DIAGNOSIS OF

TUBERCULOSIS
Emerging Trends

Dr.T.V.Rao, MD.
Robert Koch Discovers
Mycobacterium
A Global Emergency
The Tuberculosis in the beginning
of the 21st Century declared as
Global Emergency (WHO)
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

Tuberculosis is a Global Problem
 Tuberculosis - Important
communicable disease spread by
Respiratory route
 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.
 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.

 Why we fail to Diagnose
Tuberculosis.

Lack of health infrastructure.

Control is plagued with lack of
Accurate,
Robust,
and Rapid
Diagnostic methods,
Technologies.
 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.
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.
When to suspect Tuberculosis
Cough longer than 3 weeks.
Fever for 1 month, or both.
Blood stained sputum.
Nigh sweats, weight loss
Age between 14 and 70 years ( Correlates
National Tuberculosis Programme ).
DIAGNOSTIC
METHODS
 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.
 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.
X - ray examination of chest most
easily available Investigation.
 Microbiological
Investigations are essential
for definitive Diagnosis of
Tuberculosis.
 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.
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.
 Observe to identify Sputum from
Saliva.

SPUTUM SALIVA

Specimens appear mucoid Appears clear, watery, and

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

Microscopy with Ziehl –

Neelsen’s staining

A century old
procedure
 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.
 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.
Smear showing Acid Fast Bacilli.
 What is Smear Positivity
WHO

All patients who have submitted two

Specimens and found to be positive
for identification of AFB
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.

 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.
 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
 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.
Acid Fast Bacilli as seen under
Fluorescent Microscope
 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.
 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.
 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.
 Methods of Culturing.

Culturing on Lowenstein Jenson’s culture

medium remain the affordable ,economical
method in developing world.
 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.
 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.
 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.
Growth of Acid fast bacilli on
L J Medium.
 ADVANCES IN
CULTURING
TECHNIQUES.
There are emerging Modern Media
with accurate detection, are replacing
the Egg and Agar based medium.
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.
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.
Molecular Methods in
Diagnosis of
Tuberculosis
Several methods are available,
mainly used as
Research tools
Real Time PCR replacing older
Methods
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.
 Rapid Diagnostic
Methods in Tuberculosis
Past decade has seen several
emerging technologies
How far practicable ?
 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.
 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.
Non Specific Tests
Tuberculin test
( Mantoux Test )
 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.
 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.
 Dealing with
Tuberculosis in HIV /
AIDS patients.
Diagnosing Tuberculosis in HIV
infected is a priority and improve
quality of Life
 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.
 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.
 Extra pulmonary
Tuberculosis
Poses several challenges, Yet no
optimal, specific diagnostic
methods
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.
Identification of Atypical
Mycobacterium
A growing concern on infections with less
known, uncommon Mycobacterium in
immunosupreesed, an emerging infectious
disease problem
 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.
 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.
 Microscopy in Tuberculosis
TODAY

In spite of several
scientific, and
molecular advances
Microscopy in
Tuberculosis
continues to be back
bone in Diagnosis.
Created for awareness to
Medical and Paramedical
students in Developing
world
Dr.T.V.Rao MD
Email
doctortvrao@gmail.com