thank our colleagues, Professor Nadia Aït-Khaled and
Dr Jose Caminero Luna for their review and comments on the text. We
also express our admiration and respect for Drs Annik Rouillon and Karel
Styblo, from whom we learned the basics of our knowledge of this work.

IV
 TABLE OF CONTENTS

I. INTRODUCTION. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

II. TUBERCULOSIS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
A. What do we know about this disease? . . . . . . . . . . . . . . . . . . . . . . . . . 3
1. What is tuberculosis? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2. How does tuberculosis develop?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
3. How does HIV affect tuberculosis? . . . . . . . . . . . . . . . . . . . . . . . . . 4
4. What is drug resistance and how does it develop?. . . . . . . . . . 5
B. How is tuberculosis diagnosed?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
1. When is tuberculosis likely to be present? . . . . . . . . . . . . . . . . . . 6
2. Where is tuberculosis most likely to be found? . . . . . . . . . . . . . 6
3. How is a diagnosis of tuberculosis confirmed?. . . . . . . . . . . . . . 7
4. Who should be considered a “case” of tuberculosis?. . . . . . . . 9
5. How does HIV infection influence the diagnosis? . . . . . . . . . . 10
6. How do we know if a patient has drug resistance? . . . . . . . . . 10

III. TREATING THE DISEASE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

A. How is tuberculosis treated?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
1. What are the principles of treatment of tuberculosis? . . . . . . . 11
2. What if the patient has previously been treated? . . . . . . . . . . . . 12
3. What is directly observed treatment and how is it used? . . . 12
4. What do we use for treating tuberculosis? . . . . . . . . . . . . . . . . . . 13
B. What factors might affect treatment? . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
1. How does HIV affect treatment? . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
2. How does drug resistance affect treatment? . . . . . . . . . . . . . . . . . 22
3. What if the patient is pregnant or breast feeding? . . . . . . . . . . 23
C. What about those exposed to tuberculosis? . . . . . . . . . . . . . . . . . . . . . 23
1. What is preventive therapy and its role? . . . . . . . . . . . . . . . . . . . . 23
2. Can a patient on treatment infect you?. . . . . . . . . . . . . . . . . . . . . . 24

IV. CARING FOR THE PATIENTS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

A. How should the patients be followed?. . . . . . . . . . . . . . . . . . . . . . . . . . 25
1. Are all the patients on treatment? . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

V
 2. How can we encourage full participation of the patient? . . . 25
3. How do we monitor progress during treatment? . . . . . . . . . . . . 26
B. What is the most efficient way to deliver tuberculosis services? . 27
1. What is the proper structure of tuberculosis services? . . . . . . 27
2. How should the services be organised? . . . . . . . . . . . . . . . . . . . . . 28
C. How is the laboratory service organised?. . . . . . . . . . . . . . . . . . . . . . . 31
1. What is the basis of the laboratory examination? . . . . . . . . . . . 31
2. What are the aims of the laboratory service? . . . . . . . . . . . . . . . 32
D. How do we monitor care? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
1. What records are necessary? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
2. How are the results reported? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
E. What supplies are needed and how are they managed? . . . . . . . . 42
1. How are supplies of medications managed?. . . . . . . . . . . . . . . . . 42
2. How are laboratory supplies managed?. . . . . . . . . . . . . . . . . . . . . . 44
3. What other supplies are needed? . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

V. PROTECTING THE COMMUNITY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

A. What is the rationale for a tuberculosis programme?. . . . . . . . . . . 46
1. Why do we believe tuberculosis can be controlled? . . . . . . . . 46
2. Can tuberculosis be prevented by vaccination?. . . . . . . . . . . . . . 48
B. What should be done if there is no programme? . . . . . . . . . . . . . . . 48
1. Why is a programme important? . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
2. How can care be given safely?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
C. How can we ensure that activities achieve good results?. . . . . . . 50
1. How do we evaluate control measures? . . . . . . . . . . . . . . . . . . . . . 50
2. What is the size of the tuberculosis problem? . . . . . . . . . . . . . . 51
3. How will HIV affect the situation? . . . . . . . . . . . . . . . . . . . . . . . . . 52
4. Will patients respond to treatment?. . . . . . . . . . . . . . . . . . . . . . . . . . 53

VI. APPENDIX 1
Technical Guide for Smear Microscopy. . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

VII. APPENDIX 2
Forms. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91

VI
 I. INTRODUCTION

Tuberculosis is a great problem in most low income countries; it is the sin-

gle most frequent cause of death in individuals aged fifteen to forty-nine
years. For this reason, activities directed against tuberculosis as a public
health problem are the direct responsibility of government health authorities.
The International Union Against Tuberculosis and Lung Disease is the
oldest international non-governmental organisation dealing with health. It
has gained immense experience in collaborating with partners in providing
care for more than one million tuberculosis patients in some of the poor-
est countries in the world, through the vehicle of the National Tuberculosis
Programme. This Guide summarises that experience.
In setting out to combat a problem like tuberculosis, it is essential to
have a clear concept of aims and priorities. The aims of the fight against
tuberculosis are:

– for a community: to reduce the spread of tuberculous infection, and

by this means to hasten the disappearance of this disease from society.

– for individual patients: to cure their disease, to quickly restore their

capacity for activities of daily living and to preserve their position in
their family and community.

Of the priorities of tuberculosis activities, the first is the treatment and

cure of tuberculosis patients, especially those patients who are the source
of transmission of infection with tuberculosis micro-organisms. Because
tuberculosis is so frequent and is such a serious disease, it must be a high
priority for all who provide health care in low income countries, and tuber-
culosis services must be included in all health services provided.
Tuberculosis can be controlled successfully only in the context of a
National Tuberculosis Programme (NTP). Such a programme must operate
within the routine health service of each country. Paramedical personnel as
part of the many activities of the general health services usually perform
the everyday tuberculosis activities (case finding and treatment). It is essen-
tial that such personnel be properly trained, motivated and supervised.
The general population must be mobilised to participate, including
community organisations as well as groups of health professionals. It is

1
important to make clear to the population that tuberculosis is curable and
that there is no basis for discrimination or stigma. Community participa-
tion is essential to encourage individuals with symptoms suggestive of tuber-
culosis to present themselves to the health services for diagnostic exami-
nation and to ensure that tuberculosis patients continue to take their treatment
until they are cured.
In many countries, non-governmental organisations provide tuberculo-
sis services. They often work under difficult conditions in remote areas
where they provide the only medical services available. Their activities
should, nevertheless, always be undertaken in co-ordination with govern-
ment offices and must follow the guidelines of the National Tuberculosis
Programme.

2
 II. TUBERCULOSIS

A. What do we know about this disease?

1. What is tuberculosis?
Tuberculosis is an infectious disease, caused in most cases by micro-
organisms called Mycobacterium tuberculosis. The micro-organisms usually
enter the body by inhalation through the lungs. They spread from the ini-
tial location in the lungs to other parts of the body via the blood stream,
the lymphatic system, via the airways or by direct extension to other organs.

– Pulmonary tuberculosis is the most frequent form of the disease, occur-

ring in over 80% of cases. This is the form of tuberculosis which may
be infectious.

– Extra-pulmonary tuberculosis is tuberculosis affecting organs other than

the lungs, most frequently pleura, lymph nodes, spine, joints, genito-
urinary tract, nervous system or abdomen. Tuberculosis may affect any
part of the body.

2. How does tuberculosis develop?

Tuberculosis develops in the human body in two stages. The first stage
occurs when an individual who is exposed to micro-organisms from an
infectious case of tuberculosis becomes infected (tuberculous infection), and
the second is when the infected individual develops the disease (tubercu-
losis).

2.1 How are tuberculosis micro-organisms spread?

How likely it is that a patient with tuberculosis may infect another person
is determined by the concentration of micro-organisms within the lungs and
their spread into the surrounding air. Patients with pulmonary tuberculosis
in whom the micro-organisms are so numerous as to be seen on micro-
scopic examination of sputum specimens (smear positive cases) are the most
infectious cases. Those in whom micro-organisms cannot be seen directly

3
under the microscope (smear negative cases) are very much less infectious
and the severity of their disease is usually less than that of the smear pos-
itive cases. Extra-pulmonary cases are almost never infectious, unless they
have pulmonary tuberculosis as well.
The infectious tuberculosis patient expels micro-organisms into the air
in tiny droplets when coughing, laughing or sneezing. These small droplets
dry rapidly, become droplet nuclei carrying the micro-organisms, and may
remain suspended in the air for several hours. Any person entering the room
may inhale these droplet nuclei. If the micro-organisms establish themselves
in the lungs of the person who inhaled them, and begin to multiply, infec-
tion has occurred. Exposure to the micro-organisms is greatest among those
in close and prolonged contact with an infectious case (i.e., those living in
the same household).
The micro-organisms are rapidly destroyed by exposure to sunlight and
their concentration in the air is reduced by good ventilation. Except in the
event of close and prolonged contact with an infectious case of tuberculo-
sis, the chance of becoming infected from a single contact with a tubercu-
losis patient is very small. Most individuals who become infected have no
symptoms or evidence of illness in association with this infection.

2.2 What happens after infection

Among those who do become infected, most (possibly 80-90%) will never
become ill with tuberculosis unless their immunity is seriously compro-
mised. The micro-organisms remain dormant within the body and their pres-
ence is indicated only by a significant size of induration in reaction to a
tuberculin skin test. Some individuals who have become infected subse-
quently develop disease from this infection (termed tuberculosis). They are
most likely to develop disease in the period immediately following infec-
tion, but continue to experience a risk of tuberculosis throughout the remain-
der of their lives.

3. How does HIV affect tuberculosis?

Infection with the human immunodeficiency virus (HIV) leads to extensive
destruction of the immune defence mechanisms of the body. As a result,
those infected with HIV become ill with severe and often deadly diseases
to which persons without HIV infection would not usually be susceptible.
The development of tuberculosis following infection with tuberculosis micro-

4
organisms is usually prevented by the actions of the immune system; this
explains why only a relatively small proportion of those individuals who
have been infected with tuberculosis go on to become ill with the disease.
When the protection provided by the immune system is reduced by HIV
infection, the tuberculosis micro-organisms that are dormant within the body
of an individual who has been infected begin to multiply, causing tuber-
culosis.

4. What is drug resistance and how does it develop?

Clinically important resistance to medications is always a man-made prob-
lem. Because large populations of tuberculosis micro-organisms always con-
tain some mutants naturally resistant to medications, a substantial popula-
tion of resistant micro-organisms is always selected when a single medication
is used to treat a patient with a large population of micro-organisms. This
occurs because only the micro-organisms susceptible to the medications are
killed, leaving the resistant micro-organisms to multiply. When the micro-
organisms in a patient are resistant to all but one of the medications given
to that patient, the treatment has the same result as when a single medica-
tion is given alone.
There are two important types of resistance to medications in tuber-
culosis micro-organisms:

– acquired or secondary resistance is due to incorrect treatment; for

instance, treatment with a single powerful medication in patients with
smear positive pulmonary tuberculosis (this is sometimes referred to
as “monotherapy”), or administration of powerful medications to a
patient harbouring tuberculosis micro-organisms resistant to all but one
of the medications which the patient is given (this phenomenon is
sometimes referred to as “effective monotherapy”);

– primary resistance occurs when a patient develops tuberculosis after

being infected by another patient who has resistant micro-organisms.

Micro-organisms with resistance to at least the two most important med-

ications, isoniazid and rifampicin, are termed “multidrug-resistant”.

5
B. How is tuberculosis diagnosed?

1. When is tuberculosis likely to be present?

The most frequent symptoms of pulmonary tuberculosis are:

– persistent cough for 3 weeks or more; every patient presenting to a

health facility with this symptom should be designated a “tuberculo-
sis suspect”;

– sputum production which may be blood-stained (termed haemoptysis),

shortness of breath and chest pain;

– loss of appetite and loss of weight, a general feeling of illness (malaise)

and tiredness (fatigue), night sweats and fever.

A patient presenting with these symptoms who is, or was, in contact with
a person with infectious tuberculosis is all the more likely to be suffering
from tuberculosis.
Symptoms of extra-pulmonary tuberculosis depend on the organ
involved. Chest pain from tuberculous pleurisy, enlarged lymph nodes and
sharp angular deformity of the spine are the most frequent signs of extra-
pulmonary tuberculosis.

2. Where is tuberculosis most likely to be found?

Tuberculosis cases are most frequently found in the following circumstances:

– among patients who present themselves on their own initiative at a

health facility, with symptoms suggesting tuberculosis;

– among those (especially children and young adults) living in the same
household with smear positive patients;

– in those with an abnormality on a chest radiograph which has the

appearance of tuberculosis.

Tuberculosis will be detected most efficiently where health care providers

and community members are highly conscious of the symptoms suggestive
of tuberculosis.

6
3. How is a diagnosis of tuberculosis confirmed?

3.1. What is the value of bacteriology?

Every individual suspected of having tuberculosis must have an examina-
tion of sputum to determine whether or not they have infectious tubercu-
losis. This must be done prior to the commencement of their treatment. The
examination consists of microscopic examination of a specimen of sputum
that has been spread on a slide and stained by the Ziehl-Neelsen method
(smear microscopy). If micro-organisms (frequently referred to as acid-fast
bacilli, or AFB) are detected by this method, the patient is said to have
smear positive tuberculosis. Smear microscopy is the only means by which
the diagnosis of tuberculosis can be confirmed in most low income coun-
tries. It is important to carry out because it efficiently identifies the cases
that are most infectious and therefore have the highest priority for care. In
many low income countries, a different form of treatment is given to infec-
tious cases than is given to those cases who are not infectious.
Whenever tuberculosis is suspected, three specimens must be collected
for examination by microscopy. Whenever possible, they should be obtained
within twenty-four hours, as follows:

First specimen – At the first interview with the patient a spot specimen is
collected; this specimen is obtained on the spot, after coughing and
clearing the back of the throat, under the supervision of a staff mem-
ber, in a well ventilated area, preferably in the open air.

Second specimen – The patient is then given a sputum container for col-
lection of an early morning specimen (early morning specimen) before
the second interview, which should be on the next working day.

Third specimen – On the second interview with the patient, another spot
specimen is collected.

Should the first spot specimen be positive and should the patient not return
for the second interview, an immediate search must be made to find the
patient in order to prevent transmission of micro-organisms in the commu-
nity and deterioration of the patient’s condition. A diagnosis should always
be confirmed by a second positive sputum specimen. A Medical Officer
should review any patient who is positive on only a single specimen.

7
 With three consecutive early morning specimens, it has been repeat-
edly found that of those patients who are eventually demonstrated to be
positive, approximately 80% are positive on the first examination, an addi-
tional 15% are first demonstrated to be positive on the second and an addi-
tional 5% on the third examination. An early morning specimen is more
likely to be positive than a spot specimen. Thus, the yield from a third,
spot, specimen might be expected to be lower. Consequently, when the
workload of the laboratory is excessive, it might be reasonable to routinely
examine only two specimens, rather than three. In this case, should a patient
be judged to require treatment, even though the two specimens are nega-
tive, a third specimen should be examined.
Prior to commencing treatment, a Medical Officer should review all
those thought to have tuberculosis but in whom the sputum smears are neg-
ative. The Medical Officer may wish to proceed in the following manner in
order to determine whether or not the patient actually has tuberculosis. If
chest radiography is available, it may be performed. If the chest radiograph
demonstrates shadows in the lung fields consistent with a pulmonary infec-
tion, a course of broad spectrum antibiotics may be given. If the patient
continues to show symptoms after completion of the antibiotics, a second
series of 3 sputum smear examinations may be performed and, if still neg-
ative, the Medical Officer may choose to treat the patient for tuberculosis
and record the patient as a case of pulmonary tuberculosis, smear negative.

3.2 Is radiology useful?

Diagnosis by means of radiographic examination in patients suspected of
tuberculosis is unreliable. Abnormalities identified on a chest radiograph
may be due to tuberculosis or to a variety of other conditions, and the
appearance on the radiograph is not specific for tuberculosis. Some indi-
viduals who have previously had tuberculosis that is now healed (and there-
fore does not require treatment) may have a chest radiograph that resem-
bles tuberculosis requiring treatment. Chest radiographs may be helpful in
those patients who are not sputum smear positive, but they can be read
reliably only by a competent Medical Officer.

3.3 What about the tuberculin test?

A tuberculin skin test is sometimes used to help in the diagnosis of tuber-
culosis. The interpretation of a test result is often very difficult, as a pos-
itive test may not be caused by tuberculosis and a negative test does not

8
always rule it out. Furthermore, tuberculin is not routinely available in many
peripheral health institutions, it is expensive, has a very short expiry date,
must be kept protected from light and heat and requires some technical
skills in administration and reading. Thus in most instances health care
workers are forced to work without this test.

3.4 How is tuberculosis diagnosed in children?

Diagnosis of tuberculosis in children is quite difficult. It should be remem-
bered that, in the majority of instances (with the exception of disseminated
tuberculosis, tuberculous meningitis, spinal tuberculosis and tuberculosis in
immunosuppressed children), childhood tuberculosis is a mild disease that
heals on its own, even with minimal or no treatment. Nevertheless, chil-
dren with tuberculosis should be treated to prevent complications and to
ensure that they do not subsequently develop tuberculosis from reactivation
of their infection. Only a very small proportion of children have tubercu-
losis that is smear positive, and many children cannot produce sputum for
examination. Points of most importance in determining a diagnosis in chil-
dren, in order of priority, are:
– a history of contact with a case of infectious tuberculosis, particularly
in the same household;
– an abnormal chest radiograph showing unilateral lymphadenopathy
and/or shadows in the lung field indicating infiltration;
– a positive tuberculin skin test, where such a test is available.
In the absence of all of the above, it is highly unlikely that the child has
tuberculosis. Any child whose tuberculin skin test remains consistently neg-
ative over some months of observation, while the clinical condition is good
or shows improvement, does not have tuberculosis.
Any child under 5 years of age, in contact with a smear positive case
and with signs or symptoms suggesting tuberculosis, should be regarded as
having active tuberculosis and should be given a full course of treatment.
Those without signs or symptoms of disease should be considered for pre-
ventive chemotherapy.

4. Who should be considered a “case” of tuberculosis?

Any person given treatment for tuberculosis should be recorded as a case.
Those who have tuberculosis micro-organisms visible on two microscopic

9
examinations of sputum should be recorded as smear positive. All other
cases should be recorded in such a way as to distinguish them from smear
positive cases (as smear negative or as extra-pulmonary cases).

5. How does HIV infection influence the diagnosis?

Tuberculosis cases associated with HIV infection are, in the majority of
instances, indistinguishable from other cases. Smear positive cases are equally
identifiable whether or not they are infected with HIV. Some cases of tuber-
culosis associated with HIV infection may show unusual clinical features
and there may be an increase in the overall proportion of cases that are
smear negative and/or extra-pulmonary. Nevertheless, sputum smear exam-
ination remains an essential component in the diagnosis of tuberculosis in
those countries where HIV infection is frequent, because of its ability to
identify the infectious cases, and because the majority of patients with pul-
monary tuberculosis and HIV infection are found to be smear positive.

6. How do we know if a patient has drug resistance?

The confirmation of the diagnosis of tuberculosis in most low income coun-
tries is based on sputum smear microscopy. To detect resistance, it is nec-
essary to culture the micro-organisms and subsequently perform tests to
determine their susceptibility to medications. These methods are complex
and expensive, and are not routinely available in most low income coun-
tries. Treatment is given without knowledge of the susceptibility of the
micro-organisms to the medications.
If drug resistance is already present, there is a possibility that the treat-
ment might create more resistance. The recommendations put forward in
this Guide were developed specifically in order to prevent this from occur-
ring. Changes to the recommendations may compromise the balance nec-
essary to prevent resistance. On the other hand, when the recommendations
are strictly followed, tuberculosis can be successfully treated in the vast
majority of cases, without knowledge of the susceptibility patterns of indi-
vidual patients, and without promoting drug resistance.

10
 III. TREATING THE DISEASE

A. How is tuberculosis treated?

If the diagnosis of tuberculosis is made at an early stage of the disease and
the patient is not seriously ill (either from tuberculosis or from other dis-
ease), it is possible to cure virtually any case of tuberculosis. This is achieved
if they are treated properly and the micro-organisms causing their disease
are not resistant to the medications frequently used for treatment of tubercu-
losis. Patients with multidrug-resistant tuberculosis (caused by micro-
organisms that are resistant to both isoniazid and rifampicin) are difficult,
if not impossible, to cure.

1. What are the principles of treatment of tuberculosis?

1.1 What is the basis of treatment?

The basis of the treatment of tuberculosis is chemotherapy. It is also one
of the most efficient means of preventing the spread of tuberculosis micro-
organisms. The requirements for adequate chemotherapy are:

– an appropriate combination of antituberculosis medications to prevent

the development of resistance to those medications;

– prescribed in the correct dosage;

– taken regularly by the patient;

– for a sufficient period to prevent relapse of the disease after comple-

tion of treatment.

Treatment must be given to every patient confirmed as having tuberculo-

sis, and must be given free of charge to the patients.

1.2 When should treatment be started?

Treatment should not be commenced until a firm diagnosis is made.
Treatment should always be started as soon as possible after two labora-

11
tory reports are received indicating smear positive examinations, or if the
patient is severely ill and the clinical suspicion of tuberculosis is high. A
Medical Officer should determine treatment for those with only a single
positive report or with negative reports.

1.3 What are the phases of treatment?

Treatment of smear positive cases should always include an initial inten-
sive phase. An initial course of the combination of medications recom-
mended in this Guide is effective in eliminating micro-organisms and in
minimising the influence of micro-organisms that are resistant to medica-
tions. The intensive phase in those patients who are initially smear posi-
tive should be given for a minimum of 2 months and continued until they
become smear negative, but for no longer than a total of 3 months (the
majority of cases will already be negative at 2 months). The intensive phase
is a very important part of the chemotherapy.
The continuation phase is important to ensure that the patient is per-
manently cured and does not relapse after completion of treatment. The
continuation phase does not require as many medications, but does require
a sufficient duration to ensure success.

2. What if the patient has previously been treated?

Before treatment is started, it is essential to question all patients closely
and carefully to determine whether or not they have previously taken treat-
ment for tuberculosis. Sputum smear positive patients who have been pre-
viously treated for as much as one month should be suspected of having
micro-organisms resistant to one or more medications. Such patients require
a different form of treatment from those who have never been previously
treated. A Medical Officer should carefully supervise their care.

3. What is directly observed treatment and how is it used?

The regimens proposed in this Guide will cure most newly diagnosed cases
of tuberculosis. To achieve this, it is vital that the patient takes the total
quantity of medication prescribed. To ensure that this occurs, frequent and
careful supervision is necessary. Whenever rifampicin is given to a patient,
a health worker must directly observe that the patient swallows every dose
of the combination of medications given. This will require the patient to
be present for direct administration on a daily basis for the total period

12
during which rifampicin is given. This is usually accomplished on an
ambulatory basis if the patient can attend the treatment centre daily.
Occasionally it will require that the patient has accommodation arranged
at the treatment centre, in a hostel or in some other location. When the
patient is very ill, it may be necessary for the patient to be admitted to
hospital.
The continuation phase does not contain rifampicin and is usually given
in monthly supplies for daily, self-administered intake (except in the case
of retreatment, where rifampicin is given). This limits the duration of time
required for the patient to attend the health service daily, freeing the patient
to return to normal daily activities after the initial intensive phase, when
the patient is usually strong enough to do so.
When the patient has completed the prescribed duration of treatment,
the medications should be stopped. Additional chemotherapy is unneces-
sary if all the medications prescribed have been taken. Although it is dis-
tinctly unusual for tuberculosis to relapse after adequate treatment, patients
should be told to report for re-examination if symptoms suggesting tuber-
culosis recur.

4. What do we use for treating tuberculosis?

There are only a limited number of medications currently available for the
treatment of tuberculosis. For this reason they must be used with great care
in order not to create resistance to these medications. The presence of resis-
tance, and particularly of multidrug resistance, makes the treatment much
less likely to be successful.

4.1 Which medications are most effective?

The most important medications for the treatment of tuberculosis are iso-
niazid (H), rifampicin (R), pyrazinamide (Z), ethambutol (E), streptomycin
(S) and thioacetazone (T). Some medications are available in combined
preparations: rifampicin with isoniazid {RH}, thioacetazone with isoniazid
{TH}, and ethambutol with isoniazid {EH}. The duration of time after the
manufacturing date that medications may be used safely (the shelf life of
the medications), provided they are kept in proper storage conditions, is as
follows:
5 years: isoniazid, ethambutol, thioacetazone
3 years: rifampicin, pyrazinamide, streptomycin

13
The use of rifampicin and streptomycin for diseases other than mycobac-
terial diseases should be limited to very carefully considered indications.
Those medications used for treatment of tuberculosis should only be avail-
able to the community through the National Tuberculosis Programme; they
should not be available freely from the private market.

4.2 What do they contain?

There is international agreement on the recommended dosage of each
antituberculosis medication, which is calculated per kilogram body weight
(Table 1).

Table 1. Optimal dosages for essential antituberculosis medications

(the range is given in parentheses).

Daily dose Intermittent dose

Medication in mg/kg 3 times/wk in mg/kg

5 10
Isoniazid (4-6) (8-12)

10 10
Rifampicin (8-12) (8-12)

25 35
Pyrazinamide (20-30) (30-40)

15 30
Ethambutol (15-20) (25-35)

15 15
Streptomycin (12-18) (12-18)

2
Thioacetazone –
(2.5)

This guide proposes that a limited variety of preparations be available

for each drug. This will simplify the management of the supply of med-

14
ications. It will enhance safety in prescription, and will allow the correct
dosage to be given. The preparation with the lowest content is usually rec-
ommended for treatment of adults: {RH} 150 mg/75 mg; H 100 mg;
Z 400 mg; E 400 mg; {EH} 400 mg/150 mg; S 1 g. The exception is {TH},
for which the larger, single-tablet dosage of {TH} 150 mg/300 mg is more
simple to use for adults > 40 kg.

4.3 How are they used?

For both the patient and the community it is essential to prevent the devel-
opment of drug-resistant tuberculosis. A patient who fails on a first course
of treatment is more likely to have resistant micro-organisms. Resistance
to one medication may lead to the development of resistance to any other
medication when that medication is given as a sole companion to one to
which the micro-organism is already resistant.
A retreatment regimen must be available that is likely to cure any
patient who fails on the first course of treatment. This regimen must use
the best available medications to which the patient is likely to respond,
because it is the patient’s last chance for cure. The retreatment regimen
recommended by this Guide includes rifampicin plus isoniazid throughout,
supplemented with ethambutol. Patients who fail on the first-line treatment
regimen should not be taking rifampicin as a sole companion to isoniazid
at the point at which they are identified as failing the treatment, as this
may lead to development of multidrug resistance. It is for this reason that
the Guide strongly discourages the use of rifampicin in the continuation
phase of treatment for patients never previously treated for tuberculosis for
as much as one month.
A patient who is identified as failing treatment when taking isoniazid
plus thioacetazone still has a high probability of cure. In such a case, two
effective medications (rifampicin and ethambutol) are given in the continu-
ation phase of the retreatment regimen. These medications will never have
been previously used alone with isoniazid, to which the micro-organisms
may have been resistant at the outset. If the patient has been given isoni-
azid plus ethambutol in the continuation phase of the first-line regimen, the
retreatment regimen should contain pyrazinamide throughout the course,
again to ensure that the patient at all times receives at least two medica-
tions in the retreatment regimen which are likely to be effective.

15
Eight-month chemotherapy for newly diagnosed cases
of tuberculosis
Eight-month chemotherapy should be given to all smear positive cases of
pulmonary tuberculosis who were never previously treated for as much as
one month for tuberculosis, provided arrangements can be made to ensure
that every dose of medication in the intensive phase of treatment is directly
observed to be swallowed. This regimen may also be used for patients with
other forms of tuberculosis that have never been previously treated, if they
are seriously ill. Such treatment should be given under the direction of a
Medical Officer and directly observed swallowing should be used in the
intensive phase. The directions for administration of medications according
to the weight of the patient are given in Table 2.
Table 2. Number of tablets to be taken daily for adults on treatment
according to weight and content of the tablets.

Weight in kg
Month Medication
of treatment
25-39 40-55 > 55

{RH}
(R 150 mg 2 3 4
H 75 mg)
1-2 Combined tablets
Intensive
phase Z (400 mg) 2 3 4

E (400 mg) 1.5 2 3

{TH}
(T 50 mg 2 – –
H 100 mg)
3-8 Combined tablets
Continuation
phase {TH}
(T 150 mg – 1 1
H 300 mg)
Combined tablets

R = rifampicin; H = isoniazid; Z = pyrazinamide; E = ethambutol; T = thioacetazone.

16
Eight-month chemotherapy of previously treated patients
Smear positive patients who have taken medications for treatment of tuber-
culosis for as much as one month in the past must be given a retreatment
regimen (Table 3). They include:

Relapses: these are patients who become smear positive again after having
been treated for tuberculosis and declared “cured” after the comple-
tion of their treatment.

Treatment after failure: these are patients who, on initial treatment for smear
positive pulmonary tuberculosis, remained or became again smear pos-
itive at 5 months or later during the course of treatment.

Treatment after default: these are patients who return to treatment smear
positive after having left their treatment for more than 2 months. Those
who on return to treatment are smear negative should not be newly
recorded, but should continue their original treatment until the com-
pletion of the total quantity prescribed.

Chronic cases are defined here as those who continue to be smear

positive after the completion of a fully observed retreatment regimen
(failures of retreatment). Their micro-organisms are likely to be resistant
to both isoniazid and rifampicin, and such patients are virtually incurable
in most low income countries. Such patients should be registered
separately.

Twelve-month chemotherapy for newly diagnosed cases

of tuberculosis
For twelve-month chemotherapy the following regimen is used: isoniazid
plus thioacetazone (in a combined tablet) daily for 12 months. For smear
positive patients this combination must always be supplemented by
streptomycin or ethambutol daily for the first 2 months of chemotherapy
(Table 4).
This regimen is used for all cases of tuberculosis other than those that
are smear positive. It may be given to new cases of smear positive tuber-
culosis (supplemented with streptomycin in the intensive phase) if the
8-month regimen is not available or if it is impossible to directly observe
all doses of medication in the intensive phase.

17
Table 3. Number of tablets to be taken for adults on retreatment according to
the weight and content of the tablets.

Weight in kg
Month Medication
of treatment
25-39 40-55 > 55

{RH}
(R 150 mg 2 3 4
H 75 mg)
Combined tablets
1-2
Intensive
phase Z (400 mg) 2 3 4
(daily)
E (400 mg) 1.5 2 3

S 0.5 g 0.75 g 1.0 g*

{RH}
(R 150 mg 2 3 4
H 75 mg)
3 Combined tablets
Intensive
phase
(daily) Z (400 mg) 2 3 4

E (400 mg) 1.5 2 3

{RH}
(R 150 mg 2 3 4
4-8 H 75 mg)
Continuation Combined tablets
phase
(three times
weekly)** H (100 mg) 1 1 2

E (400 mg) 2 3 4

R = rifampicin; H = isoniazid; Z = pyrazinamide; E = ethambutol; S = streptomycin

* Patients aged 45 years and over should receive 0.75 g; streptomycin should not be given to pregnant
women.
** When E is used routinely in new cases in the continuation phase, Z (400 mg) must be continued
during months 4-8 in the retreatment phase, 3 tablets for those < 40 kg, 3 tablets for those 40-55 kg
and 4 tablets for those > 55 kg.

18
Table 4. Dosage of medications to be taken for patients on twelve-month treat-
ment according to weight.

Weight in kg
Medication
10-24 25-39 40-55 >55

{TH}
(T 50 mg 1 2 – –
H 100 mg)
Combined tablets

{TH}
(T 150 mg – – 1 1
H 300 mg)
Combined tablets

S 0.25 g 0.50 g 0.75 g 1.00 g

E (400 mg) 1 1.5 2 3

T = thioacetazone; H = isoniazid; S = streptomycin; E = ethambutol

4.4 What adverse effects can they have?

Adequate treatment of each case for the full duration of the prescribed reg-
imen is very important if success in treatment is to be achieved. Any change
to the treatment regimen due to what appear to be side effects must be
made only after careful consideration.
Treatment of tuberculosis is prolonged over a number of months. During
such a period of time in anyone’s life, some events might occur which, if
they occur in someone taking medications, may be thought to have been
caused by these medications. Particularly frequent events of this type include
skin rashes and abdominal complaints. In studies of the use of isoniazid,
in which the comparison group was given no active medication, it was
noted that of episodes that doctors considered were caused by reactions to
medications, approximately half were caused by something else. How, then,
do we know if a reaction is due to the medications?

When to stop medications without further consideration

These are very infrequent reactions that require the medications to be
stopped, and frequently that the patient be hospitalised for management.
They include the following:

19
– Skin irritation or rash in any patient on thioacetazone. The med-
ication must be stopped immediately and never given again (it should
be replaced with ethambutol).

– Generalised reactions including shock, purpura and fever. This is very

rare but may be caused by rifampicin, pyrazinamide or streptomycin.
The medication thought to be responsible for the reaction should never
be given again.

– Impairment of vision in a patient on ethambutol. Patients develop-

ing impaired vision should report immediately for examination. If
ethambutol is thought to be responsible, it should never be given again.

– Patients who are pregnant must never be given streptomycin due to

risk of vestibulo-cochlear damage to the foetus.

What to do when you think there may be an adverse effect

– Dizziness may be caused by vestibular damage due to streptomycin.
This is most frequent in older individuals. Correct dosage and dura-
tion of treatment is important to prevent occurrence of these side effects.

If a patient develops the following symptoms, medications may need to be

stopped while the cause is investigated.

– Jaundice or severe abdominal discomfort may be caused by hepati-

tis. It is most frequently due to isoniazid, but may also be caused by
rifampicin and pyrazinamide. Any patient with these symptoms should
be referred to the Medical Officer for further consideration.

– Skin rash in a patient not on thioacetazone. This is most frequently

due to isoniazid, streptomycin or pyrazinamide. If the patient is clin-
ically well (does not suffer from advanced tuberculosis or serious forms
such as meningitis or disseminated disease), it is best to stop all med-
ications and recommence them when the reaction has subsided. If the
symptoms recur, the patient should be referred to the Medical Officer.

Reactions not requiring interruption of treatment

– Numbness or tingling may be caused by isoniazid. When it occurs,
it can be treated by supplementing the isoniazid with vitamin B6 at a
dose of 5 mg daily.

20
– Joint symptoms may be caused by pyrazinamide. Check the dosage
by weight; it is usually caused by overdosage. It may be easily alle-
viated with acetyl salicylic acid.

– All patients on rifampicin: inform each patient to expect a red/orange

colour to body fluids (tears, saliva, sputum, urine and sweat) which is
not dangerous.

Determine if the patient is taking birth control medications, anti-epileptic

medications, corticosteroids, oral treatment for diabetes, or oral anticoagu-
lants. These may require adjustment of dosage, or the use of alternative
methods in the case of birth control.

B. What factors might affect treatment?

1. How does HIV affect treatment?

Patients infected with HIV usually have a response to treatment similar to
that of patients who are not infected with HIV, with a few exceptions:

– they are more likely to die during the course of treatment, usually from
causes other than tuberculosis;

– they may be more likely to experience toxic reactions to medications

(and particularly to thioacetazone) than those who are not HIV
infected, and their treatment must be adjusted for this reason;

– they are more likely to relapse if treated with the twelve-month

regimen.

HIV infection is spread most frequently by sexual intercourse, through

exchange of blood or blood products and from mother to child. Because
of the association between tuberculosis and HIV infection, great care must
be taken in tuberculosis programmes and in health services in general to
prevent the spread of both of these infections. The highest standards of
hygiene must be observed, particularly when there is a risk of exposure
to blood or blood products, when caring for tuberculosis patients. The use
of injections should be limited as much as possible. Where they cannot

21
be avoided, every health care worker should strictly adhere to the princi-
ple: a sterilised needle and syringe for each injection in each individual
patient.
A health care worker who is HIV seropositive should avoid exposure
to tuberculosis patients because of the greatly increased risk of developing
tuberculosis if infected. Any patient who is infected with HIV should be
carefully protected from exposure to other patients with tuberculosis.
Moreover, wherever HIV positive patients come together (in hospital wards,
hospices and community support groups), a great deal of attention should
be paid to any possibility of the occurrence of tuberculosis in these patients
and every effort should be made to quickly diagnose and treat tuberculo-
sis which may occur.
Patients known or suspected of having HIV infection should never be
given thioacetazone. The best alternative is to provide them with treatment
consisting of appropriate doses of ethambutol and isoniazid, according to
their weight, given in the form of combined tablets containing 400 mg
ethambutol plus 150 mg isoniazid {EH}: 3 tablets for those > 55 kg,
2 tablets for those 40-55 kg, and 1.5 tablet for those 25-39 kg.

2. How does drug resistance affect treatment?

Large populations of tuberculosis micro-organisms, such as those in patients

who are sputum smear positive, always contain some mutants naturally
resistant to medications. If a correct combination of medications is pre-
scribed and is taken by the patient, this resistance is overcome and does
not pose a problem. This is the reason for using a greater number of med-
ications during the intensive phase of treatment until the population of
micro-organisms has been rapidly reduced. This important principle must
be respected in order to prevent development or extension of clinically
important resistance to medications.
Once developed, resistance to antituberculosis medications can have
an influence on the impact of treatment of tuberculosis cases, by causing
the emergence of further resistance (where an insufficient combination of
medications is used) or by rendering the patients incurable (where resis-
tance to isoniazid and rifampicin coincide in an individual patient). The
recommendations put forward in this Guide propose the steps most likely
to be successful in preventing multidrug-resistant tuberculosis and thus pre-
venting the development and spread of incurable tuberculosis.

22
3. What if the patient is pregnant or breast feeding?
Pregnant women with tuberculosis should start or continue their treatment
for tuberculosis in the same way as other patients. However, streptomycin
should not be used because of the risk of toxicity to the unborn child. When
the patient has a nursing infant, it is of particular importance to continue
breast feeding, as its discontinuation poses a serious risk for the develop-
ment of the infant.

C. What about those exposed to tuberculosis?

Those who live in the same household with any patient who is smear pos-
itive have a higher risk of having tuberculosis themselves. If they have any
symptoms, they should be requested to attend for a medical examination.
Any child in the household under 5 years of age who has symptoms that
suggest tuberculosis should be given treatment as a case of tuberculosis.
All of the other children under 5 years of age should be given preventive
chemotherapy, even if they have previously been vaccinated.

1. What is preventive therapy and its role?

Preventive therapy is the treatment of those infected with Mycobacterium
tuberculosis (tuberculous infection) who do not have the disease (tubercu-
losis). The infection can be identified with a tuberculin skin test. The risk
of developing tuberculosis in those who are tuberculin skin test positive is
relatively low unless the infection has been acquired relatively recently or
the person is also HIV positive. Preventive therapy in such persons can
prevent the development of tuberculosis to an important extent.
This Guide recommends preventive treatment with isoniazid daily for
a period of 6 months at a dose of 5 mg/kg body weight.
Often tuberculin is not available. The most important group that can
be identified as needing preventive therapy are children under the age of
5 years who are living in the same household as a newly discovered smear
positive tuberculosis patient. The chance that the child has been infected is
high, as is the chance of the development of tuberculosis. New smear pos-
itive patients must be questioned carefully to determine if there are chil-
dren in their household. These children must then be examined and treated
as outlined above.

23
2. Can a patient on treatment infect you?
Treatment is effective in rapidly diminishing the infectiousness of any patient
with susceptible micro-organisms. This is because the medications rapidly
reduce the number of micro-organisms, and the patient’s cough rapidly sub-
sides, resulting in fewer micro-organisms expelled into the air. In most set-
tings, no special precautions for preventing the spread of infection need be
taken once the patient is on treatment; the best prevention is to ensure that
the medication is being taken regularly.
This is not true, however, if the micro-organisms are multidrug-
resistant, in which case great care must be taken to avoid transmission to
those around the patient. Where multidrug-resistant tuberculosis is frequent,
great care must be taken to avoid contact, as much as possible, between
those who have (or are likely to have) tuberculosis and any person
likely to have HIV infection. Good ventilation must be provided wherever
tuberculosis patients (or those likely to have tuberculosis) gather. Institution-
alisation of tuberculosis patients should be avoided, whenever possible, and
where they are institutionalised, the best ventilation possible for the area
should be provided. Moreover, tuberculosis patients (and those likely to
have tuberculosis) should be given accommodation in an area away from
other patients.

24
 IV. CARING FOR THE PATIENTS

The quality of the care given to patients and the thoroughness with which
it is followed are important determinants of successful treatment and of
reduction in the risk of becoming infected. Poor treatment increases the
number of infectious cases in a community.

A. How should the patients be followed?

Because tuberculosis treatment is prolonged, great care must be taken to
ensure that the treatment is taken as prescribed.

1. Are all the patients on treatment?

A periodic review, usually once a week, should be performed to compare
the names of the patients identified as smear positive in the laboratory with
the list of patients commenced on treatment, to ensure that no patients iden-
tified in the laboratory go without treatment. Moreover, the laboratory results
of all those patients who have been commenced on treatment who are not
smear positive should be confirmed in the laboratory to ensure that their
examinations have been performed and are negative.

2. How can we encourage full participation of the patient?

The successful treatment of the patient requires that the patient understand
what is happening. When the patient understands the nature of the disease
and its treatment, the patient is more likely to follow the treatment required
to achieve cure. The relationship developed between the patient and the
care giver is key to achieving success in treatment, and requires investment
of time and energy.
The patient and, if possible, at least one member of the patient’s fam-
ily, should clearly understand the answers to the following questions:

– What is tuberculosis?
– How is the disease spread?
– What measures can be taken to limit its spread?

25
– How is it treated?
– Can the disease be cured?
– Can I get treatment free of charge?
– What medications are used and for how long?
– How is the treatment followed?
– What are the possible side effects of the medications?

If the patient understands the disease and its treatment, this information
will be passed on to the community and, as a result, other individuals with
tuberculosis will be encouraged to come forward to seek diagnosis and
treatment.

3. How do we monitor progress during treatment?

The results of sputum smear examination should be recorded for all adult
patients prior to the commencement of treatment. Priority is given to treat-
ment of smear positive patients, as these are the most potent sources of
infection in the community.
Bacteriological follow-up examinations in smear positive patients are
the most important means of assessing progress. After 2 months of inten-
sive phase treatment the sputum in these patients must be examined. Those
patients found to have no tuberculosis micro-organisms on sputum exami-
nation should start the continuation phase. If the examination is positive,
the intensive phase should be prolonged, but only up to a total of 3 months.
At this time the continuation phase is started without additional microscopic
examination. This is a safe practice where regimens using {TH} in the con-
tinuation phase are used. Should other medications (R or E) be given alone
with H in the continuation phase, this practice may carry the risk of extend-
ing drug resistance in a patient with micro-organisms that are already resis-
tant to H.
In all smear positive patients, a microscopic examination is done at
5 months. If the result is negative, treatment should be continued. If
any micro-organisms are identified (whatever the grade of the positive
result – see Appendix 1, Technical Guide for Smear Microscopy), the
result should be confirmed by a second positive result before declaring
the patient a treatment failure who must be given the re-treatment
regimen.
Sputum microscopy examination is repeated on the final visit, one
month prior to completing the treatment course. If negative, the patient is

26
given the last one-month supply of medications and declared cured. Patients
with positive smears on this examination, confirmed by a second exam-
ination, are declared treatment failures and must be given the re-treatment
regimen.
The total duration of treatment is always 8 months or 12 months,
depending on the regimen, unless the patients are treatment failures. Patients
who do occasionally miss an appointment during the continuation phase
should have the time of treatment missed added to the originally planned
duration. If irregular treatment lasts longer than 15 months, the outcome of
treatment of the patient is recorded as “defaulted”.

B. What is the most efficient way to deliver

tuberculosis services?

Because tuberculosis is a widespread disease and its prevention is depen-

dent on good quality medical care of individual patients, the organisation
and system through which this care is given is an important element in
achieving success in tuberculosis services.

1. What is the proper structure of tuberculosis services?

The responsibility for activities directed at controlling tuberculosis rests
with the government. These activities must be organised in the form of a
National Tuberculosis Programme. The aims of a National Tuberculosis
Programme are as follows:

– to quickly diagnose and cure as many of the infectious cases of tuber-

culosis as possible, and in this way, to rapidly and greatly reduce the
rate of spread of tuberculosis micro-organisms;

– to maintain vigilance in the detection of all new infectious cases which

will continue to arise during the entire lifetime of that group in the
population which has already been infected prior to the application of
control measures;

– to limit the excess transmission of tuberculosis micro-organisms result-

ing from the presence of HIV infection in the community.

27
To achieve these aims, a National Tuberculosis Programme must be:

– country-wide, with a focus on areas where the greatest proportion

of the population lives. It requires a strong urban component (urban-
isation is increasing rapidly and tuberculosis is a particularly serious
problem in overcrowded urban areas);

– permanent, ensuring that the cases continuously arising from those

already infected with tuberculosis micro-organisms will be rapidly iden-
tified and rendered non-infectious (a process that must continue through
the lifespan of the last heavily infected group in the population);

– adapted to the realities of each community within which it operates,

taking note of the characteristics of the population, the accessibility
of health facilities, and the attitudes of health care personnel;

– integrated within the general health services of the community, as

tuberculosis is one of the most important causes of ill health in low
income countries and patients present with their symptoms at every
level of the health service.

The modern National Tuberculosis Programme is based on the DOTS strat-

egy (Directly Observed Treatment, Short-course), promoted by the World
Health Organization. This strategy has been based on the experience of the
IUATLD and its partners, which is described in this Guide. The elements
of the DOTS Strategy are:

– political commitment on the part of the government;

– a secure supply of essential medications and diagnostic materials;

– diagnosis and follow up based on sputum smear microscopy;

– treatment using short-course chemotherapy for at least smear positive

cases, and directly observed treatment (DOT) when rifampicin is used;

– monitoring through proper recording and reporting of activities.

2. How should the services be organised?

The structure of the tuberculosis service should be based upon the “unit of
management” serving, on average, a population of 50,000 to 150,000.

28
2.1 At the unit of management
Each unit of management should have a Unit Co-ordinator. This person,
usually a paramedical, is responsible for ensuring that tuberculosis activi-
ties (case-finding and treatment) are correctly applied within the unit of
management, along with the other responsibilities normally carried out
by the health workers. This level should be the most peripheral site of
sputum microscopy, as services that are more peripheral than this cannot
be sure of maintaining proficiency in carrying out their activities, and the
organisation of supplies and supervision becomes impossible. The Unit
Co-ordinator is responsible for:

– ensuring that the correct treatment regimen is applied to all patients

(in particular, ensuring that those patients eligible for the retreatment
regimen are correctly identified and treated);

– ensuring that all rifampicin-containing treatment is given directly

observed by a health worker;

– determining that all patients commenced on treatment have had a spu-

tum smear examination performed;

– comparing the Tuberculosis and Laboratory Registers (see Appendix 2,

Forms 2 and 4) to make sure that all patients are enrolled on treatment;

– ensuring action aimed at preventing defaulting (such as patient edu-

cation and communication) and initiating early action to trace all
patients who do not appear at regular appointment times;

– keeping the Tuberculosis Register in order and up-to-date;

– reporting the results of tuberculosis activities in the unit;

– maintaining supplies of materials (including treatment supplies such as

medications, and diagnostic supplies such as reagents) within the unit.

2.2 At the intermediate level

In order to maintain a good quality of service, a system of training and
supervision must be in place to support the Tuberculosis Co-ordinator in
the management unit. For this reason, each group of 5-10 districts should
have an individual (the Provincial/Regional Co-ordinator) responsible for

29
ensuring that this occurs. In most instances, this individual is a physician
or medical assistant who acts as an “expert” in the area to determine what
is to be done when problems arise. This individual carries out the tuber-
culosis activities in addition to other responsibilities (often providing spe-
cialist services for chest diseases or other communicable diseases). The
Provincial/Regional Tuberculosis Co-ordinator is responsible for:

– nominating health workers to manage the care of tuberculosis patients;

– supporting and supervising the activities of the Co-ordinator in the

management unit, who must be visited at least quarterly (or more fre-
quently in the case of poor performance);

– providing training for all new personnel and refresher training of those
who require special attention in the light of their performance;

– maintaining a continuous supply of materials and a system of quality

control of sputum smear microscopy;

– organising the treatment of patients, including living arrangements for

patients who must remain away from home to receive treatment;

– reviewing the reports of tuberculosis activities and discussing them

with the Co-ordinators of the management units each quarter;

– co-ordinating with officials at the central level to ensure regular super-

vision, training, supply and reporting, and with colleagues respon-
sible for other programmes such as AIDS, Leprosy and Laboratory
Services.

2.3 At the central level

Within the Ministry of Health, there must be a Central Tuberculosis Unit
with a full time director and support staff to ensure that the National
Tuberculosis Programme functions appropriately. The Director must have
responsibility for all activities in the country. The functions of the Central
Tuberculosis Unit include the following:

– planning, implementing, monitoring and evaluating the National

Tuberculosis Programme, including work plans, budgets, reports and
administration;

30
– co-ordinating with the Division of Laboratories to ensure that the net-
work of laboratories is properly supervised, that quality control activ-
ities are carried out correctly and that training is appropriate;

– ensuring, in collaboration with those responsible for logistics and sup-

ply, the regular supply of materials throughout the country, including
monitoring consumption based on reports of case-finding results, co-
ordination with the supply system in the Ministry of Health, estima-
tion of requirements for supplies of materials and medications;

– regularly supervising and supporting the Provincial/Regional Co-

ordinators;

– collaborating with the AIDS Programme to ensure that patients who

are affected by both tuberculosis and HIV are properly cared for and
that exposure of HIV infected individuals to tuberculosis micro-
organisms is avoided;

– ensuring that authorities are fully aware of the priority that should be
given to tuberculosis programme activities, including allocation of mate-
rial and human resources.

2.4 External evaluation

Periodic external evaluation by recognised experts in tuberculosis control
should be undertaken in all countries for review of technical aspects of the
programme and their implementation. Such reviews provide an independent
critique of the programme and give support to programme personnel in their
attempts to gain a hearing from decision makers in order to make neces-
sary changes.

C. How is the laboratory service organised?

A well-functioning laboratory is the first requirement for successful man-
agement of tuberculosis. If the diagnosis is not made reliably and if fol-
low-up of treatment is not trustworthy, all other activities will be affected.

1. What is the basis of the laboratory examination?

Every patient requires sputum smear examination to correctly determine the
treatment that the individual needs. Because of this, it is necessary to have

31
laboratory services that reach the entire population served by the health ser-
vice. Such laboratory services should be provided within the context of the
already existing health service structure, and the duties of sputum smear
examination should be included among the other duties of the laboratory
technicians already present within the health service. There is no need for
specialised personnel for performance of sputum smear microscopy within
the general health service. Because tuberculosis contributes to such an extent
to the health problems of most countries, every general laboratory techni-
cian within the health service should have the skills to perform this diag-
nostic procedure.
The recommended method for routine confirmation of a diagnosis of
tuberculosis is the microscopic examination of smears of sputum specimens
stained using the Ziehl-Neelsen method. For this purpose, a good quality
binocular microscope with an electrical light source (or a mirror where elec-
tricity is unavailable) is essential. The microscope must be equipped with
an oil immersion objective (x 100) in order to carry out the examination,
and should have a movable stage.
The technical aspects of sputum smear examination are provided in
the Technical Guide for Sputum Examination for Tuberculosis by Direct
Microscopy (Appendix 1).

2. What are the aims of the laboratory service?

The aims of the laboratory service with respect to tuberculosis are:

– the confirmation of tuberculosis diagnosis (including the correct clas-

sification of cases upon which the treatment regimen is determined);

– the monitoring of the treatment of sputum smear positive cases; and

– the surveillance of the tuberculosis situation in the community.

2.1 The network of microscopy centres

Diagnosis must be made as close as possible to the residence of the patients,

while maintaining the proficiency of the testing procedures. To accomplish
the first two of the above aims, a network of laboratory centres carrying out
sputum smear microscopy at a high technical level must be maintained.

32
 The laboratory technicians in the general health services must be com-
petent to carry out sputum smear microscopy; this requires that they be
trained, motivated and properly supervised.
It is important to plan services in such a way that they are accessible
to the population and yet maintain an adequate degree of technical profi-
ciency. To accomplish this, it is generally recommended that one microscopy
centre should be developed for each unit of population containing between
50,000 and 150,000 inhabitants, according to the incidence of tuberculosis
and the geographic distribution of the population. Great care should be
taken not to extend the network to a more peripheral level, as this results
in deterioration of technical proficiency and an inability to properly super-
vise the activities. In principle, the microscopy centre should be located at
the same site as the treatment centre (which should serve a similar sized
population).

2.2 Assuring quality of smear microscopy

Quality assurance of all laboratory investigations (including sputum smear
microscopy) is essential if the tests are to be meaningful and useful in the
care of the patient. Because the network of sputum smear microscopy cen-
tres is so important in the care of the patients, quality assurance is an indis-
pensable component of any tuberculosis programme. Quality assurance of
smear microscopy is the responsibility of the National Public Health Service
Tuberculosis Reference Laboratory. A regular system of quality assurance
must be part of the supervision process, and retraining of technicians who
perform their duties in a deficient manner must be undertaken.
Quality assurance consists of three components. Internal quality con-
trol includes all means by which the laboratory itself controls its operation.
Proficiency testing (often called external quality control) is undertaken by
selecting a sample of slides for rereading outside the laboratory by a dif-
ferent individual than the one who performed the original examination, with-
out the second individual knowing the result of the first examination.
Improvement in the quality of work results from identification, through
internal quality control and proficiency testing, of weaknesses or errors.
Quality assurance of sputum smear microscopy is more than a laboratory
exercise: it is a method of ensuring the quality of diagnosis and classifi-
cation of the tuberculosis patients within the health service.
Various methods may be chosen for proficiency testing. Immediately
following training of a laboratory technician, slides of known content may

33
be given to the trainee for re-examination. This allows the capabilities of the
newly trained individual to be assessed. To ascertain the routine performance
of a laboratory, slides might be randomly selected following identification
in the Laboratory Register. To determine the quality of patient classifica-
tion, slides from patients registered in the Tuberculosis Register, comprising
a representative sample of all patients cared for in the health service, may
be examined. With each approach, the sample should include negative slides
(or slides from patients classified as sputum smear negative) and positive
slides (or slides from patients classified as sputum smear positive).
Detailed information on establishing and carrying out these activities
is included in the publication “The Public Health Service National
Tuberculosis Reference Laboratory and the National Laboratory Network”,
available from the IUATLD.

2.3 Culture and susceptibility testing

Sputum smear microscopy must have the highest priority in the care of
tuberculosis patients. More sophisticated tests (such as culture and suscep-
tibility testing) should not be undertaken until an adequate network of lab-
oratories for smear microscopy has been developed which serves the whole
community and which has a good system of quality control. It is usually
not possible to provide a diagnostic service based upon culture and sus-
ceptibility testing. The role of culture and susceptibility testing is to accom-
plish another aim of the laboratory service, the surveillance of the tuber-
culosis situation in the community.
The determination and surveillance of resistance of M. tuberculosis to
medications is useful as a means of monitoring the adequacy of a tuber-
culosis programme. Clinically important resistance of micro-organisms to
medications is always a man-made problem and is frequently a reflection
of individual or programmatic malpractice (the prescription or provision of
inappropriate or inadequate treatment regimens, resulting in effective
monotherapy). The development and/or promotion of resistance to medica-
tions reflects such malpractice. When it is detected using a system of sur-
veillance, prompt action must be taken to prevent further occurrence. The
development of resistance to medications (and particularly to isoniazid and
rifampicin) severely compromises the ability of the health services to cure
the individual patient and to bring the tuberculosis problem under control.
Technical guidelines for the determination and surveillance of resis-
tance to medications in National Tuberculosis Programmes are available
upon request from the IUATLD.

34
D. How do we monitor care?
The adequate care of tuberculosis cases requires that records be kept on
each individual patient, with periodic reporting of the results of case-find-
ing and of treatment. This is essential to ensure that the patient is correctly
treated and that adequate supplies of essential materials are provided. In
addition, the information that is routinely collected and reviewed allows
problems that may arise with the management of the patients and of the
system to be identified. The documents used to record and report the care
of the patients should be simple, clear and kept to the absolute minimum
that is required for adequate care. The following description provides a
guide for the recording of patients as they appear to the health facility, and
comprises the minimum number of records and reports necessary to ensure
the proper care of the patients.

1. What records are necessary?

1.1 Records of diagnostic examinations

All individuals who present themselves to the general health service who
are likely to have tuberculosis are required to have a sputum smear exam-
ination. The initial sputum sample is obtained during the first consultation
with the health care worker, at which time a Request for Sputum Examination
(Appendix 2, Form 1) is completed. When the sputum sample is received
in the laboratory, the information on the individual patient is entered in the
Tuberculosis Laboratory Register (Appendix 2, Form 2). As indicated in
the sample form, each patient examined for diagnosis will have at least
two sputum examinations that are entered on a single line in the laboratory
register.

1.2 Records of cases of tuberculosis

If the patient is designated a tuberculosis case, a Patient Identity Card will
be completed which will be kept by the patient. This card contains the
name, age, sex and address of the patient. It identifies the health service
identification number (where this is routinely used), the tuberculosis regis-
ter number and the name of the health service. The type of tuberculosis as
well as the date and results of bacteriological examination at the time of
diagnosis, the date the treatment was commenced, the regimen prescribed

35
and spaces for the dates of follow-up appointments and results of follow-
up sputum examinations are recorded. At the same time a Tuberculosis
Treatment Card (Appendix 2, Form 3) is completed, which is kept at the
health service where the patient receives treatment. The information from
this card is entered into the Tuberculosis Register (Appendix 2, Form 4)
within the unit where the patient’s care is managed. If the patient is diag-
nosed in a referral facility (such as a hospital or other institution), the loca-
tion in which the patient’s care will be managed after leaving the institu-
tion should be determined, and the patient should be entered into that
register.
In completing the Tuberculosis Register, great care should be taken to
ensure that the information is correctly recorded and regularly updated.
When a patient is newly detected, precise information should be entered.
Patients should be recorded in numerical order as they become known to
the health worker responsible for the Tuberculosis Register. Numbering
commences with number one at the beginning of each calendar year, regard-
less of when the patient was diagnosed or commenced treatment. Particular
attention should be taken to ensure that the patient is correctly recorded
and treated. This includes identification of the correct “disease site”:
– Pulmonary cases are those with tuberculosis of the lungs including
those who are sputum smear positive and those who are sputum smear
negative (provided a minimum of three sputum examinations have been
performed).
– Extra-pulmonary cases are all other patients, including those with tuber-
culous pleurisy and miliary tuberculosis (the specific site should be
recorded).

The correct “category of patient” necessary to determine the proper treat-

ment is one of the following:
– A new case is one who has never previously been treated for as much
as one month.
– A relapse case is one who, having previously been treated, was declared
cured prior to becoming once again sputum smear positive.
– Treatment after failure is a patient who, while on treatment, remained
or became again smear positive at 5 months or later during the course
of treatment.

36
– A patient is recorded as treatment after default on returning to the
health service sputum positive after having interrupted treatment for
more than 2 months.

– A patient who is a transfer in is any patient who has been registered

for treatment in another tuberculosis register and has been transferred
to the unit to continue treatment. The results of treatment of all such
patients should be reported to the location in which the patient was
initially registered, for inclusion in the regular reports of treatment
results. The results of such patients should never be reported to the
central authorities from the location to which the patient has been trans-
ferred to continue treatment.

– All other patients are entered under the column other.

It is extremely important to determine correctly, for all patients with smear

positive pulmonary tuberculosis, whether or not they have previously been
treated for as much as one month. The incorrect designation of such patients
will result in incorrect treatment of the patient. The correct assignment of
patients to the three categories relapse, treatment after failure, and treat-
ment after default, allows precision in evaluation. However, the correct des-
ignation of these three categories may not be feasible in all locations. In
this case, the three categories must be merged into the single category
retreatment. The Treatment card (Form 3), the Tuberculosis register
(Form 4) and the Quarterly report on case-finding (Form 5) have to be
modified accordingly.
A patient is classified as having sputum smear positive tuberculosis if
the microscopic examination shows acid-fast bacilli on two occasions, in
one of which the result must be at least 1+ positive. All other patients who
have at least three sputum examinations may be treated for tuberculosis
(depending on the assessment of a Medical Officer), but are classified as
sputum smear negative cases.
The results of all sputum smear examinations must be entered in the
correct column, with the laboratory serial number taken from the Request
for Sputum Examination form (Form 1). Sputum smear results should be
recorded as negative (written “neg”), scanty (“1-9”), positive (“+”, “++”,
or “+++”), or not done (“ND”). All positive sputum results should be entered
into the register in red ink for ease of identification. The laboratory iden-
tification number and the date the examination was performed should be
entered in the column next to that for the result of the examination.

37
1.3 Records of treatment
Some information (i.e., sputum examination results, direct observation of
medication taking) should be recorded immediately. At the end of each
work day, the health worker responsible must collect all tuberculosis treat-
ment cards of individuals cared for during the day and transcribe the rel-
evant information into the Tuberculosis Register. Once each week, the per-
sonnel responsible for the treatment of patients must meet with those
responsible for carrying out sputum smear microscopy in the laboratory, to
ensure that all patients recorded in the Tuberculosis Laboratory Register as
sputum smear positive have been enrolled on treatment.
The results of treatment for each individual patient should be recorded
as they become available, as follows:

– Smear negative (cured) indicates an individual who was smear negative

at the last month of treatment and on at least one previous occasion.

– Smear not done (treatment completed) indicates those patients who

have completed treatment but whose smear results do not meet the
criteria to be classified as smear negative or smear positive.

– Smear positive (failure) designates any new patient who remains or

becomes again smear positive at 5 months or later during treatment,
or a retreatment case who remains positive at the end of treatment.

– Died is recorded for patients who die for any reason during the course
of their treatment, entered into the register at the time the death is
reported.

– Defaulted is recorded for any patient who has failed to collect med-
ication for more than 2 consecutive months after the date of the last
attendance during the course of treatment. This should be entered into
the register when the 2 months since the last appointment have lapsed.
Those who are still on treatment at the time the treatment results are
evaluated (15 months after the close of the quarter in which the patient
was entered into the tuberculosis register) should be recorded as
defaulted.

– Transferred indicates any patient for whom treatment results are

unknown, who was to continue treatment at another centre to which
the patient was transferred to continue treatment.

38
The first of these events to occur is the event recorded as the result of
treatment.

2. How are the results reported?

Reporting the results of case-finding and of treatment every three months
(quarterly) permits the tuberculosis activities to be evaluated and allows
early identification of problems in the health services providing care for
the patients. While more frequent reports may be required for certain other
conditions, reports on activities related to tuberculosis are not necessary
at intervals of less than every 3 months; the reports are used primarily
for planning future activities and/or calculating supply requirements. All
quarterly reports are prepared from the Tuberculosis Register and are only
as accurate as the information recorded in that register. The quarterly
reports should be prepared the week following the end of each quarter
(the first week of April, July, October and January) for the quarter being
evaluated. These reports should be submitted to the authorities (in this
case the Provincial/Regional Tuberculosis Co-ordinator) no later than the
end of the month in which they were completed (end of April, July,
October and January). They should be forwarded to the central level,
where they are tabulated, no later than the end of the quarter in which
they are prepared.

2.1 Quarterly Report on Case-Finding (Appendix 2, Form 5)

This report is completed systematically by counting the number of cases
recorded in the Tuberculosis Register within the quarter that has just ended.
Any case classified as “Transfer in” or “Other” is not reported. The order
in which the cases are counted is noted in Table 5.
The next step in preparing the quarterly report on case-finding is to
determine the number of new cases of smear positive pulmonary tubercu-
losis by age category and sex. This can be done using the method illus-
trated in Table 6. Using this method, identify cases whose pre-treatment
smear result is recorded as positive and then look across to be sure that
the patient is recorded as “new” under the heading “Category of patient”.
If the patient is smear positive and “new”, determine the age and sex. Then
record the case on a separate sheet of paper using the method outlined in
Table 6. For example, for a woman aged 33 years, you would add a “l”
to the right side of the age group 25-34; for a man aged 51, add a “l” to

39
the left of the age group 45-54. The sum of the cases should be the same
as the number of new smear positive cases listed in Table 5.

Table 5. Preparing the Quarterly Report on Case-Finding: the order of counting

the cases in each quarter.

Pre-treatment
Disease site sputum smear result Category of patient

1. Pulmonary + New
2. Pulmonary + Relapse
3. Pulmonary + Treatment after failure
4. Pulmonary + Treatment after default
5. Pulmonary Neg/ND New < 15 years of age
6. Pulmonary Neg/ND New ≥ 15 years of age
7. Extra-pulmonary Neg/ND New

Table 6. Determining the distribution of new smear positive cases by age group
and by sex.

Males Age group Females

l l l 0-14 l l l l
l l l 15-24 l l
l l l l l l 25-34 l l l l l l
l l l l 35-44 l l l l
l l l 45-54 l l
l l l 55-64 l
l l l l 65+ l
28 total 21

The final step (optional), used to determine medication requirements

in programmes (such as those that are newly introduced into an area) in
which a proportion of the new smear positive patients are not yet enrolled
on the 8-month regimen, is to complete the section “Enrolment on treat-
ment”. The number of patients enrolled on each of the different regimens
during the quarter, as indicated under the appropriate column of the regi-
men that applies, is entered in this section.

40
2.2 Reporting the Results of Treatment
At the same time as the report on case-finding is completed (the first week
of January, April, July and October), a Quarterly Report on the Results of
Treatment (Appendix 2, Form 6) should also be completed. The quarter for
which the report should be prepared is that quarter ending 15 months prior
to the time at which the report is being prepared. For this report, only the
results of treatment of sputum smear positive cases are included. It is com-
pleted separately for new cases enrolled on eight-month treatment, new
cases enrolled on twelve-month treatment, and relapse cases, treatment after
default and treatment after failure enrolled on retreatment. Those cases
recorded as “Transfer in” must not be included in the report, as the results
of treatment of such cases should be sent to the unit from which the patient
was transferred and reported in that unit.
When preparing the Quarterly Report on the Results of Treatment, the
Quarterly Report on Case-finding for the same quarter should be consulted.
From this report, the number of cases reported should be entered in the
appropriate location on the Quarterly Report of Results of Treatment (indi-
cated by the asterisk). The total number of cases evaluated within each cat-
egory (according to the type of case and treatment regimen) should be equal
to the number entered in this section, obtained from the Quarterly Report
on Case-finding. Where the number is different, an explanation must be
provided.
In completing the report, the information should be obtained from the
Tuberculosis Register under the section entitled “Results of treatment”. The
result for every case should have been recorded at this point. Where more
than one result occurs for a single patient, the result that will be recorded
is that event which occurs first. That is to say, if an individual remained
smear positive at 5 months but subsequently died (or defaulted or was trans-
ferred) the patient must be evaluated as smear positive (failure). At the time
of preparation of the report, if no other result is recorded, the patient must
be evaluated as having defaulted. When a patient has been transferred to
another unit to continue treatment, the outcome of the treatment at the unit
to which the patient was transferred should be obtained and entered into
the register. Those patients evaluated as “Transferred” should be those in
whom the outcome of treatment is not known.
When completed, the report must be forwarded to the relevant author-
ities as noted above.

41
E. What supplies are needed and how are they
managed?
In order to have the best success in the management of tuberculosis patients,
it is necessary to ensure that supplies are continuously available. This is
even more important for tuberculosis patients than might be the case with
some other types of illnesses.

1. How are supplies of medications managed?

Because successful treatment of patients depends upon their regularly tak-
ing medications, without interruption, for a long period of time, an uninter-
rupted supply of medications is essential. A system of supply management
also permits proper accounting of supplies and materials, when consump-
tion is compared with requirements, estimated by reports of case-finding.

1.1 How are supplies ordered?

The ordering described here is for the “unit of management” serving a pop-
ulation of 50,000 – 150,000. At this level it is most convenient to order
supplies once every quarter and to allow for an additional “reserve” stock
in case of irregularity of supply.
Ordering and maintaining supplies of medications is determined from
the results of case-finding which are recorded on the Quarterly Report on
Case-Finding. The ordering of these supplies is done at the same time as
case-finding is reported (on the first week after the end of the quarter: the
first week of April, July, October and January). Supplies of medications
are ordered using the Order Form for Treatment Supplies (Appendix 2,
Form 7). The quantity of materials required for the treatment of patients
each quarter is determined as follows:

– The number of patients to be treated is determined from the Quarterly

Report on Case-Finding that has just been completed for the preced-
ing quarter.

– The number of patients is entered under the column headed “cases”.

New smear positive cases are in the first column, the sum of new
smear negative and extra-pulmonary cases in the second column (“other
new cases”), and the sum of the retreatment cases (relapse, treatment
after failure, treatment after default) in the third column.

42
– The quantity of each type of medication that is to be ordered is deter-
mined by multiplying the number of cases by a “factor”. This factor
is the average number of tablets to be taken by a patient during treat-
ment, and taking the sum of all the numbers in the three columns
(A+B+C).

When the section of the Quarterly Report on Case-finding entitled

“Enrolment on treatment” has been completed, the number of cases to be
entered on the Order Form for Treatment Supplies may be taken from this
section.

1.2 How are supplies maintained?

In many countries, the transfer of information (post and telecommunica-
tions) is difficult and transportation of supplies may be delayed. As a result,
supplies may not reach health institutions in a timely fashion if they are
situated a long distance from the store where the supplies are kept. It is
therefore very important to make allowances for the problems of commu-
nication and transportation. This can be accomplished by maintaining a
“reserve” stock of medications within the health services system. In this
way, every patient can be assured of receiving all the medications neces-
sary to be cured of tuberculosis.
The quantity of reserve stock to be maintained is determined using the
second section of the Order Form for Treatment Supplies. The total quan-
tity advisable to be kept at each unit where a Co-ordinator is assigned is
equivalent to the quantity of medications that are consumed in one single
calendar quarter. This is calculated using the form as follows:

– The quantity of medications in the store (pharmacy) at the time of

completion of the form must be counted and the quantity entered under
“G”. The expiry date of the medications should also be noted to ensure
that supplies are used correctly. Medications that have passed their
expiry date should not be counted, but should be returned to the Central
Store.

– The total quantity needed for the patients for a quarter has been cal-
culated under the column “D” (A+B+C).

– This figure (D) is entered into the second table on the form, under E
and under F.

43
– It is possible to arrive at the total quantity required for the next quar-
ter by adding the quantity of medications required for the patients dur-
ing the quarter (E) and the quantity of “reserve” stock needed (F).
Then subtract from this sum the quantity of medications presently on
hand in the store (G), to arrive at the quantity to be ordered (E+F-G).
Maintaining the reserve stock ensures that all patients receive regular treat-
ment. As is apparent, the numbers of cases requiring different forms of
treatment which have been determined from the Quarterly Report on Case-
Finding are not exact figures. Some seriously ill patients with smear neg-
ative or extra-pulmonary tuberculosis may require the eight-month regimen.
Some new smear positive patients will be given the twelve-month regimen
because it is not possible to provide direct observation of the swallowing
of medications. The numbers of patients transferred into and out of the dis-
trict while on treatment and the number of patients who die or default also
influence the requirements. However, the reserve stock ensures that suffi-
cient medications will be available for the coming period, and the correc-
tion for the differences noted will automatically occur when the next order
for medications is completed in the following quarter.

2. How are laboratory supplies managed?

The treatment of each patient depends on the results of the laboratory exam-
ination of sputum samples (and therefore every patient must have an exam-
ination). Requirements for laboratory supplies can be determined by the
same method as for medication requirements, based upon the results of
case-finding. However, the materials required for the laboratory are in small
quantities and for this reason are ordered every half year (every other quar-
ter) rather than every quarter, and the reserve requirement is estimated to
be equivalent to one half year of supplies.
The basis for calculating the needs is the number of reported sputum
smear positive patients. Because many tuberculosis suspects must be exam-
ined to find one patient with sputum smear positive tuberculosis, this must
be taken into account. Experience from IUATLD collaborative programmes
shows that between 10 and 20% of all examined suspects are sputum smear
positive cases. Thus, if 10% are cases, then 10 suspects need to be exam-
ined for each case; if the proportion is 20%, then five suspects must be
examined. As each suspect should also have three sputum examinations,
and each case should have three additional examinations during follow-up,
the number of slides that need to be examined for each case is (1 x 10 x

44
3) + 3 = 33, where the proportion of cases among suspects is 10%. Where
it is 20%, the calculation is (1 x 5 x 3) + 3 = 18. The actual proportion
of cases among suspects varies in different locations, but can be calculated
easily from the Laboratory Register by counting several hundred suspects
(those recorded as examinations for diagnosis) and determining the num-
ber found positive amongst them. In the examples in this Guide, it is
assumed that the proportion of smear positive cases among suspects is 10%.
The calculation of the requirements is made using the Order Form for
Laboratory Supplies (Appendix 2, Form 8). The calculation is performed
as follows:
– the sum of smear positive patients (new cases and relapses) recorded
on the Quarterly Report on Case-Finding of the previous two quar-
ters is entered under the column headed “No. of patients”.
– The requirements for the half year (A) are calculated by multiplying
the number of patients by the factor (the quantity required for a sin-
gle examination).
– The “reserve” stock requirement (B) is equal to the quantity required
for one half year.
– The quantity of materials presently available (C) is determined by
counting the materials in the store at the unit.
– The total order (D) is the sum of the quantity required for the half
year (A) plus the quantity required for “reserve” stock (B), minus the
quantity in the store (C) at the time that the order form is completed.
In some locations, it is more practical for the intermediate level to prepare
the reagents for staining, which are then distributed to the peripheral
laboratories. An example order form for the peripheral level is found in
Appendix 2, Form 8b.

3. What other supplies are needed?

To maintain the regular care of patients, other materials are also required.
In particular, a regular supply of forms, registers and other recording materi-
als is needed to ensure that the patients are correctly managed. Determination
of the volume of materials required is based on the regular reports on the
results of case-finding. A list of items required should be prepared and a
stock kept at the unit.

45
 V. PROTECTING THE COMMUNITY

A. What is the rationale for a tuberculosis programme?

Tuberculosis is one of the frequent fatal infectious diseases for which
effective interventions exist, but which is presently not yet under control
in many countries.

1. Why do we believe that tuberculosis can be controlled?

Tuberculosis can be controlled world-wide. The reasons why this is possi-
ble are the following:

– the source of the infection is nearly exclusively a person who is sick

with the disease and who can thus be relatively easily identified;

– the rate of spread of infection can be quickly reduced if the infectious

cases are identified and effectively treated;

– the transmission of micro-organisms is relatively inefficient, so that

any reduction in the number of sources of infection and the period of
time each is infectious will inevitably improve the epidemiological
situation;

– the tools required to carry out the tasks (sputum smear microscopy
and chemotherapy) exist and can be applied efficiently even in diffi-
cult socio-economic conditions.

1.1 How does HIV affect the situation?

The introduction of HIV infection into the community has upset the bal-
ance between tuberculosis micro-organisms and the human host by com-
promising the immune system which, under usual circumstances, makes the
progress from tuberculous infection to tuberculosis relatively inefficient in
the individual. As a result, individuals, once infected, are much more likely
to develop disease and to become infectious themselves. The number of
infectious cases in the community increases considerably, thereby increas-
ing the risk of exposure for as yet uninfected members of the community.

46
Where individuals infected with HIV gather together (often in health care
facilities), exposure to infectious tuberculosis cases is more likely.
The impact of HIV on interventions for the control of tuberculosis, how-
ever, may not be as great as may be imagined if the available interventions
are applied efficiently. The following points should be considered:

– identification of infectious cases is unchanged as sputum smear

microscopy remains the most efficient means of determining the infec-
tious potential of individuals;

– cure of infectious cases is still possible in the presence of HIV infec-

tion as the treatment regimens remain as powerful, regardless of the
presence of HIV;

– the basis of tuberculosis control (case-finding and treatment) is there-

fore unchanged.

The urgency of applying widespread control methods cannot be over-

emphasised, as without these measures the transmission of tuberculosis
micro-organisms will increase rapidly.

1.2 How does drug resistance affect the situation?

An important objective of the management of tuberculosis is to avoid mak-
ing things worse than they already are. Inadequate management of tuber-
culosis patients is the most frequent cause of drug resistance. If tuberculo-
sis patients are managed as recommended in this Guide, development of
drug-resistant tuberculosis will be prevented and the extent of existing drug
resistance will be reduced. If there is already widespread multidrug resis-
tance, the outcome of treatment of patients will be affected depending on
the extent of the problem. In most low income countries the impact will
be unlikely to be great, if good case management is applied.
A poorly organised or managed National Tuberculosis Programme is
a frequent cause of drug resistance and often reflects misdirected priorities
for the Programme. Where the emphasis (and resources) are directed pri-
marily to specialised services for chronic resistant cases, a frequent occur-
rence where these cases are numerous, the problem is compounded. This
is because new drug-resistant cases are created faster than they can be cured.
Treating the unfortunate cases of multidrug-resistant tuberculosis without
addressing the reasons why they occurred in the first place (poor case man-

47
agement) will never succeed in overcoming the problem. The first priority
for allocation of resources must always be the proper case management of
those cases known to be curable, thus diminishing the creation of new resis-
tant cases.

2. Can tuberculosis be prevented by vaccination?

It is generally accepted that BCG provides a certain degree of protection
(particularly in young children) against serious forms of tuberculosis such
as miliary tuberculosis and tuberculous meningitis. Vaccination in child-
hood has little impact on controlling the spread of tuberculosis micro-organ-
isms in the community because the type of tuberculosis prevented by it is
usually not the infectious form (smear positive pulmonary tuberculosis), as
this form is infrequent in childhood.
BCG vaccination is included in the Expanded Programme on
Immunization (EPI) in most countries. BCG vaccine is usually given at
birth. The vaccine is injected intradermally on the upper portion of the left
arm, at a dose of 0.05 ml for those up to one year of age (and at a dose
of 0.1 ml for those more than one year of age). There is no scientific jus-
tification for revaccination with BCG, and this practice is a waste of
resources.

B. What should be done if there is no programme?

In some countries, particularly when the political situation is unstable, there
is no existing National Tuberculosis Programme. Very often, voluntary and
other non-governmental organisations are given the challenge of dealing
with tuberculosis patients as part of the general health service they provide.
On humanitarian grounds, health care personnel may be required to treat
patients even when conditions are far from ideal. Nevertheless, some
important considerations need to be taken into account.

1. Why is a programme important?

Research has clearly shown that poor treatment of tuberculosis patients can
have a harmful impact on the tuberculosis situation. This is due to the fact
that, while it is relatively easy to prevent death due to tuberculosis, it is
more difficult to cure a patient permanently and thus to prevent the patient
from spreading infection to uninfected members of the community.

48
 In addition to increasing the number of sources of infection in the com-
munity (by keeping alive a patient who would otherwise have died but fail-
ing to cure the patient and therefore failing to stop the spread of tubercu-
losis micro-organisms), poor treatment has another, very serious consequence.
Patients who are treated for tuberculosis but fail to be cured are at a high
risk of developing chronic and resistant tuberculosis. Thus, in addition to
increasing the risk of transmission of tuberculosis micro-organisms, the type
of tuberculosis that is being spread is resistant and, when resistant to mul-
tiple medications, is also incurable with the types of treatment currently
available. If you cannot ensure that a patient will be treated properly, you
must carefully consider whether it is ethical to treat such a patient at all.

2. How can care be given safely?

In the light of humanitarian concerns, it is often not possible to do noth-
ing, even when the action is potentially harmful. Certain guidelines should
therefore be followed under such circumstances:

– Treatment of tuberculosis patients should not be initiated until a

microscopy service is established, and then priority must be given to
those who are sputum smear positive.

– Rifampicin-containing regimens should be used only if it is certain that

the patient will remain available to have the medications directly
observed throughout the period of treatment during which rifampicin
is given. Failure to follow this principle will increase the risk of devel-
opment of resistance to medications, will threaten the life of the patient,
and will create a danger to the community.

– Patients who have been previously treated for as much as one month
should be given a retreatment regimen.

– If a previously untreated patient, at the time of diagnosis, is unable to

ensure availability throughout the period in which treatment with
rifampicin is to be given, the patient should be given the 12-month
regimen described previously.

– Where regular supplies cannot be assured, a full treatment regimen

should be set aside for each patient enrolled on treatment. This will
ensure that every patient who commences treatment is able to com-

49
 plete it. Treatment should not be commenced if the full supply of all
medications cannot be assured from the outset of treatment.

– Medications should, as much as possible, be in combined preparations.

Isoniazid can be obtained in combination with thioacetazone (the pre-
ferred method of treating a patient in the absence of a structured pro-
gramme) or with ethambutol. Rifampicin should always be used in
combination with isoniazid. The preparations used must be of proven
bioavailability. Failure to follow this advice is professional negligence.

– Rifampicin should never be used in situations where its use cannot be

strictly controlled (administered under direct observation of swallowing
of medications and its disappearance into the market place prevented).
The greatest risk of using uncontrolled rifampicin is the creation of
resistance to both isoniazid and rifampicin, which means that the patient
is no longer curable and members of the community may become
infected with incurable micro-organisms.

C. How can we ensure that activities achieve good

results?
Evaluation of tuberculosis entails evaluation both of the tuberculosis situ-
ation and of the control measures (interventions) applied. Evaluation of the
control measures applied (the process whereby the tuberculosis situation is
modified) is accomplished through the quarterly reports on case-finding and
on treatment results, as described previously. Targets for control measures
have been established at a global level and may be modified for the local
situation during the implementation of a programme. Targets should focus
as much as possible on items that it is possible to do something about. A
comparison of the results achieved with the targets that have been estab-
lished forms the basis of evaluation of the control measures applied.

1. How do we evaluate control measures?

The most important evaluation is the regular review of treatment results.
Several results are of particular importance:

– The proportion of all cases that have defaulted from treatment reflects
the organisation of the services. The only way to achieve acceptable

50
 results of treatment (the ultimate target of the treatment programme)
is by reducing the ratio of patients who default. This indicates whether
patients find the service accessible and appropriate, and often reveals
the attitude of the health services personnel in providing care for
patients. This is the most important target.

– The proportion of patients whose treatment result is transfer should be

very low. Those who are transferred to facilities in the same general
area should have their results obtained from that facility.

Other outcomes to monitor over the course of time include:

– The proportion of cases who are smear positive at 5 months or more

after starting treatment (treatment failures) may be an indication of the
efficacy of the regimen utilised, and indirectly indicates the level of
resistance to medications in the patients. If this ratio persistently remains
above 5% several years after the programme has commenced, it may
indicate a need for investigation.

– The proportion of patients who died has indicated, in some settings,

the impact of the HIV epidemic in the community.

The review of regular reports allows the treatment activities to be evalu-

ated. Progress in approaching the targets can be determined by comparing
various geographical areas of a country to identify problem areas. Trends
can be defined (both positive and negative) in the results of treatment in a
single location which indicate the quality of care in that location.

2. What is the size of the tuberculosis problem?

Evaluation of the tuberculosis situation (epidemiological surveillance) is
important to enable planning and budgeting and to evaluate and adjust the
programme in relation to the challenges identified. Several epidemiological
indices have been utilised for measuring the extent of the tuberculosis prob-
lem in a given community.

2.1 Rate of reported smear positive cases

This index can only be used in countries where case-finding and reporting
of smear positive pulmonary tuberculosis is relatively complete. The com-
pleteness of notification is dependent upon the coverage of the diagnostic

51
service (sputum smear microscopy), its reliability, and upon the efficiency
of the reporting system. This is the most practical means of surveillance in
most countries, and its accuracy and completeness should have a high pri-
ority among surveillance activities.

2.2 Prevalence of smear positive pulmonary tuberculosis

A well-conducted prevalence survey gives reliable information on the num-
ber of infectious cases in a community at a given point in time. It is, how-
ever, expensive and complicated to undertake reliably.

2.3 Tuberculin surveys

The measurement of the prevalence of significant reactions to the tuber-
culin skin test and the derivation of the average annual risk of infection
has been used to monitor the epidemiological situation of tuberculosis in a
community. It is not easy to conduct a reliable tuberculin survey, as it must
be based on a sample of the population that is representative of the gen-
eral population. Moreover, certain technical aspects of the performance of
the test and of the determinants of test reactions (vaccination coverage, the
occurrence of infection with other Mycobacterium species) may lead to dif-
ficulties in interpretation. Information on the conduct of a tuberculin sur-
vey may be obtained from the IUATLD upon written request.
The determination of trend in tuberculin skin reactivity is probably
more informative than a single determination of prevalence. Repeated
surveys of representative samples of a population 5 to 10 years apart may
give a reliable estimation of the trend of the tuberculosis problem in a
community.

3. How will HIV affect the situation?

Because HIV infection may have a dramatic impact on the trend in tuber-
culosis rates, where the two infections coincide in a population it is impor-
tant to determine the level and trend of HIV infection in tuberculosis cases.
The determination of HIV seroprevalence and of its trend is accomplished
relatively easily in a representative sample of at least all the new smear
positive cases of tuberculosis within a country because tuberculosis has a
“magnifying” effect in the determination of HIV prevalence. HIV has already
caused a dramatic rise in the numbers of tuberculosis patients in a number
of countries, and has resulted in tremendous constraints on health services.

52
Proper planning and budgeting can only be carried out when there is knowl-
edge of how the two infections coincide in the community.
A standardised approach to the determination of HIV seroprevalence
in tuberculosis patients is recommended. This protocol can be obtained on
written request from the IUATLD.

4. Will patients respond to treatment?

Because resistance to antituberculosis medications is a man-made problem,
it may result from poorly organised or managed National Tuberculosis
Programmes. Thus, the trend in resistance to antituberculosis medications
is a means of identifying deficiencies in the application of a National
Tuberculosis Programme.
In order for it to be a reliable reflection of programme performance,
the determination of prevalence and trend in resistance must be undertaken
with a representative sample of the tuberculosis cases in a defined area in
which the programme has been implemented. Certain specific sampling
and technical aspects must be taken into account in planning such a deter-
mination. For this reason, a standardised protocol has been developed for
the conduct of such surveys and is available upon written request from the
IUATLD.

53
 VI. APPENDIX I

TECHNICAL GUIDE

55
 TECHNICAL GUIDE

Sputum Examination
for
Tuberculosis by Direct Microscopy
in Low Income Countries

Fifth edition
2000

International Union Against Tuberculosis and Lung

Disease
68 boulevard Saint Michel, 75006 Paris, France

57
 WRITING COMMITTEE
Mohammed Akhtar
Gisela Bretzel
Fadila Boulahbal
David Dawson
Lanfranco Fattorini
Knut Feldmann
Thomas Frieden
Marta Havelková
Isabel N de Kantor
Sang Jae Kim
Robert Küchler
Frantz Lamothe
Adalbert Laszlo
Nuria Martin Casabona
A Colin McDougall
Håkan Miörner
Graziella Orefici
C N Paramasivan
S R Pattyn
Ana Reniero
Hans L Rieder
John Ridderhof
Sabine Rüsch-Gerdes
Salman H Siddiqi
Sergio Spinaci
Richard Urbanczik
Véronique Vincent
Karin Weyer

On a draft document prepared by Adalbert Laszlo, for the International Union Against
Tuberculosis and Lung Disease

Graphic design: Edik Balaian

59
 PREFACE
A technical guide for sputum smear microscopy, based on one initiated in 1969
by Dr J Holm, the then Director of the International Union against Tuberculosis,
was first published in 1978 by the IUAT as the Technical Guide for Sputum
Examination for Tuberculosis by Direct Microscopy. The guide was included in
the third and fourth editions of the IUATLD’s Tuberculosis Guide for Low Income
Countries. It was designed to be a simple reference standard for the collection,
storage and transport of sputum specimens and for the examination of sputum
smears by direct microscopy. It was meant to address the needs of health care
workers in low income, high prevalence countries which represent the bulk of the
global tuberculosis caseload.

More than twenty years have elapsed since its first publication, and the guide
has remained unchanged throughout that time. Today, tuberculosis is one of
the main causes of death from a single infectious agent among adults in low
income countries, where it remains a major public health problem. The basic
tool for TB diagnostic services, i.e., sputum smear microscopy, has not changed
in its technical details in spite of major advances in modern diagnostic tech-
nologies. However, the context in which it is applied, the National Tuberculosis
Programme, has been refined to a considerable extent in the last two decades.

The field use of the guide over the years has revealed omissions and inac-
curacies that needed to be addressed. Furthermore, biosafety and quality assur-
ance aspects of sputum smear microscopy were not sufficiently well covered in
the previous edition. It was therefore felt that the IUATLD Technical Guide needed
revision so it could better reflect its public health essence and keep up to date with
modern TB control strategies. This document was carefully revised by members
of the Bacteriology and Immunology Section of the IUATLD, by directors of the
WHO/IUATLD Supranational TB Reference Laboratory Network and by other dis-
tinguished professionals in the field of tuberculosis control.

DR ADALBERT LASZLO
Ottawa 2000

60
 TABLE OF CONTENTS

1. SPUTUM MICROSCOPY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

1.1 Aims of tuberculosis laboratory diagnostic and follow-up services . . . . . . . . . . . 63

1.2 “Spot”, “morning” and “spot” sputum specimens . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

1.3 “Morning” specimens for follow-up. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

1.4 Sputum containers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

1.5 Collection of sputum specimens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

1.6 Transportation of sputum specimens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

2. THE SPUTUM SMEAR MICROSCOPY LABORATORY. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

2.1 Role of the laboratory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

2.2 Physical environment of the laboratory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68

3. THE STAINING METHOD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

3.1 Preparation of Ziehl-Neelsen reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

3.1.1 Ziehl’s carbol fuchsin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
3.1.2 Aqueous phenol solution. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
3.1.3 Decolourising agent solutions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
3.1.4 Methylene blue counterstaining solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70

3.2 Smear preparation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70

3.2.1 Smearing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
3.2.2 Fixing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
3.2.3 Staining. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
3.2.4 Decolourising. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
3.2.5 Counterstaining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
3.2.6 Quality of smearing and staining. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74

4. MICROSCOPIC EXAMINATION OF SPUTUM SMEARS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74

4.1 The microscope. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74

4.2 Operating the microscope . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74

4.3 Microscopic examination of smears. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76

4.4 Grading of sputum smear microscopy readings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76

4.5 Preservation of smears for quality assurance testing . . . . . . . . . . . . . . . . . . . . . . . . . . 77

5. RECORDING AND REPORTING OF SMEAR MICROSCOPY RESULTS . . . . . . . . . . . . . . 77

5.1 The laboratory register . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77

61
6. QUALITY ASSURANCE OF SPUTUM SMEAR MICROSCOPY . . . . . . . . . . . . . . . . . . . . . . . 79
6.1 Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
6.2 Procedures. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79

7. DISINFECTION, STERILISATION AND DISPOSAL OF CONTAMINATED MATERIALS 80

8. BIOSAFETY IN THE TB MICROSCOPY LABORATORY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

8.1 General aspects. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
8.2 Specific aspects. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

9. MATERIALS MANAGEMENT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82

REFERENCES. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84

SUGGESTED READING . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
Annex 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
• Prevention of false positive results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
• Consequences of false positive results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
• Prevention of false negative results. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
• Consequences of false negative results. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
Annex 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
• Care of the microscope . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
Annex 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
• Trouble-shooting guide for microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87

62
 1. SPUTUM MICROSCOPY
1.1 Aims of tuberculosis
laboratory diagnostic
and follow-up services
In low income and high tuberculosis preva-
lence countries, sputum smear microscopy
is, and is likely to remain for the foreseeable
future, the only cost-effective tool for diag-
nosing patients with infectious tuberculosis
and to monitor their progress in treatment.
Sputum smear microscopy is a simple, inex-
pensive, appropriate technology method
which is relatively easy to perform and to
read. It yields timely results with a very high
sensitivity of detection of tubercle bacilli
transmitters, and provides most of the essen-
tial laboratory-epidemiological indicators
needed for the evaluation of the National
Tuberculosis Programme (NTP).
The aims of TB laboratory diagnostic ser-
vices within the framework of an NTP are:
– diagnosis of cases
– monitoring of tuberculosis treatment
1.2 “Spot”, “morning” and “spot”
sputum specimens for diagnosis
Under NTP conditions, the IUATLD recom-
mends collecting three sputum samples “on
the SPOT – early MORNING – on the SPOT”,
preferably within two days, from each per-
son presenting at health centres, out-patient
clinics, etc., with respiratory symptoms of
more than 3 weeks’ duration. These samples
are to be examined by smear microscopy in
the nearest laboratory. Under these condi-
tions, a case of sputum smear positive tuber-
culosis is usually defined as a person pre-
senting with respiratory symptoms with at
least two positive sputum smear microscopy
examinations.
This approach, also known as passive
case finding, detects about 80% of TB sus-
pects ultimately positive on sputum smear
examination with the first specimen, an addi-
tional 15% with the second and a final
5% with the third. “SPOT” specimens are
obtained when the TB suspects present at
63
the health centre; “MORNING” specimens
consist of all sputum produced within one or
two hours after rising.
1.3 “Morning specimens” for follow-up
There are two phases in the treatment of
tuberculosis: the intensive phase, usually 2
to 3 months, and the continuation phase,
which is 4 to 10 months, depending on the
type of treatment. Regardless of the treat-
ment regimen, one “MORNING” sputum
specimen is collected for follow-up at the end
of the intensive phase of treatment to deter-
mine whether the patient can proceed to the
continuation phase if the smear is negative
or, if the smear is positive, continue the inten-
sive phase. Another sputum specimen must
be taken during the continuation phase to
check patient evolution and to detect possible
treatment failure, and another upon comple-
tion of chemotherapy to verify cure. Sputum
specimens at the end of treatment are often
difficult to obtain, as many patients have
stopped expectorating. The exact schedule
of follow-up sputum examinations varies
according to the drug regimen, and should
be set out in the NTP Manual.
The patient is said to have completed
treatment even if sputum specimens are not
examined during and at the end of treatment.
The cure rate is the proportion of initially spu-
tum smear-positive patients who are declared
cured based on negative sputum smear
results on at least two occasions, including
one at the end of treatment. The objective of
the NTP is to achieve at least 85% cure rate
among new sputum smear positive TB cases
registered.
The IUATLD recommends:
– The examination of three sputum spec-
imens – “SPOT” + “MORNING” +
“SPOT” – for the diagnosis of tubercu-
losis cases.
– The examination of single “MORNING”
sputum specimens on three occasions
for follow-up of treatment: one at the
end of the intensive phase, one during
the continuation phase, and one at the
end of treatment.

1.4 Sputum containers
The use of two kinds of sputum containers is
recommended. One, available from UNICEF
(Figure 1 A), is a rigid wide-mouthed screw-
capped container made of unbreakable trans-
parent plastic, readily disposable by burning,
which is used for most routine diagnostic
work. Its screw cap can be hermetically
sealed to prevent desiccation of the sample
and leakage.
The other, a screw-capped heavy glass
container, such as the Universal bottle (Figure
2 A), is used for specimen transportation in
custom-made boxes that can be made of
metal, wood or styrofoam. A wooden box is
a sensible compromise in terms of sturdiness
and weight (Figure 2 B, C). The Universal
bottle is reusable after disinfection by auto-
claving for 30 minutes at 121oC and careful
cleaning. If an autoclave is unavailable, a
domestic pressure cooker is recommended.
1.5 Collection of sputum specimens
The risk of infection for health care workers is
highest when TB suspects cough; sputum
specimens should therefore be collected in
the open air and as far away as possible from
other people. Failing this, a separate, well-
ventilated room should be used.
64
Figure 1
The health care worker should reassure
persons suspected of having tuberculosis by
explaining the reasons for the examination,
and give instructions on how to cough so that
the expectoration is produced from as deep
down in the chest as possible. As an adjunct,
written instructions can be handed out if the
person is literate.
The health care worker should make sure
that the specimen is of sufficient volume (3 to
5 ml) and that it contains solid or purulent
material, the presence of which increases the
sensitivity of detection, and not just saliva.
However, if only saliva is obtained or, as fre-
quently happens in “spot” sputum, volumes
of less than 3 ml are produced, the specimen
should nevertheless be processed, as it is
sometimes likely to yield positive results. A
sputum specimen can be classified by macro-
scopic examination as “salivary” when it
consists mainly of saliva, “mucous” when it
is mainly mucus, “purulent” when it appears
yellow as pus, “muco-purulent” when there
are visible yellowish particles in the mucus
and “bloody” when it contains blood. The
presence of blood should always be noted
because it is indicative of severe disease and
could interfere with the reading of the smear.
The health care worker should provide a
sputum container with the health centre code
and the TB suspect or patient’s identification

B
A specimens
C portation should be selected. Contaminating
Figure 2
must be written on the side of the container,
never on its lid. (Figure 1 C). The health care
worker must ask the person being examined
to bring it close to the mouth and expectorate
into it (Figure 1 B). This specimen is called a
“SPOT” specimen.
If expectoration is not produced, the spu-
tum container must be considered as used
and must be properly disposed of. Sputum
containers must be closed securely and if
they are to be sent to a nearby laboratory,
they should be placed in the appropriate
transport box. Collected specimens should
be kept in a cool place, batched and trans-
ported without much delay, i.e., at least twice
weekly, and processed as soon as possible.
Sputum can also be processed in the health
65
centre, and fixed smears can be sent to the
nearest laboratory. This procedure is dis-
couraged, however, because fixed smears
are often of poor technical quality, having
been prepared by untrained personnel, and
tend to decompose quickly in warm and
humid climates.
The health care worker should provide
the TB suspect with a new, pre-labelled spu-
tum container, explain how it should be used
in the morning to collect the “MORNING”
specimen and demonstrate how it should be
securely closed before it is brought back to
the health centre.
1.6 Transportation of sputum
In countries lacking laboratory facilities
that rely on specimen collecting units,
transportation of specimens is required.
Transportation is also required when opera-
tional research projects of interest to the NTP
are undertaken, such as a survey of TB drug
resistance, etc. If culturing of specimens is
required, the specimens should reach the lab-
oratory within 3-4 days and should be refrig-
erated while waiting for shipment. The most
rapid and cost-effective means of trans-
flora do not affect the acid-fastness of
mycobacteria but may liquefy the sputum,
making smear preparation difficult and read-
ing of slides unreliable.
A list identifying the sputum specimens
contained in the transport box and a com-
pleted Request for Sputum Examination
Forms (Figure 3) for each specimen must
accompany the shipment. Before the ship-
ment from the health centre, the health care
worker must verify for each transport box
that:
– the total number of sputum containers in
the box corresponds to that on the accom-
panying list and that on the Request for
Sputum Examination Forms;
– the identification number on each sputum
container corresponds to that on the accom-
panying list and that on the Request for
Sputum Examination Forms;
– the accompanying Request for Sputum
Examination Forms contain the requested
information for each of the TB suspects.
REQUEST FOR SPUTUM EXAMINATION

Figure 3. Request for sputum examination form

66
 When this verification is completed, the
health care worker:
– dates the accompanying list;
– puts the list and the Request for Sputum
Examination Forms in an envelope which
will be attached to the outside of the trans-
port box.
A laboratory code, a serial number and
a specimen sequence identifier, i.e., 1 for
2. THE SPUTUM SMEAR MICROSCOPY LABORATORY
2.1 Role of the laboratory
In developing countries, most of the bacteri-
ological diagnosis of tuberculosis is carried
out in peripheral or local laboratories, whose
major responsibility is to provide diagnostic
microscopy for the NTP based on sputum
smear examination by Ziehl-Neelsen (ZN)
staining. These laboratories, located in health
centres, health posts, hospitals, etc., usually
have qualified technical personnel capable of
performing – among other duties – sputum
smear microscopy. They should be able to
carry out the following functions:
Figure 4
67
first, 2 for second, 3 for third (Figure 12),
will be assigned to each specimen by labo-
ratory staff. Results of the examination
will be entered on the bottom half of the
Request for Sputum Examination Form. The
Laboratory Serial Number begins with 1 on
1 January each year and increases by one
with each patient until 31 December of the
same year.
– perform all sputum smear microscopy
requested in their catchment area, usually a
district (50,000-100,000 inhabitants);
– act as referral center for specimen collect-
ing units;
– co-ordinate with Regional (intermediate)
Laboratories the referral of specimens
requiring culture and drug susceptibility test-
ing;
– receive specimens during the opening
hours of the health centre;
– send information to the Regional Labo-
ratory;
– comply with national quality assurance
guidelines;
– order, manage and store laboratory sup-
plies.

2.2 Physical environment

of the laboratory
The detailed arrangement of the microscopy
laboratory varies greatly depending on local Figure 5
conditions. It is difficult to generalise about
the design of such laboratories since TB diag-
nostic services have, over time, been inte- – a sink (D) with running water for washing
grated into existing general laboratory diag- hands;
nostic services in many countries. Ideally, the – a bench area (E) for microscopy reading
TB microscopy laboratory should include the directly below a window (Figures 4 and 8);
following distinct sections (Figure 4), adapted – a bench area or a table (F) for the labora-
from Collins et al:1 tory register books and slide storage space
– a bench space or a table (A) for incoming (Figures 4 and 9);
specimen (Figures 4 and 5);
– one well-lit work bench (B) for smear
preparation (Figures 4 and 6);
– a staining sink (C) with running water
(Figures 4 and 7);

Figure 6
1. Slide-holder for the preparation of smears 5. Alcohol lamp / Bunsen burner
2. Slide dryer 6. Forceps
3. Sputum container placed as close as possible to the 7. Metal waste receptacle with lid to receive infectious
slide-holder on the right material
4. Wooden applicators 8. Box of engraved slides for the smears

68

Figure 7
Figure 8
– a locker (G) for the technicians’ clothing
(Figure 4).
If the work bench is made of porous
material, a non-porous surface plate, such as
formica, a marble slab, or a galvanised metal
or aluminum sheet, should cover the work
3. THE STAINING METHOD
3.1 Preparation of Ziehl-Neelsen
reagents
The method of choice for sputum smear
microscopy is the Ziehl-Neelsen (ZN) stain-
ing technique because it is the only one that
provides consistently good results without
the need for special equipment. Preparing the
necessary reagents requires a weighing scale
that is not always available in a peripheral
laboratory, and preparing the reagents in the
National Reference Laboratory or in the near-
est intermediate laboratory is therefore a fre-
69
Figure 9
bench: this plate should be about 80 cm wide,
with borders 5 cm high. The front edge must
be bent down at an angle of 90o to meet the
edge of the table, thus facilitating manipula-
tions (Figure 6). These must be conducted
strictly over the surface plate, which should
be decontaminated every day after use by
soaking with a TB germicide (e.g., 5% phe-
nol, 0.1% solution of sodium hypochlorite*
[NaClO], also known as common household
bleach, Chlorox, Javex, etc.).
NOTE: If the technician is left-handed, it
may be more convenient to arrange all (or
most) items in Figure 6 in exactly the oppo-
site position on the table (i.e., in a mirror
image).
* Household bleach contains 5% of NaClO (50 g/litre);
a 0.1% solution containing 1 g of NaOCl/litre is pre-
pared by diluting 20 ml of household bleach in 1 litre
of water. This solution is used as an all-purpose dis-
infectant for “clean conditions”.2
quently used option. The advantages of this
option, i.e., better standardisation and qual-
ity assurance, outweigh the disadvantages of
long term storage. Cold staining procedures
such as the Kinyoun and Tan Thiam Hok
methods are not recommended, as evidence
shows that they have difficulty detecting acid-
fast bacilli (AFB) in paucibacillary samples
and the staining fades rapidly. Fluorescence
microscopy, which is recommended when
the daily workload exceeds 50 specimens,
has no place in most peripheral laboratories
of low-income countries.
3.1.1 Ziehl’s carbol fuchsin
3% fuchsin alcoholic stock solution (solution A)
Basic fuchsin*. . . . . . . . . . . . 3 g†
95% alcohol‡ . . . . . . . . . . . . . up to 100 ml
Place the required amount of fuchsin in a
volumetric flask or measuring cylinder and,
adding enough ethanol or methylated spirit
to obtain a total volume of 100 ml, shake well
until completely dissolved. Small quantities
of this solution should be filtered prior to
staining.
3.1.2 Aqueous phenol solution
(solution B)
Phenol§ cristals . . . . . . . . . . . 5 g
Distilled water, if possible . . up to 90 ml
Before adding water, liquefy the phenol
crystals in a flask by gentle heating.
To prepare the 0.3% Ziehl’s carbol fuchsin
working solution, mix 10 ml of Solution A
with 90 ml of Solution B.
3.1.3 Decolourising agent solutions
– Acid-alcohol solution
Alcohol 95% . . . . . . . . . . . . . . . . . . 970 ml
Concentrated (35%)
hydrochloric acid** . . . . . . . . . . . . 30 ml
Or, when alcohol is unavailable:
– 25% aqueous sulfuric acid solution
Distilled water if possible . . . . . . . 300 ml
Concentrated sulfuric acid†† . . . . . 100 ml
Pour 300 ml of water into a 1 litre
Erlenmeyer flask. Slowly add 100 ml of con-
centrated sulfuric acid, allowing it to flow
along the side of the flask. The mixture will
heat up. Never pour water into concentrated
sulfuric acid – explosive spills may occur.
3.1.4 Methylene blue counterstaining
solution 0.3%
Methylene blue chloride‡‡ . . 0.3 g
Distilled water, if possible . . up to100 ml
3.2 Smear preparation
Sputum containers are arranged in sequential
order. Laboratory serial numbers must
match the corresponding information on the
70
accompanying Request for Sputum Exam-
ination Form. The use of new slides is rec-
ommended; however, because they are often
greasy, they tend to cling together and must
be cleaned with alcohol and then carefully
air-dried. When alcohol is not available the
slides may be held over a flame to remove
oils. Under weather conditions prevalent in
most low income countries, the use of slides
in tropical packaging (each slide separated
from the next by a strip of impermeable
paper) is recommended. Laboratory code,
serial number and sequence identifier can be
engraved with a diamond marker on the
smear side and at one end of the slide. When
diamond markers are not available, a dis-
carded round-tipped dental drill inserted in
the tapered end of a discarded plastic pen
can be used.3 An ordinary lead pencil can be
used if frosted-end slides are available.
3.2.1 Smearing
– Verify that the numbers on slides and con-
tainers match.
– Take sputum container corresponding to
the number on the slide.
– Open container carefully to avoid aerosol
production.
– Break a wood or bamboo stick applicator
(Figure 10), select yellow, purulent particle of
sputum with jagged end of the broken wood
or bamboo stick applicator. Use the broken
* Pararosaniline chloride, Minimum dye content 88%
(C19H18NCl) Sigma P1528 or equivalent.
† Staining powders are seldom pure, so a corrected
weight should be used to ensure proper staining. The
percentage of available dye content is frequently listed
on the original container label. The corrected weight
is determined by dividing the desired amount of dye
by the decimal equivalent of the available dye. So, if
the desired amount of dye is 3 g and the per cent avail-
able dye is 75%, the actual amount of dye to be
weighted is 3/0.75 = 4 g of impure dye. If the avail-
able dye content is 88% or more, there is no need to
correct the weight.
‡ Ethanol 95% (C H OH) – United States Pharmacopeia
2 5
XVIII, 20, 1067 (1970). Can be of industrial grade.
§ Phenol approx. 99% (C H O) – Sigma P 3653 or
6 6
equivalent.
** Concentrated hydrochloric acid (HCl) – can be of
industrial grade.
†† Concentrated sulfuric acid (H SO ) – can be of indus-
2 4
trial grade.
‡‡ Methylthionine chloride, minimum dye content 82%
(C16H18CIN3S) – Sigma M 9140 or equivalent.

Figure 10
end of the two pieces of the applicator to
break up larger particles.
– Spread the sputum evenly over the central
area of the slide using a continuous rotational
movement (Figure 11); the recommended
Figure 11
size of the smear is about 20 mm by 10 mm
(Figure 12).
Figure 12
– Place slides on dryer with smeared surface
upwards, and air dry for about 30 minutes.
– Re-cap sputum container, which should not
be discarded before results are read and
recorded.
71
Applicators are only used once. Discard
by placing them in a waste receptacle
containing 5% aqueous phenol solution or a
0.5% solution of sodium hypochlorite,* then
autoclave or incinerate. CAUTION: Vapours
are very toxic.
3.2.2 Fixing
Fix dried smears by holding them with for-
ceps and passing them smear side up over
the flame 5 times for about 4 seconds (Figure
13). Do not heat-fix moist slides, and do not
overheat.
Figure 13
3.2.3 Staining
– Place fixed slides on the staining rack in
serial order, smeared side up. Slides should
be separated by a 1 cm gap, and should never
touch one another.
– Cover slides individually with filtered 0.3%
Ziehl’s carbol fuchsin working solution
(Figure 14). Placing a strip of absorbent paper
such as filter or even newspaper will hold the
staining solution and prevent deposits of
fuchsin crystals on the smear.
– Heat slides from underneath with the flame
of a Bunsen burner, an alcohol lamp or an
alcohol soaked cotton swab until vapour
starts to rise. Staining solution should never
be allowed to boil. Do not allow the stain to
dry (Figure 15).
* Sodium hypochlorite is a strong oxidizing agent
which is corrosive to metal. A 0.5% solution contain-
ing 5 g of NaClO/litre, prepared by diluting 100 ml of
household bleach in 1 litre of water, is recommended
for dealing with “dirty conditions”.2
 – Rinse slides gently with water to remove
excess carbolfuchsin (Figure 16).
– Drain off excess rinsing water from slides
(Figure 17). Sputum smears appear red in
colour.

Figure 14

Figure 17

3.2.4 Decolourising
– Cover slides with 25% sulfuric acid or
acid-alcohol solution and allow to stand for
3 minutes, after which the red colour should
have almost completely disappeared (Figure
18). If needed, repeat sequence until the
Figure 15 red colour disappears, but do not over-
decolourise.

– Keep slides covered with hot, steaming car-

bolfuchsin for 5 minutes by re-flaming as
needed.

Figure 18

– Gently wash away the sulfuric acid or acid

alcohol and the excess stain with water
(Figure 19). Drain off excess rinsing water
Figure 16 from slides (Figure 20).

72
 – Rinse slides individually with water
(Figure 22).

Figure 19

Figure 22

– Drain water off the slides, which are then

allowed to air dry (Figure 23).

Figure 20

3.2.5 Counterstaining
– Cover slides individually with 0.3% meth-
ylene blue counterstaining solution and allow
to stand for 1 minute (Figure 21).

Figure 23

The Ziehl-Neelsen staining procedure

requires:
– Staining for 5 minutes
– Decolourising for 3 minutes
– Counterstaining for 1 minute
Figure 21

73
3.2.6 Quality of smearing and staining
– A properly stained smear should show a
light blue colour due to methylene blue. If
dark blue, i.e., a newspaper cannot be read
Figure 24
4. MICROSCOPIC EXAMINATION OF SPUTUM SMEARS
4.1 The microscope
A binocular microscope with two objectives
– a regular 40 x magnification objective and
an oil immersion 100 x magnification objec-
tive – and eyepieces of moderate magnifica-
tion (8x or 10x) is required for the examina-
tion of smears (Figure 26).
Microscopes equipped with the light-col-
lecting mirror option are strongly recom-
mended, as they are useful in the event of
power failures and necessary in laboratories
that lack electricity. The mirror has one plane
surface for artificial light and another con-
cave surface for natural light. An illuminator
is built into the base of the microscope; a
halogen bulb provides good illumination.
Halogen lamps have higher luminosity and
longer life than tungsten lamps.
When not in use, microscopes should be
kept in their case protected from dust, heat
and humidity. Fungus growth is a constant
threat to the microscope’s optical system: it
can be inhibited by fitting the storage case
with a 20-40 watt lamp, which is kept lit dur-
ing the storage of the microscope. The objec-
tive, eyepiece, condenser and light source are
74
when held underneath the slide, the smear
is too thick.
– Example of a good smear (Figure 24).
– Examples of bad smears (Figure 25)
Figure 25
kept clean by wiping with lens paper on a
daily basis.
4.2 Operating the microscope
– A drop of immersion oil is placed on a dry
stained slide to increase the resolving power
of the objective. To prevent cross contami-
nation by AFB, the immersion applicator
should not touch the slide. Cedarwood
immersion oil should never be used, as it
forms a thick paste upon drying that could
damage the lenses of the microscope.
“Makeshift” use of other oils, such as linseed,
palm, olive, liquid paraffin, etc., is completely
unsatisfactory. Some immersion oils can dis-
solve fuchsin stain,4 a circumstance that
might accelerate the fading of the ZN stain.
Synthetic hydrocarbons and advanced poly-
mers with a refractive index of 1.5, non-
drying and non-hardening, with no solvent
capability, are recommended.*
* Type A or B immersion oil (R. P. Cargille Labs, Inc.
Cedar Grove, NJ. Catalogue No. 16484, or VWR brand
Immersion Oil, Resolve, Catalogue No. 48218, or
equivalent).
 Figure 26

1) eye piece; 2) Diopter ring; 3) objective; 4) stage; 5) condenser; 6) diaphragm lever;

7) coarse focus knob; 8) fine focus knob; 9) light source.

75
– With the condenser raised to the upper-
most position, the stained slide is placed on
the stage and the light source is adjusted for
optimal light by looking through the eyepiece
and the regular 40 x objective.
– An area containing more leukocytes (pus
cells) than epithelial cells (more frequent in
saliva) is selected before placing the drop of
immersion oil.
– By slowly changing to the immersion
objective, a thin film of oil will form between
the slide and the lens. The fine adjustment
knob is used to focus the field; the lens
should not be allowed to touch the slide.
For more on the use and operation of the
microscope, see reference 5.

4.3 Microscopic examination
of smears
– Acid-fast bacilli appear bright red or pink
against the blue counterstained background.
They vary greatly in shape, from short, coc-
coid to elongated filaments; they can be uni-
formly or unevenly stained, and can even
appear granular. They occur singly or in vari-
able sized clumps, and typically appear as
long, slender curved rods.
– The microscopic examination must be sys-
tematic and standardised. It can start at the
left end of the smear. The reading begins at
the periphery of the field and ends at the cen-
tre (Figure 27). When the field is read, the
slide is moved longitudinally to examine
adjacent fields. The slide can be moved ver-
Figure 27
tically so that a second length can be read
from right to left. There are about 100 immer-
sion fields in the 2 cm long axis of a smear.
4.4 Grading of sputum smear
microscopy results
The information on the number of bacilli
found is very important because it relates to
the degree of infectivity of the patient as well
as to the severity of the disease. For this rea-
son, the report of the results of sputum smear
microscopy must be not only qualitative but
also semi-quantitative. The IUATLD recom-
mends the following grading of results of
smear microscopy (Table 1).

Table 1 IUATLD-recommended grading of sputum smear microscopy results

AFB counts Recording/reporting

No AFB in at least 100 fields 0/negative
1 to 9 AFB in 100 fields* Actual AFB counts†
10 to 99 AFB in 100 fields‡ +
1 to 10 AFB per fields in at least 50 fields † ++
> 10 AFB per field in at least 20 fields ‡ +++

* A finding of 1 to 3 bacilli in 100 fields does not correlate well with culture positivity. The interpretation of the significance
of this result should be left to the NTP and not to the microscopist. It is recommended that a new smear be prepared from
the same sputum specimen and be re-examined.
† The reporting of actual AFB counts is recommended to allow a competent authority to determine whether the number fits

the TB case definition of the NTP.

‡ In practice most microscopists read a few fields and confirm the finding by a quick visual scan of the remaining fields.

76
 The microscopist should initial the smear
result as well as other result entries in the
laboratory register.
The microscopist should take at least 5
minutes to read 100 fields, and should never
be expected to process and read more than
25 ZN-stained sputum specimens per day
when working full time. No more than 10 to
12 specimens should be processed at one
time. However, this situation seldom occurs
even in the peripheral laboratories of high
incidence countries. When TB sputum smear
microscopy is fully integrated in to the gen-
eral primary health care services, the real
challenge is to reach a workload high enough
to maintain testing proficiency.
4.5 The preservation of smears
for quality assurance testing
Before storage of the slides, immersion oil
must be washed from the smears. Cleaning
5. RECORDING AND REPORTING
OF SMEAR MICROSCOPY RESULTS
A positive sputum smear is like a document
upon which the diagnosis of pulmonary
tuberculosis is based. Results must be
recorded and copies of these records must
be kept in the laboratory. If possible, positive
readings should be confirmed by a second
reader. Examined slides should be kept in the
laboratory for the period of time prescribed
by the NTP for the purposes of supervision
and proficiency testing (see chapter 6).
5.1 The laboratory register
The IUATLD laboratory register (Figure 28)
has two essential and useful features: it
distinguishes between diagnostic sputum
smear examination and treatment follow-up
microscopy, and allots a single line to each
tuberculosis suspect examined, and not to
each sputum specimen examined. This per-
mits the rate of smear positive cases among
77
the immersion oil from the smear by blotting
with lens paper is discouraged because the
smear might be scraped off the slide and
the oil will never be thoroughly removed.
Cleaning the slides of immersion oil by dip-
ping them in xylene (xylol)* and drying them
before storing them in slide boxes until the
next supervision is recommended. Positive
and negative slides should be kept in sepa-
rate slide boxes. Filled slide boxes should be
stored closed and as far removed from heat
and humidity as possible until they are sam-
pled for re-reading. Slides should not be dried
and stored under direct UV light. The sam-
pling and re-reading of slides should be done
as soon as possible, because long term stor-
age under tropical climatic conditions will
cause fading of the ZN staining.
* Xylene, mixed ACS Reagent Sigma X 2377 or equiv-
alent. A safer, less toxic, less flammable xylene sub-
stitute is available.6
suspects to be evaluated, which in turn
allows laboratory supply requirements to be
planned based on the number of reported
smear positive cases.
– The laboratory code, serial and sequence
number on the slide must be the same as that
in the results section of the Request for
Sputum Examination Form. The upper por-
tion of the Form must be accurately com-
pleted. The results of the smear examination
should be recorded according to the IUATLD
grading scale (Table 1). The report form is
then dated and signed by the laboratory’s
responsible officer.
– All information from the laboratory form
should be entered in full in the appropriate
spaces of the Laboratory Register. All the
information requested in the laboratory reg-
ister must be entered, i.e., a blank space is
not a negative result but a missing record.
Positive results are entered in red ink.
Figure 28. Laboratory register for sputum smear microscopy

78
– Completed Request for Sputum Examin-
ation Forms must be sent back to the treat-
ment centre or the treating physician within
two working days. In case of a referral from
another health unit, the patient should
receive a copy of the completed form and the
original must be sent to the treatment centre.
Results should never be given to the patient
only. If the patient fails to take the results to
the treatment centre, he or she may not
receive treatment.
6. QUALITY ASSURANCE
OF SPUTUM SMEAR MICROSCOPY
6.1 Definitions
Quality assurance of sputum microscopy is
an indispensable part of an effective TB
Control Programme. It encompasses the
whole process of sputum collection, smear
preparation, smear staining, microscopy,
recording and reporting.
The purpose of quality assurance pro-
grammes is the improvement of the effi-
ciency and reliability of smear microscopy
services. A quality assurance programme has
three main components:
– Quality control: Quality control is a process
of effective and systematic internal monitor-
ing which aims to detect the frequency of
errors against established limits of accept-
able test performance. Although it is not usu-
ally feasible to determine error frequencies
accurately, it is nevertheless a mechanism by
which tuberculosis laboratories can at least
validate the competency of their diagnostic
services.
– Proficiency testing: Also known as External
Quality Assessment, this is a programme
designed to allow participant laboratories to
assess their capabilities by comparing their
results with those obtained with the same
specimens in other laboratories of the net-
work, e.g., Regional and National Reference
Laboratories.
– Quality improvement: Quality improve-
ment is a process by which the components
of smear microscopy diagnostic services are
79
– Upon completion of the examination of
each batch of submitted specimens, the date
of examination is recorded on the dispatch
list which is returned along with the trans-
port box to the originating health centre
as soon as possible. Transport boxes are
cleaned with a cloth wet with a TB germicide
(5% phenol or 0.1% sodium hypochorite) and
also returned to the health centre. Caution:
both of these solutions are extremely corro-
sive – protective gloves should be used.
analysed with the aim of looking for ways to
permanently remove obstacles to success.
Data collection, data analysis, identification
of problems and creative problem solving are
key components of this process. It involves
continued monitoring and identification of
defects, followed by remedial action to pre-
vent recurrence of problems.
6.2 Procedures
Internal quality control of staining is manda-
tory. New lots of staining solutions need to be
tested. This usually involves the staining of
known, unstained, positive and negative
smears. The inclusion of known, unstained
smears each time staining is carried out in
the laboratory is also strongly recommended.
The re-reading of positive smears by another
technologist is highly desirable; in practice
however, very few peripheral laboratories
employ two TB microscopists. Direct obser-
vation by an experienced observer of labo-
ratory technicians performing their routine
tasks at all stages is an essential aspect of
quality assurance.
There are four principal methods of pro-
ficiency testing of smear microscopy:
– Sending smears from the Reference
Laboratory to the peripheral laboratory for
checking reading and reporting.
– Monitoring the quality of sputum smear
microscopy in all its stages during super-
visory visits in the field.
– Sending smears from the peripheral
laboratory to the Reference Laboratory for
re-reading.
– Sampling smears of registered patients
found in the District Tuberculosis Register
All four methods have distinct advan-
tages and disadvantages; it is therefore
advisable to implement them according to
the needs and the circumstances of each
NTP.
7. DISINFECTION, STERILISATION AND DISPOSAL
OF CONTAMINATED MATERIALS
After the smears are examined, the lids of all
used sputum containers are removed. Used
containers, lids and applicators are placed in
a waste receptacle containing 5% phenol or
Figure 29
80
In the present context, quality improve-
ment consists of correcting deficiencies in
smear microscopy performance and reading
by taking appropriate remedial action. It is
the responsibility of the higher level labora-
tories of the network, i.e., Regional and
Central Reference Laboratories, to retrain
technologists who demonstrate less than
optimal performance. For a more detailed dis-
cussion of quality assurance programmes in
TB microbiology, see references 7 and 8.
0.5% sodium hypochlorite solution and are
fully submerged. Thereafter, these materials
can be disposed of by autoclaving. If an auto-
clave is not available, all materials should be
burned in an incinerator, an open pit or an
empty oil drum (Figure 29). NB: If large num-
bers of plastic containers are being burnt, the
fumes produced are toxic.
In the event that both burnable materials
and glass sputum bottles are used, the latter
should be discarded into a separate container
so they may be boiled and washed for re-use.
Other items such as slide holders, the dryer
and the work surface should be soaked in 5%
phenol or 0.5% sodium hypochlorite solution.
After the slides have been quality con-
trolled, the positive slides should be broken
and disposed of like other “sharps”. Negative
slides can either be disposed of, or if neces-
sary, washed clean and re-used for non-TB
work (e.g., malaria, haematology).
8. BIOSAFETY IN THE TB MICROSCOPY LABORATORY
8.1 General aspects
Laboratory workers are responsible for their
own safety and that of their co-workers.
Transmission of Mycobacterium tuberculo-
sis results essentially from micro-aerosols,
i.e., tubercle bacilli contained in droplet
nuclei, 1 to 5 microns in diameter, which are
sufficiently small to reach lung alveoli, yet
sufficient large to adhere to the lining of the
lung alveoli.
Infection control in the laboratory must
aim at reducing the production of aerosols.
Good ventilation is necessary for the protec-
tion of the laboratory staff from airborne
infectious droplet nuclei. An easy way to
ensure ventilation and directional airflow is
by judiciously locating windows and doors
so that airborne particles are blown away
from the laboratory worker (see Figure 4).
Where electricity is available, extractor fans
can be used to remove air from the labora-
tory.
Each time the technologists enter or leave
the laboratory they must wash their hands.
Staff should wear protective clothing such as
laboratory coats while exercising their duties,
returning them to the lockers before leaving
the laboratory. Access to the laboratory
should be restricted to laboratory staff only.
The wearing of disposable gloves for
smearing and staining is desirable; however,
because they are meant to be discarded after
each laboratory manipulation, their use rep-
resents a major expense for peripheral labs.
Disposable gloves are for single use only, but
in many laboratories they tend to be re-used
81
Slides with negative smears are boiled
for half an hour in soap or detergent solution
(dishwashing liquid), washed under running
water, wiped with cotton or cloth, air dried,
examined to confirm absence of scratches,
cleaned with an alcohol soaked cotton swab
and stored for re-use.
TB slides, whether negative or positive,
should never be re-used for TB work.
until torn. This improper use affords a sense
of false security and carelessness that often
impacts negatively on the biosafety condi-
tions of the laboratory – contaminated gloves
are used to handle or to operate laboratory
equipment that would otherwise never
become contaminated. As the use of gloves
is impractical in most settings where this
guide will be used, soaking hands in 70%
alcohol followed by washing with a detergent
solution, rinsing with water and drying with
paper, is highly recommended.
Wearing conventional surgical masks
does not significantly reduce the risk of infec-
tion by aerosol inhalation. The emphasis
again is to be placed on the reduction of
aerosols produced during laboratory proce-
dures by adopting and strictly enforcing
Good Laboratory Practices.8
Eating, drinking and smoking are not per-
mitted in the laboratory.
8.2 Specific aspects
Laboratory procedures differ considerably in
their potential to create aerosols:
– Specimen collection
Sputum from tuberculosis suspects is often
collected in the laboratory for sputum collec-
tion. This practice exposes laboratory work-
ers to a high risk of contagion by aerosols
and should not be allowed under any cir-
cumstance. As mentioned in Chapter 1, pre-
cautions to lower this risk can be taken by
instructing the tuberculosis suspects to cover
their mouths while coughing and by having
them produce the specimen outdoors, where
the aerosols will be diluted and sterilised by
the UV light of direct sunlight.
– Smear preparation
While opening sputum containers and the
smearing of slides may produce aerosols,
these manipulations entail less risk of trans-
mission than the unprotected coughing of a
smear positive patient. There is little evidence
that preparing sputum smears is correlated
with an increased risk of tuberculosis infec-
tion. However, absence of evidence is not evi-
dence of absence, and laboratory workers
must be careful and remain vigilant at all
times.
9. MATERIALS MANAGEMENT
To ensure the continuous flow of laboratory
supplies, programmes must budget rationally
for requirements. The only quantifiable basis
for planning is the number of patients
recorded and reported. The number and per-
centage of smear positive patients can be
determined from the Laboratory Register.
Assuming that the smear positivity rate is
15%, that each tuberculosis suspect requires
three sputum examinations and that each
case of smear positive tuberculosis has three
follow-up examinations, the number of
microscope slides and sputum containers
needed for each sputum smear positive case
detected is (1 / 0.15) x 3 + 3 = 23.
Laboratory material requirements are rel-
atively small and for this reason are ordered
every 6 months rather than every 3 months
and the reserve requirement is estimated at
one year’s supply (Figure 30).
The amounts of basic fuchsin, methylene
blue, ethanol and phenol are calculated from
the IUATLD recommended method for ZN
staining, assuming that 5 ml of each of the
82
Expensive and sophisticated equipment is no
substitute for good microbiology laboratory
practice. Moreover, commercial type bio-
safety cabinets (BSCs) require expert and
extensive yearly maintenance, an expense
that is seldom considered at the time the
equipment is purchased. Commercial type
BSCs which are not properly maintained give
a false sense of protection, and the same
applies to the home made variety. Twenty
years of field experience in low income coun-
tries have demonstrated the impracticality of
the design proposed in the first edition of this
Guide. Therefore, BSCs are not mandatory in
peripheral laboratories that perform smear
microscopy only.
solutions are needed for each slide. It is fur-
ther assumed that 2 drops or 1/10 ml of oil are
used for each slide.
The calculation is performed as follows:
– the total number of smear positive patients
(new patients and retreatment cases)
recorded on the previous two Quarterly
Reports on Case-finding is entered under the
column headed “No. of patients”;
– the requirements for the next half year (A)
are calculated by multiplying the number of
patients by a predetermined factor, based on
the assumption that 10 suspects of TB need
to be examined for each smear positive case;
– the reserve stock requirements (B) are
equal to twice the amount requirements for 6
months (A x 2);
– the amount of materials inventoried (C) in
the district store;
– the total order (D) is the sum of the amount
required for the next semester (A) plus the
amount required for “reserve” stock (B)
minus the inventoried amount (C) at the time
the order form is completed.
 CALCULATING THE REQUIREMENTS FOR PERIPHERAL LABORATORIES AND COST OF MATERIAL PER IDENTIFIED CASE.

Fraction Require-
Require- Annual Running Reserve
of cases ment per No Total Cost per case (FOB)
ment depre- require- require- In stock
among identified of cases ment order identified Per cent
Item Unit per slide ciation ment
suspects case of cost

A B C D=(1/Bx3+3) E F=DxE G=2xF

xAxC H I=F+G–H US$ FF

Slides 1 1 0.15 1.00 23 1000 23,000 46,000 19,550 49,450 0.667767 4.006600 30.5
Sputum containers 1 1 0.15 1.00 23 1000 23,000 46,000 19,550 49,450 0.909092 5.454554 41.6
Methylene blue 1g 0.01500 0.15 1.00 0.34500 1000 345 690 293 742 0.002216 0.013294 0.1
Basic fuchsin 1g 0.01500 0.15 1.00 0.34500 1000 345 690 293 742 0.002625 0.015748 0.1
Immersion oil 1 mL 0.10000 0.15 1.00 2.30000 1000 2,300 4,600 1,955 4,945 0.015859 0.095157 0.7
Sulfuric acid 1 mL 1.25000 0.15 1.00 28.75000 1000 28,750 57,500 24,438 61,813 0.213038 1.278225 9.7
Phenol 1g 0.25000 0.15 1.00 5.75000 1000 5,750 11,500 4,888 12,363 0.036216 0.217298 1.7
Xylene 1 mL 1.00000 0.15 1.00 23.00000 1000 23,000 46,000 19,550 49,450 0.315561 1.893364 14.4
Methanol 1 mL 0.50000 0.15 1.00 11.50000 1000 11,500 23,000 9,775 24,725 0.024731 0.148388 1.1
Filter paper sheets 1 box 0.00007 0.15 1.00 0.00153 1000 2 3 1 3 0.000005 0.000029 <0.1
Lens tissue 1 pack 0.00010 0.15 1.00 0.00230 1000 2 5 2 5 0.000007 0.000040 <0.1
Slide storage box 1 0.00140 0.15 0.10 0.00322 1000 3 6 3 7 0.000125 0.000748 <0.1
Wire loop holder 1 0.00040 0.15 0.10 0.00092 1000 1 2 1 2 0.000004 0.000025 <0.1
Ni-Cr wire 1 reel 0.00020 0.15 0.15 0.00069 1000 1 1 1 1 0.000003 0.000019 <0.1
Microscope 1 0.00020 0.15 0.10 0.00046 1000 0 1 0 1 0.000138 0.000829 <0.1

Total cost FOB 2.19 13.12

Insurance, freight, storage, and distribution (30%) 0.66 3.94
Total cost, including product, insurance, freight, storage, and distribution 2.84 17.06

Figure 30. Taken from Reference 7 (Table V.6)

References
1. Collins C H, Grange J M, Yates M D.
Organization and practice in tuberculosis
bacteriology. London: Butterworths, 1985.
2. Laboratory Biosafety Manual. 2nd ed.
Geneva: WHO, 1993: pp 60-61.
3. McDougall A C. An inexpensive slide
marker made from a dental bur and a
plastic pen. Lep Rev 1992; 63: 79-80.
4. Smithwick R C. Laboratory manual for
acid-fast microscopy. 2nd ed. US
Department of Health, Education, and
Welfare, Public Health Service. Atlanta,
GA: Centers for Disease Control,
Bacteriology Division, 1976.
5. The Microscope. A Practical Guide. WHO
Project: ICP TUB 001. New Delhi, India:
WHO Regional Office for South-East Asia,
1999.
6. McDougall A C. The use of xylene (xylol)
in medical laboratories. Lep Rev 1989; 60:
67.
7. Woods G L, Ridderhof J C. Quality assur-
ance in the mycobacteriology labora-
tory. In: Clinics in Laboratory Medicine.
Vol 16, Number 3. Philadelphia, PA: W B
Saunders, 1996.
8. Kumari S, Bathia R, Heuck C C. Quality
assurance in bacteriology and immunol-
ogy. WHO Regional Publication, South-
East Asia Series No 28. New Delhi, India:
WHO Regional Office for South-East Asia,
1998.
Suggested reading
1. Bacteriology of tuberculosis. The speci-
men. Microscopy examination. Technical
note no. 26. Washington, DC: Pan
American Health Organization, 1984.
2. Minamikawa M. Laboratory Manual for
the National Tuberculosis Programme of
Nepal. National Tuberculosis Centre.
JICA/HMG National TB Control Project
(II). March 1998.
84
3. De Kantor I N, Kim S J, Frieden T, Laszlo
A, Luelmo F, Norval P Y, Rieder H L,
Valenzuela P, Weyer K. Laboratory ser-
vices in tuberculosis control. WHO Global
Tuberculosis Programme. WHO/TB/
98.258. Geneva: WHO, 1998.
4. Manual of norms and technical proce-
dures for tuberculosis bacteriology. Part 1
Smear microscopy. Technical note 26.
Washington, DC: Pan American Health
Organization, 1984.
5. Manual for Laboratory Technicians.
Revised National Tuberculosis Control
Programme (RNTCP). Nirman Bhavan,
New Delhi, India: Central TB Division,
Directorate General of Health Services,
Ministry of Health and Family Welfare,
1997.
6. Module for Laboratory Technicians.
Nirman Bhavan, New Delhi, India: Central
TB Division, Directorate General of
Health Services, Ministry of Health and
Family Welfare, 1997.
7. Rieder H L, Chonde T M, Myking H,
Urbanczik R, Laszlo A, Kim S J, Van Deun
A, Trébucq A. The Public Health Service
National Tuberculosis Reference Labor-
atory and the National Laboratory
Network. Minimum requirements, role
and operation in a low income country.
Paris: IUATLD, 1998.
8. Fujiki A. TB microscopy. Tokyo, Japan:
The Research Institute of Tuberculosis,
Japan Anti-Tuberculosis Association,
Japan International Cooperation Agency,
Hachioji International Training Centre,
1998.
9. Tuberculosis control: a manual of meth-
ods and procedures for integrated pro-
grams. Scientific Publication no. 498.
Washington, DC: Pan American Health
Organization, 1986.
10. Enarson D A, Rieder H L, Arnadottir T,
Trébucq A. Management of tuberculosis:
a guide for low income countries. 5th ed.
Paris: IUATLD, 2000.
 ANNEX 1

PREVENTION OF FALSE-POSITIVE SPUTUM RESULTS

• Use new slides
• Use a new applicator stick for each sample
• Use filtered carbolfuchsin
• Keep slides separate from one another while staining
• Do not use staining jars
• Do not allow carbolfuchsin to dry on the slide
• Do not allow oil immersion applicator to touch the smear
• Do not allow oil immersion lens to touch the smear
• Label sputum containers, slides and laboratory forms completely and accurately
• Cross check the number on the Request for Sputum Examination Form and sputum container
before recording
• Record and report results accurately

CONSEQUENCES OF FALSE POSITIVITY

• Unnecessary treatment - wastage of drugs

• Loss of confidence in the NTP

PREVENTION OF FALSE-NEGATIVE SPUTUM RESULTS

• Make sure sample contains sputum, not just saliva
• Make sure there is at least 3 ml of sputum
• Select thick, mucopurulent particles for smearing
• Smears should not be too thick nor too thin
• Stain smears for 5 minutes
• Decolourise smears for 3 minutes
• Counterstain for 1 minute
• Read all 100 fields before declaring the slide to be negative
• Known positive control smears should show well stained AFB
• Label sputum containers, slides and laboratory forms carefully
• Cross check the number on the Request for Sputum Examination Form and on the sputum
container before recording
• Record and report results accurately

CONSEQUENCES OF FALSE-NEGATIVE RESULTS

• Patient remains untreated, resulting in suffering, spread of TB and death
• Intensive phase treatment may not be extended, leading to inadequate treatment
• Loss of confidence in the NTP

85
 ANNEX 2

CARE OF THE MICROSCOPE

The microscope is the centrepiece of the TB diagnostic services of the NTP. Proper
handling and maintenance of the microscope by laboratory staff is essential to pro-
long its useful life. The following points should be observed:

• When not in use the microscope should be stored in a dry, dust and vibration free
environment

• Avoid exposing the microscope to direct sunlight and moisture and humidity

• Use silica gel in the microscope storage box; restore by heating when silica gel
becomes pink

• Clean the microscope with lens paper before and after use

• Wipe the surface of immersion lens with a piece of clean cotton before and after
use. Do not use alcohol for cleaning lenses

• Oil immersion lens should never touch the smear

• Use fine focusing knob only while using the oil immersion lens

86
 ANNEX 3

TROUBLE-SHOOTING GUIDE FOR MICROSCOPY

PROBLEM POSSIBLE CAUSES SOLUTION

Condenser too low Raise condenser

Field is dim
Diaphragm closed Open diaphragm

Eye piece dirty Clean eyepiece

Dark shadows in the field Eyepiece or objective A new eyepiece may be
that move with eye piece contaminated with fungus needed
when it is rotated Surface of eyepiece A new eyepiece may be
scratched needed

Smear portion of slide Turn slide over

upside down
Air bubble in the oil Move immersion oil
The image is not clear objective from side to side
Poor quality oil Change oil
Lens dirty Clean lens

Oil on the lens Clean lens

Low power image
Dust on upper surface of lens Clean lens
not clear
Broken lens A new lens is needed

87
 COMPOGRAVURE
IMPRESSION, BROCHAGE
IMPRIMERIE CHIRAT
42540 ST-JUST-LA-PENDUE
AOÛT 2000
DÉPÔT LÉGAL 2000 N° 9481

IMPRIMÉ EN FRANCE

89
VII. APPENDIX 2

FORMS

91
 TUBERCULOSIS PROGRAMME FORM 1

REQUEST FOR SPUTUM EXAMINATION

Treatment Unit Date

Patient’s Name

Age Sex (check one): M [ ] F [ ]

Address (precise)

Reason for examination (check one): diagnosis [ ] follow-up examination [ ]

Signature of person requesting examination

RESULTS (to be completed in laboratory

Laboratory Serial No.

Result (check one)

Date Specimen Appearance* neg 1-9 + ++ +++

* visual appearance of sputum (blood-stained, muco-purulent, saliva)

Date Examined by (Signature)

The completed form (with results) should be sent promptly to the treatment unit
TUBERCULOSIS PROGRAMME

TUBERCULOSIS LABORATORY REGISTER Year

Reason Results
Lab Name Address –
Sex for examination* of specimen
Serial Date Name Age of treatment patient Signature Remarks
M/F
No. unit for diagnosis
diagnosis follow up 1 2 3

* Check the appropriate category from the Request for Sputum Examination
FORM 2
TUBERCULOSIS PROGRAMME TUBERCULOSIS TREATMENT CARD District TB No.

Name: Disease site (check one):

Pulmonary [ ] Extra-pulmonary [ ] Site (specify)

Address:
Category of patient (check one):

New [ ] Treatment after failure [ ]

Treatment Centre Age Sex (check one): M [ ] F [ ] Relapse [ ] Treatment after default [ ]
Date Transfer in [ ] Other [ ] (specify)

I. INITIAL INTENSIVE PHASE Date

Prescribed regimen and number of tablets: Month Date / Lab no. Smear result Weight (kg)
Date of next
appointment
STH RHZE SRHZE 0

{TH} S {RH} Z E {RH} S Z E 5

7
{TH} = thioacetazone/isoniazid; S = streptomycin; {RH} = rifampicin/isoniazid;
E = ethambutol; Z = pyrazinamide
> 7

Day
Month 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

Enter X on day when medications were swallowed under direct observation please turn over
FORM 3
II. CONTINUATION PHASE
regimen and number of tablets: new cases (daily) retreatment
(2 S {TH}/10 {TH} = 10 months; {TH} 3 times a week {RH} E H
2 {RH} ZE/6 {TH} = 6 months) (5 months)
Day 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
Month
Enter X on day of supervised administration or when medications are collected. Draw a horizontal line through the days to indicate number
of days’ supply given.
Remarks:
FORM 4
TUBERCULOSIS PROGRAMME TUBERCULOSIS

Category of patient**
Disease
Date Unit Sex Treatment Treatment Treatment Treatment
Name in full Age Address in full Regimen* site Transfer
registered TB No. M/F Unit start date New Relapse after after Other
P/EP in
failure default

* New case: HRZE = 8-month or STH = 12-month ** New: never previously treated for as much as 1 month Treatment after failure: positive 5 or more months after starting treatment, commenced on retreatment
* Retreatment: SHRZE ** Relapse: previously treated, declared cured, Treatment after default: returned smear positive after leaving treatment for 2 months or more, commenced on retreatment
* P = pulmonary returns smear positive Transfer in: registered and started treatment in another unit
* EP = extra-pulmonary
REGISTER Year FORM 4

Results of smear examination according to duration of treatment Result of treatment*** and date of result (check one)
Before treatment 2 months 5 months 7 months 11 months Smear result at completion:
Remarks
Lab Lab Lab Lab Lab Died Defaulted Transferred
Result Result Result Result Result Negative Not done Positive
no./date no./date no./date no./date no./date

*** Smear negative (cured): negative smear at last month of treatment and on one previous occasion Died: died from any cause while on treatment
*** Smear not done (completed): completed treatment, but sputum examination insufficient (not done) for classification as smear negative Defaulted: failed to collect medications for more than 2 months after date last seen
*** Smear positive (failure): positive smear at 5 months or later during treatment, confirmed by a second positive smear Transferred: sent to another unit for continuation of treatment and result of treatment is unknown
 QUARTERLY REPORT ON TUBERCULOSIS CASE-FINDING

Name of Unit Unit Tuberculosis Co-ordinator

Patients registered in quarter of 20 Signature Date

ALL CASES REGISTERED IN THE QUARTER

TUBERCULOSIS PROGRAMME

SMEAR-POSITIVE SMEAR-NEGATIVE
EXTRA-
TOTAL
Treatment Treatment PULMONARY
New cases Relapses < 15 yrs 15 + yrs
after failure after default

NEW SMEAR-POSITIVE CASES ONLY

Age group (years)

TOTAL
0-14 15-24 25-34 35-44 45-54 55-64 65 +

M F M F M F M F M F M F M F Male Female Total

Definitions to use when completing the form:

Quarters 1st quarter – 1 January to 31 March 3rd quarter – 1 July to 30 September
2nd quarter – 1 April to 30 June 4th quarter – 1 October to 31 December

Optional ENROLMENT DURING THE QUARTER ON TREATMENT REGIMEN

2RHZE/6TH 2STH/10TH 12TH 2SRHZE/1RHZE/5R3H3E3

FORM 5
TUBERCULOSIS PROGRAMME

QUARTERLY REPORT ON THE RESULTS OF TUBERCULOSIS TREATMENT

PULMONARY SMEAR POSITIVE CASES REGISTERED IN THE QUARTER ENDING 15 MONTHS PRIOR TO REPORTING DATE

Name of Unit Unit Tuberculosis Co-ordinator

Patients registered in quarter of 20 Signature Date

Smear Smear not done

Smear positive
Type of case Regimen negative (treatment Died Defaulted Transferred Total
(failure)
(cured) completed)

New

n° enrolled* [ ] 2{RH}ZE/6{TH}

2S{RH}/10{TH}

Retreatment 2S{RH}ZE/1{RH}ZE/5{RH}3E3

n° enrolled* [ ] Relapse

n° enrolled* [ ] Treatment after failure

n° enrolled* [ ] Treatment after default

* from Quarterly Report on Tuberculosis Case-finding for that quarter

FORM 6
TUBERCULOSIS PROGRAMME

ORDER FORM FOR TREATMENT SUPPLIES AT UNIT LEVEL

Enter the number of cases enrolled in the previous three months (from the Quarterly Report on Case-Finding)

2{RH}ZE/6TH 2(S){TH}/10{TH} 2S{RH}ZE/1{RH}ZE/5{RH}3E3

Total
Cases Factor Total Cases Factor Total Cases Factor Total
Item A B C A+B+C= D
{RH} 150/75 ~ 210 = ~ 0 = 0 ~ 540 =
Z 400 ~ 210 = ~ 0 = 0 ~ 320 =
S 1 g ~ 0 = 0 (~ 60) = ~ 60 =
{TH} 150/300 ~ 180 = ~ 360 = ~ 0 = 0
H 100 ~ 0 = 0 ~ 0 = 0 ~ 100 =
E 400 ~ 150 = ~ 0 = 0 ~ 450 =

Running requirement Reserve requirement Currently in stock Total order

Item E (= D from above) F (= E) G E+F– G
{RH} 150/75
Z 400
S 1 g
{TH} 150/300
H 100
E 400
{TH} 50/100
Date: {EH} 400/150
Syringes/Needles
Name and signature:
Water for injection (5 ml)

NB: For each patient for whom {TH} is replaced by {EH} due to side effects, 360 tablets must be ordered. Factor is the number of tablets taken by each patient
FORM 7
TUBERCULOSIS PROGRAMME

ORDER FORM FOR LABORATORY SUPPLIES AT UNIT LEVEL

Enter the number of sputum smear positive patients enrolled in the previous half year (from the Quarterly Report on Case-finding)

6-month running 1 year reserve Currently Total Order

No.
Material Factor* requirement requirement in stock
of patients
A B = A ~ 2 C D= A +B– C

Basic fuchsin ~ 0.5 g =

Methylene blue ~ 0.5 g =

Immersion oil ~ 3.3 ml =

Sulphuric acid ~ 41 ml =

Phenol ~ 8.3 g =

Methanol ~ 17 ml =

Slides ~ 33 =

Sputum containers ~ 33 =

* The calculations are based on the assumption that 5 ml saturated fuchsin solution, 5 ml 25% sulphuric acid, and 5 ml methylene blue solution are needed for
one slide. These quantities serve as a basis for calculating the needs for one diagnosed case, assuming that 10% of examined tuberculosis suspects are found
to be smear positive.
FORM 8
TUBERCULOSIS PROGRAMME

ORDER FORM FOR LABORATORY SUPPLIES AT UNIT LEVEL

(example for the peripheral level)

Enter the number of sputum smear-positive patients enrolled in the previous half year (from the previous two Quarterly Reports on Case-finding)

6-month running 1 year reserve Currently Total Order

No.
Material Factor* requirement requirement in stock
of patients
A B = A ~ 2 C D= A +B– C

Staining solution ~ 165 ml =

Decolouration solution ~ 165 ml =

Counterstaining solution ~ 165 ml =

Immersion oil ~ 3.3 ml =

Slides ~ 33 =

Sputum containers ~ 33 =

* The calculations are based on the assumption that 5 ml each of staining, decolouration, and counterstaining solution are needed for one slide.
These quantities serve as a basis for calculating the needs for one diagnosed case, assuming that 10% of examined tuberculosis suspects are positive.
FORM 8b