Tuberculosis Course for

English-speaking students

2. THE METHODS OF
TUBERCULOSIS
DIAGNOSTICS
2.1. SETTING QUESTIONS
2.2. SYMPTOMS OF TUBERCULOSIS
2.3. TUBERCULIN TESTING
 Impotance of TB diagnosis

• Both the 1994 CDC guidelines on

tuberculosis control for health care
facilities and the 1997 proposed OSHA
rule on tuberculosis control emphasize the
fact that prompt identification, isolation,
and treatment of people with infectious
tuberculosis is critical to an effective
tuberculosis control program.
 2.1. SETTING QUESTIONS
• The majority of cases of
tuberculosis generally
discovered when the patient
visits his/her general
practitioner.
• The patient feeling some type
of discomfort does not
immediately refer to the doctor.
Patient complains of a
somewhat constant sub-febrile
temperature up to 37,5 °C.
• Dry cough with occasional
presence of sputum appears in
case of advanced TB.
Heavy smokers do not pay
adequate attention to their
coughing and relates their
coughing to smoking rather
than TB.
Medical professionals whatever their specialty
must be aware of the TB prevalence.
• Here is a set of questions that are to be addressed in the case a
doctor is faced with a tuberculous patient:
•
1. Whether the given patient was prior infected by TB?
• 2. Whether his/her relatives were infected by TB?
• 3. Whether the patient had contact with TB patients or animals?
• 4. Whether the patient is registered in a tuberculosis dispensary.
5. When the patient had the X-ray examination?
• 6. Whether the patient was invited after the X-ray examination for
additional research?
• 7. Whether he was in a prison or lived with someone who was in a
prison.
• 8. Whether the patient is homeless, a refugee, migrant or being in
unfavorable social conditions?
 AIDS
• In the recent years, AIDS has become one
of the most powerful increasing factors, of
TB infection. Patients simultaneously
infected with HIV and MBT, have a 50%
increased chance of developing TB.
 2.2. SYMPTOMS OF TUBERCULOSIS

• If a patient has any of the following, consider him a

'Tuberculosis Suspect':
• 1. Cough for over 3 weeks.
• 2. Haemoptysis.
• 3. Pain in the chest for over 3 weeks.
• 4. Fever for over 3 weeks.
_______________________________
All these can be due to some other diseases but sputum
must be tested if any of the symptoms are present.
 Some guidelines for the proper diagnosis
of pulmonary tuberculosis.
• General Symptoms: • Respiratory Symptoms:
• ++ Loss of weight. • +++ Cough.
• ++ Fever and sweating. • +++ Sputum.
• + Loss of appetite. • ++ Blood-spitting.
• + Breathlessness. • + Tiredness.
• + Chest wall pain.
• + Localized wheeze in
lungs.
+ Frequent colds.
 Anamnesis
• Upon gathering anamnesis, it is also
necessary to find out, when the
tuberculin test reacted positive for the
first time.
• After a well-collected anamnesis, it is
easier for the doctor to confirm his
assumption of presence of tubercular
process.
 Physical signs. Often these do not help much. But do
examine the patient carefully. The pathogenous signs could
be revealed.

• 1. General condition. Sometimes the patient appears to

be in good health despite advanced disease.
• 2. There may be different type of fevers.
3. Pulse is usually raised in proportion to fever.
• 4. Finger clubbing. This symptom may be encountered,
especially in patients with extensive disease. One should
keep in mind that clubbing is common in lung cancer.
• 5. Examination of chest. Often there are no abnormal
signs. The commonest is fine crepitations (crackles) in
the upper part of one or both lungs.
Erythema nodosum
• This picture shows reddish-
purple, hard (indurated),
painful nodules (Erythema
nodosum) that occur most
commonly on the shins. These
lesions may be anywhere on
the body and may be
associated with tuberculosis
(TB), sarcoidosis,
coccidioidomycosis, systemic
lupus erythematosis (SLE),
fungal infections, or in
response to medications.
 5. Examination of chest.
• Often there are no abnormal signs.
The commonest is fine crepitations (crackles) in the
upper part of one or both lungs. These are heard
particularly on taking a deep breath after coughing.
Later there may be dullness to percussion or even
bronchial breathing in the upper part of both lungs.
Occasionally there is a localized wheeze due to local
tuberculous bronchitis or pressure by a lymph node on a
bronchus. In chronic lung tuberculosis, with much
fibrosis (scarring), the scarring may pull the trachea or
the heart over to one side. At any stage the physical
signs of pleural effusion may be present.
 2.3. TUBERCULIN TESTING

• Tuberculin test is a valuable

supplementary method for clinical
diagnosis of tuberculosis.
• It specifies the presence of specific
sensitivity caused by virulent MBT or
BCG vaccine.
Purpose of the tuberculin test application

• Tuberculin test as the specific test is

applied:

- in mass examination of the population for

tuberculosis (mass tuberculin testing),

- in clinical practice for diagnosing the stages of

tuberculosis (individual tuberculin testing).
 Tuberculin applies for tuberculin
tests.
• Tuberculin was isolated from products of
mycobacterium tuberculosis by R. Koch in 1890.
Tuberculin is water-glycerin extraction from
broth culture of mycobacterium tuberculosis.
• Tuberculin has incomplete antigenic
properties i.e. it is not sensitize healthy
organism and form anti-tubercular immunity.

Its effective agent is tuberculoproteid.

Tuberculin delayed local allergic reaction

• When tuberculin is injected into the skin, a

delayed local allergic reaction develops within
24-48 hours.
• Pathomorphology of the infiltration is
characterized by edema of all layers of the skin
with mononuclear and histiocyte reaction.

The reaction characterizes the degree of

allergy and NOT the degree of immunity.
In Russia 2 kinds of PPD-L tuberculin are
produced

1. In the form of solution, ready to use liquid form

of tubercular allergen purified in standard solution
for intradermal application (purified tuberculin in standard
dilution)

2. Dry tubercular purified allergen (dry purified

tuberculin)
*discontinued
• Tuberculin – liquid allergen represents a solution of
tuberculin in 0,85 % solution of NaCl with phosphate
buffer, with the twin – 80 as the stabilizer and phenol as
a conservator.

• The preparation is produced in ampoules as a solution

containing 2 TU (tuberculin units) PPD-L in 0,1 ml,
colorless transparent liquid.
•
 Purified tuberculin in standard solution

• Purified tuberculin in standard solution is used for

performance of uniform intradermal tuberculin test (test
Mantoux).

• The industrial production of PPD-L solution allows usage

of standard preparations for mass tuberculin testing.

• It is already diluted and therefore avoids errors of

tuberculin dilution.
 National standards of the
appropriate types of tuberculin

• The specific activity of tuberculin preparations is

established and supervised according to the
national standards of the appropriate types of
tuberculin.

• The WHO and the International Union Against

Tuberculosis and Lung Disease recommend
using PPD-RT23. This is purified tuberculin.
 Mantoux test

• The Mantoux test is

performed as follows: the
inner third surface of the
forearm’s skin is disinfected
with 70% ethanol and is dried
by sterile cotton.
• A thin needle penetrates the
skin intradermally parallel to
skin surface with its aperture
facing upward. After
introducing the needle into the
skin, immediately inject 0,1 ml
of tuberculin solution - one
dose, containing 2 TU PPD-L
Рис. 2-1. A correctly performed Mantoux test.
Figure 2-2. Positive Mantoux test.
• The estimation of
Mantoux reaction is
carried out after 72
hours.
It is necessary to
distinguish an infiltrate
from hyperemia.
For this purpose, in
the beginning palpate
the depth of skin fold of
forearm in healthy skin,
then – in the place of
tuberculin introduction.
Figure 2-3- Measurement infiltration size after
tuberculin introduction

• Result of the Mantoux

test is read after 72
hours.

• Measure the infiltrate in

mm with transparent ruler
and register the diameter
of infiltrate
 The response to a tuberculin can be:
• 1) negative – absence of the
infiltration and hyperemia or
otherwise pricked reaction (0-
1 mm);
• 2) doubtful –infiltrate in the size
of 2-4 mm or only hyperemia of
any size;
• 3) positive –infiltrate in the size of
5 mm and more.
• Positive reactions on
tuberculin are differentiated
according to the size of
infiltrate in diameter:
• – weakly positive –5-9 mm;
• – medium intensity – 10-14
mm;
• – marked (prominent) – 15-
16 mm;
• – hyperergic for adult 21 mm
and more, and also vesicular-
necrotic reactions
 TABLE 2-2. CDC Recommendations for Interpreting
Reactions to the Tuberculin Skin Test

• I. Classify induration of ≥5 mm as positive

for•HIV-positive persons•Recent contacts
of individuals with tuberculosis•Persons
with fibrotic changes on chest radiograph
consistent with prior tuberculosis•Organ
transplant recipients and others with
conditions or treatments that suppress
their immune systems
 TABLE 2-2. CDC Recommendations for Interpreting
Reactions to the Tuberculin Skin Test

• II. Classify induration of ≥10 mm as positive for•Recent

immigrants (within 5 years) from high-prevalence
countries•Injection drug users•Residents and employees
of high-risk congregate settings: prisons and jails,
nursing homes and other long-term care facilities for
elderly, individuals, hospitals and other health care
facilities; residential facilities for AIDS patients, homeless
shelters*•Mycobacteriology laboratory
personnel•Persons with diabetes and other clinical
conditions (other than those identified in category I) that
place them at high risk•Children under 4 years of age or
children and adolescents exposed to adults in high-risk
categories
 TABLE 2-2. CDC Recommendations for Interpreting
Reactions to the Tuberculin Skin Test

• III. Classify induration of ≥15 mm as

positive for•Persons with no known risk
factors for tuberculosis*For employees
who are otherwise at low risk and who are
tested upon hiring, an induration of ≥15 is
considered positive. SOURCE: Adapted
from ATS/CDC (2000a).
2.4. LABORATORY METHODS OF
MYCOBACTERIUM TUBERCULOSIS
DETECTION
 The histologic hallmark of
tuberculosis
• The histologic hallmark is caseating granulomata with
Langhan's type giant cells. The granuloma is a rounded
collection of macrophages and lymphocytes containing
multinucleated giant cells, the nuclei of which are
arranged at the periphery in a horse-shoe shape.
• Acid-fast bacilli can sometimes be demonstrated by the
Zeihl-Neelson stain on tissue sections. Cultures are
much more sensitive.
• Secondary TB has a much high incidence of large areas
of caseating necrosis. Otherwise, primary and secondary
TB are histologically similar.
 The laboratory MBT identification consists of
the following methods:

1) sputum collection and processing;

2) microscopic identification of MBT in secretions or
tissues;
3) culture techniques;
4) drug susceptibility testing;
5) serological testing;
6) performance of new molecular biological methods of
MBT identification, including polymerase chain reaction
(PCR) and restriction fragment length polymorphism
(RFLP).
Collection of sputum specimens
• Collection of sputum specimens with
MBT performs in specially equipped
medical institutions or in ambulatory
services. The collected specimens
immediately send for laboratory
examination.
There are two acceptable types of container.

• One available from UNICEF (UN Children’s fund), is

plastic with a black bottom, a translucent lid, and is
readily destroyed by burning; the patient's identification
must be made on the container (not on the lid).

• The other is a heavy glass, screw-capped jar that may

be reused after disinfection by boiling (10 min.) and
thorough cleaning.
 The risk of infection

• The risk of infection is very high when

the patient coughs, therefore
specimens must be collected as far
away as possible from other people in
specially prepared room.
 Additional procedures for MBT
collection
Laryngeal swabs.
Bronchial flush waters.
Gastric suction (Gastric flush waters).
Examination of cerebrospinal fluid.
Bronchoscopy.
Pleural fluid.
Pleural biopsy.
Lung biopsy.
 Sputum microscopy

• Main techniques that are still employed today –

the carbol-fuchsin methods of Ziehl–Neelsen.
The fundamental principles of this method relate
to the ability of the cell wall to absorb carbol-
fuchsin dye. The mycobacterial cell walls
absorbing the red carbol-fuchsin are becoming
so impregnated, that they resist decolorization
even with a potent hydrochloric acid-ethanol
solution (acid-alcohol). So, when the slide is
counterstained with methylene blue, the MBT
appear as red rods on the blue background.
• Ziehl-Neelsen acid-fast staining procedure:
• Heat fix cells on glass microscope slide.
• Flood the slide with carbol fuchsin stain.
• Heat the slide gently until it steams (5 min).
• Pour off the carbol fuchsin.
• Wash slide thoroughly with water.
• Decolourize with acid-alcohol (5 min).
• Wash slide thoroughly with water.
• Flood slide with methylene blue counterstain for 1 min.
• Wash with water.
• Blot excess water and dry in hand over bunsen flame.
 This sputum smear micrograph shows Mycobacterium
tuberculosis stained using the Ziehl-Neelsen technique; Magnified

1000X.
The acid fast stain of Mycobacterium tuberculosis (MTB)
in sputum.
Fluorescence microscopy
• Mycobacteria can also be
stained with auramine
and viewed with
fluorescence microscopy,
in which acid fast bacilli
now appear as glowing
yellow rods. This method
is easier to use to screen
for mycobacteria and is
the method routinely used
in sputum specimens
sent to the laboratory.
 ACID FAST STAINS - ZIEHL-NEELSEN
All at 400X original magnification

Single bacillus in necrotic area

Several bacilli in necrotic area
 ACID FAST STAINS - ZIEHL-NEELSEN
All at 400X original magnification

Numerous bacilli in necrotic area Single bacillus in giant cell

 ACID FAST STAINS - ZIEHL-NEELSEN
All at 400X original magnification

Single bacillus in giant cell Numerous bacilli in giant cell

Drug susceptibility test on Mycobacterium
tuberculosis bacteria
• These thin agar culture
plates reveal the results of a
drug susceptibility test on
Mycobacterium tuberculosis
bacteria. The agar medium is
imbibed with various drugs to
determine to which
medications the bacteria are
susceptible. Though in this
case it is not known what
drugs are being tested, it is
apparent that each had a
different effect on the
organism’s survival.
 BАСTEC MGIT-960

The BACTEC Mycobacteria Growth Indicator Tube (MGIT) 960 System is the
mycobacteria testing instruments; it is an automated system that exploits the
fluorescence of an oxygen sensor to detect growth of mycobacteria in culture. It is
specially designed to accommodate Mycobacteria Growth Indicator Tube (MGIT)
and incubate them at 37°C.