DIAGNOSIS OF

TUBERCULOSIS
Emerging Trends

Dr.T.V.Rao, MD.

Dr.T.V.Rao
 Robert Koch Discovers
Mycobacterium

Dr.T.V.Rao
 A Global Emergency

The Tuberculosis in the

beginning of the 21st Century
declared as Global Emergency
(WHO)

Dr.T.V.Rao
 Why Tuberculosis is a Important
Disease.
 Tuberculosis continues to be a
Important communicable disease.
 A leading cause of morbidity and

mortality in Developing world.

 Most Important communicable

disease in Bangladesh, China,

Indonesia, Africa, and Pakistan.

But it is Curable Disease

Dr.T.V.Rao
 Tuberculosis is a Global Problem

Dr.T.V.Rao
 Tuberculosis - Important
communicable disease spread by
Respiratory route

Dr.T.V.Rao
 Why Everybody Concerned.

 Tuberculosis kills young adults.

 Premature death of the infected a
prominent future.
 Today many are co infected with HIV.
 The open cases of Tuberculosis infects a
few around his/her environment.
 A social burden to the family, society and
Nations.

Dr.T.V.Rao
 Tuberculosis in the era of
HIV / AIDS.

HIV / AIDS epidemic led to large

increase of Smear negative
pulmonary tuberculosis which in turn
has led to poor treatment out comes,
and early mortality

Frequently involves Lower lobes of

Lungs.
Dr.T.V.Rao
 Why we fail to Diagnose
Tuberculosis.

 Lack of health infrastructure.

 Control is plagued with lack of
Accurate,
Robust,
and Rapid
Diagnostic methods,
Technologies.

Dr.T.V.Rao
 Why we failed ( Cont )

 Diagnostic services are poor, and so

we failed at Individual and
community levels.
 Patients are diagnosed late.

 Many patients are never diagnosed

before death.
Early deaths are burden to
Social Infrastructure and
Economic loss.
Dr.T.V.Rao
 Importance of Clinical services

Early diagnosis rests with clinicians,

whose contribution is immense in
prompt treatment.

A clinicians knowledge, proper

documentation are immense help in
Developing countries.

Dr.T.V.Rao
 When to suspect Tuberculosis
 Cough longer than 3 weeks.
 Fever for 1 month, or both.

 Blood stained sputum.

 Night sweats, weight loss

 Age between 14 and 70 years

( Correlates National Tuberculosis

Programme ).

Dr.T.V.Rao
 DIAGNOSTIC METHODS

Dr.T.V.Rao
 Diagnosis.

 Tuberculosis is a diversified disease.

 Any organs can be involved.
 Any age group, gender no bar for
Tuberculosis.
 Involvement of Lungs contribute to
majority of tuberculosis.
 And involvement of Lungs is designated as
Pulmonary tuberculosis.

Dr.T.V.Rao
 Diagnosis of Pulmonary
Tuberculosis
 Majority of Adults suffer with pulmonary
tuberculosis.
 Microbiological examination of Sputum
continues to be a Gold standard in proving
the Diagnosis.
 Sputum examination in Children is not
sensitive in Diagnosis.
 Radiological examination of Lungs, most
commonly prescribed investigation.

Dr.T.V.Rao
 X - ray examination of chest most
easily available Investigation.

Dr.T.V.Rao
 Microbiological
Investigations are essential
for definitive Diagnosis of
Tuberculosis.

Dr.T.V.Rao
 Importance of Optimal Specimens

Pulmonary Tuberculosis is the

commonest presentation of
Tuberculosis
Sputum is the Most important
specimen for identification and
isolation of Acid fast bacilli.
The developing countries suffers the
most important step in getting an
ideal sample.
Dr.T.V.Rao
 Sputum specimens
Train the staff to obtain the appropriate
specimen
A few minutes of education to patients on
importance of ideal sample make a great
difference and improves the Diagnosis.

Dr.T.V.Rao
 Observe to identify Sputum from
Saliva.

SPUTUM SALIVA

Specimens appear mucoid Appears clear, watery,

even, blood stained. and frothy.
Contains many
Contains many squamous epithelial
Polymorphoneutrophils. cells
Absence of
Polymorphoneutrophils.

Dr.T.V.Rao
Role of Microscopy in Tuberculosis.

 Microscopy for Diagnosis of Tuberculosis is

initiated in 1880
 The conceptions have not changed since
then.
 Best efforts should be put to obtain
sputum,
 Processing of saliva loses all valuable clues
to diagnose.

Dr.T.V.Rao
 Microscopy and Tuberculosis

Microscopy with
Ziehl – Neelsen’s
staining

A century old
procedure

Dr.T.V.Rao
 Why Microscopy
 Only we need Microscope, and few
stains.
 Most rapid, economical,

 Can detect bacterial load.

 A Diagnostic, and Prognostic tool.

 A little of sputum 0.2 µl is adequate.

 A prompt diagnosis after searching

as few as 100 fields.

Dr.T.V.Rao
 Limitation of Microscopy for
Tuberculosis.
 Repeated sample examinations. load on
technical staff.
 Training and dedication of Microscopist.
 The load of bacilli must be more than
10,000 / 1 ml of sputum.
 Low in sensitivity < 50 %
 Repeated requests for samples
 High drop out by patients, for repeated
samples.
 Not dependable in pediatric age group.
Dr.T.V.Rao
 Smear showing Acid Fast Bacilli.

Dr.T.V.Rao
 What is Smear Positivity
WHO

All patients who have submitted

two
Specimens and found to be
positive
for identification of AFB
Dr.T.V.Rao
 Processing Direct smear negative
specimens
 Sputum Microscopy can be improved with
Sputum liquefaction, concentration and
gravity sedimentation.
 Popular solvents
Sodium hypochlorite.
Sodium hydroxide.
Ammonium sulphate

N-acetyl-L-cysteine –sodium
hydroxide.
Dr.T.V.Rao
 Benefits of Liquefaction and
Concentration
 Major studies showed processing of
sputum with chemicals and centrifugation
improved sensitivity up to 18 %.
 Incremental yield ( positive with bleach
minus positives with Ziehl – Neelsen stain)
up to 9 %.
 Treating specimens with Sodium
hypochlorite is Mycobactericidal and also
kills HIV and improves the safety and
acceptability by technical staff.
Dr.T.V.Rao
 When Microscopy fails
 Smear negative tuberculosis.
 In HIV infected patients, on many

occasions prove negative. in spite of

presence of bacilli, ( as few bacilli are
expectorated).
 Needs concentration and liquefaction

with chemicals.
 Time consuming, needs more

technical manpower
Dr.T.V.Rao
 Growing role of
Fluorescent Microscopy

 There is a growing need for screening for

AFB by Florescent Microscopy.
 Several studies prove, Florescent
Microscopy in Diagnosis of Tuberculosis is
a priority,
 Developing world should opt and initiate
florescent microscopy.

Dr.T.V.Rao
 Acid Fast Bacilli as seen under
Fluorescent Microscope

Dr.T.V.Rao
 Why we need Florescent
Microscopy
 Useful when few bacilli are present.
 Increases the sensitivity in HIV patients with
tuberculosis.
 Reduces the time needed for testing.
 About 15 times as many fields of view can be
scanned by fluorescent microscopy than by Ziehl
– Neelsen’method in the same period.
 Increases the sensitivity by 10 %
 Better conclusions with one or two specimens,
unlike Ziehl Neelsen’s method needing 3 or > 3
specimens.

Dr.T.V.Rao
 Culturing Mycobacterium

 Culturing for isolation of

Mycobacterium spp continues to be a
Gold standard, particularly in
Developing countries.
 Need only 10 – 100 bacilli / 1 ml of

sputum.

Dr.T.V.Rao
 Culturing
Most useful in
 Surveillance,
 Drug sensitivity testing patterns.
 Identify treatment failures.
 Useful in Patients presenting with
respiratory symptoms, X- ray’s
suggestive, but smear negative. Can prove
culture positive.
 Cultures remain suggestive and helpful in
early treatment periods, failed drug
regimes.

Dr.T.V.Rao
 Methods of Culturing.

 Culturing on Lowenstein Jenson’s

culture medium remain the
affordable ,economical method in
developing world.

Dr.T.V.Rao
 Limitation in Culturing

 Mycobacterium spp are slow

growing.
 Need 6 – 8 weeks for growing.

 Specimens can be contaminated

while growing, needs repeated

specimens, in turn patients loose
confidence in Laboratories.

Dr.T.V.Rao
 Recent facts on Culturing

 Useful in HIV infected patients with

Tuberculosis.
 As even few bacilli can be grown in

spite of smear negativity.

 But the specimens to be incubated

for longer time as few bacilli are

present.

Dr.T.V.Rao
 Pitfalls in Culturing

 Specificity is lost due to

contamination.
 Can yield false positive results in 1 –

4 % of the cases.
 Cultures may be negative in spite of

x rays are suggestive of tuberculosis.

Dr.T.V.Rao
 Growth of Acid fast bacilli on
L J Medium.

Dr.T.V.Rao
 ADVANCES IN
CULTURING
TECHNIQUES.
There are emerging Modern Media
with accurate detection, are
replacing the Egg and Agar based
medium.

Dr.T.V.Rao
 Emerging methods in Culturing
 MGIT – Mycobacterium growth
incubator tube method.
 Growth occurs in shorter than egg

medium.
 Usefulness in HIV patients

established.
 Contamination is less

 But expensive to people in

Developing world.
Dr.T.V.Rao
 Blood culturing for Mycobacterium

 Useful in HIV patients, and children.

 Effective in isolation of Atypical

mycobacterium.
 But not cost effective.

 May be important tool in future for

diagnosing Tuberculosis in HIV

infected.
Dr.T.V.Rao
 Molecular Methods in
Diagnosis of Tuberculosis
Several methods are
available, mainly used as
Research tools

Dr.T.V.Rao
 Real Time PCR replacing older
Methods

Dr.T.V.Rao
 PCR How useful to our Patients?

 PCR ( Polymerase chain reaction ) used by

several investigators.
 However most cases can be diagnosed
with simple methods if effectively used.
 The definite role of PCR continues to be
controversial
 Above all not cost effective to Developing
countries.

Dr.T.V.Rao
 Rapid Diagnostic Methods
in Tuberculosis
Past decade has seen several
emerging technologies
How far practicable ?

Dr.T.V.Rao
 Emerging Rapid Methods.

1. Fast Plaque TB uses phage amplification

technology.

2. ELISA ( QuantiFERON – TB )

3. Enzyme-Linked immunospot
( ELISPOT )
ELISPOT proved highly useful to detect active
tuberculosis in Adults and children.

Dr.T.V.Rao
 Emerging Technology
MODS
 Microscopic observation drug susceptibility
assay. ( MODS )
 A new method gained importance in
several reviews.
 Use a tissue culture plate based assay
with use of Middle Brook 7HG.
 Needs a inverted light microscope.
 Even the drug resistance can be tested
with Rifampicin,and Isoniazid.
 Safe to work with cultures.

Dr.T.V.Rao
 Non Specific Tests

Tuberculin test
( Mantoux Test )

Dr.T.V.Rao
 Tuberculin Test
( Mantoux Test )
 Test to be interpreted
in relation to clinical
evaluation.
 Even the induration of
5 mm to be
considered positive
when tested on HIV
patients.
 Lacks specificity.

Dr.T.V.Rao
 Serology in Tuberculosis.

 Several serological methods were

evaluated.
 But never gained the acceptance of

the majority of the clinicians.

 Serological tests are low sensitivity.

 Many physicians depend on serology

in extra pulmonary tuberculosis.

Dr.T.V.Rao
 Dealing with Tuberculosis
in HIV / AIDS patients.
Diagnosing Tuberculosis in
HIV infected is a priority and
improve quality of Life

Dr.T.V.Rao
 HIV/AIDS - Tuberculosis
 Consider the HIV status
 Identify the severity of Tuberculosis.
 Early use of chest radiography.
 Maximal number of sputum smear
examinations.
 Sputum concentration methods to be
encouraged even by smaller laboratories.
 Explore the use of Florescent Microscopy.
 All smear negative specimens should be
cultured.
Dr.T.V.Rao
 Limitations of Rapid Tests

 The testing needs advanced and

sophisticated infrastructure.
 These tests are known for their

inability to diagnose between active

disease and latent infection.
 Exclusively used in Developed

nations.

Dr.T.V.Rao
 Extra pulmonary
Tuberculosis
Poses several challenges, Yet
no optimal, specific diagnostic
methods

Dr.T.V.Rao
 Extra pulmonary Tuberculosis

 A real challenge to Clinicians and

Laboratories.
 Optimal specimen collection a priority,
 Molecular Methods are growing need.
 Clinicians start drug regimes on empirical
basis.
 Several serological tests for antibody
determinations are evaluated.

Dr.T.V.Rao
 Identification of Atypical
Mycobacterium

A growing concern on infections with

less known, uncommon Mycobacterium
in immunosupreesed, an emerging
infectious disease problem

Dr.T.V.Rao
 Atypical Mycobacterium

 Needs the help of reference

laboratories.
 Needs different drug regimes, unlike

typical Mycobacterium isolates.

 Now a gowning concern in the era of

AIDS.

Dr.T.V.Rao
 Future perceptions
 It is highly essential to explore and discover
rapid, simple, and accurate tuberculosis
diagnostic tools.
 A massive investment, greater scientific interest,
political commitment a top priority,
 Man power development, Human resource
utilization a greater concern.
 Microscopy and Florescent Microscopy utilization
should be immediate concern, and strengthening
of treatment initiation protocols.
 Effective methods in diagnosing smear negative
patients a growing priority.

Dr.T.V.Rao
 Microscopy in Tuberculosis
TODAY

In spite of several
scientific, and
molecular
advances
Microscopy in
Tuberculosis
continues to be
back bone in
Diagnosis.
Dr.T.V.Rao
 Specific detection of active
Tuberculosis cases in
patients with HIV infection or
AIDS is feasible and
improves the rate of early
diagnosis and successful
treatment of Tuberculosis.

Dr.T.V.Rao
Dr.T.V.Rao
 Created for Medical graduates and
Health care workers in Developing world

Dr.T.V.Rao, MD.

e mail; doctortvrao@gmail.com

Dr.T.V.Rao