ESTABLISHING

MYCOBACTERIOLOGY
LABORATORY SERVICES
Dr.T.V.Rao MD

TUBERCULOSIS A TRIBUTE TO

Dr.T.V.Rao MD

TUBERCULOIS IS NOT ONLY A DISEASE OF POOR

BUT RICH AND POWERFUL TOO GET

Dr.T.V.Rao MD

India Profile
No of Govt. hospitals 12760,
CHCs 4510, PHCs 23391, Sub-centers 145894
Beds in Government Sector, 576793;
Population per Government Hospital Bed 2012.
No of medical colleges 314 +; Blood banks - 2445, Eye Banks 586,
Diverse socio-economic, cultural, political conditions

Large unregulated private sector in health care

Dr.T.V.Rao MD

WHY NEED FOR ESTABLSHING

MYCOBACTERIOLOGY LABORATOREIS
A high-quality laboratory system that uses
modern diagnostics is a prerequisite for
early, rapid and accurate detection of TB.
Lack of diagnostic capacity has been a
crucial barrier preventing an effective
response to the challenges of HIVassociated and drug resistant tuberculosis
(TB)
Dr.T.V.Rao MD

PRIMARY STEP IN CARING THE

TUBERCULOSIS PATIENTS
Care of patients with TB
starts with a quality assured
diagnosis, obtained by
identifying Mycobacterium
tuberculosis from clinical
specimens and conducting
DST of the organism to
confirm or exclude
resistance.
Dr.T.V.Rao MD

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 MD

WHO MONITORS THE DIAGNOSTIC TOOLS

Research on new TB diagnostic tools
has been accelerated over the last few
years and the diagnostic pipeline has
been growing rapidly as a result. At
the same time, an unprecedented
effort to improve and expand TB
laboratory capacity is currently being
lead by WHO and the Stop TB
Partnership Global Laboratory
Initiative (GLI) together with the GLI
network of international collaborators

Dr.T.V.Rao MD

Enhancement of Diagnostic Capacity

Enhancement of
diagnostic capacity for
TB and MDR-TB is
urgently needed to
scale-up access to care
and treatment of
MDR-TB. To help meet
this challenge
Dr.T.V.Rao MD

Development of laboratory norms and

standards
Including WHO policy
recommendations on the use
of new diagnostics,
specifications for TB laboratory
equipment, laboratory
biosafety, standard operating
procedures for TB laboratories,
and technical manuals for firstand second-line drug
susceptibility testing
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Prioritization of human resource

development and training,
Including development
of comprehensive
training and retention
strategies, and
proposals to train
different cadres of
laboratory consultants
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Dr.T.V.Rao MD

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Laboratory accreditation
Involving a WHO-led task
force with international
experts and key partners, to
develop a framework for a
voluntary laboratory
accreditation programme for
national and regional TB
reference laboratory
networks
Dr.T.V.Rao MD

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Laboratory biosafety guidance

Under WHO and CDC

leadership, to develop
risk-based biosafety
standards for
laboratories in resourceconstrained settings,
supported by appropriate
biosafety manuals and
training packages
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Core elements of laboratory services

Laboratory infrastructure, appropriate biosafety measures and
maintenance
Equipment validation and maintenance
Specimen transport and referral mechanisms
Management of laboratory commodities and supplies
Laboratory information and data management systems
Laboratory quality management systems
Appropriate, adequate strategies and funding for laboratory human
resource development
Coordination of technical assistance
Integration of diagnostic algorithms in laboratory strengthening plans.
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TB diagnostics and
laboratory strengthening

Microscopy and Tuberculosis

Microscopy with Ziehl Neelsens
staining
A century old
procedure

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Diagnosis of Pulmonary Tuberculosis

Three specimens optimal
Spot specimen on first visit; sputum
container given to patient
Early morning collection by patient on next
day
Spot specimen during second visit
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WHO and IUATLD

Positive and Negative Report

Negative Report:

Negative for AFB

where no organisms observed in 100 oil
immersion fields

Positive Report:

Positive for acid-fast

bacilli; provide AFB quantification
Dr.T.V.Rao
19 MD

WHO/IUATLD Quantification scale

Ziehl Neelsen
Number of AFB

Number of fields* examined

No AFB in 100 fields

100 fields

No Acid Fast Bacilli detected

19 AFB in 100 fields

Record exact figure

(1 to 9 AFB per 100 fields)

100 fields
10 99 AFB in 100 fields

1+

100 fields
1 10 AFB in each field

2+

50 fields
More than 10 AFB in each field

3+

20 fields

* Oil immersion fields

What to report

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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 MD

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False Negatives and Consequences

False-negative means that the results that were
reported negative were truly positive

Patients with TB may not be treated resulting in ongoing disease, disease transmission, or death.
Intensive phase treatment may not be extended,
resulting in inadequate treatment and potential drug
resistance
Dr.T.V.Rao
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False Positives and

Consequences
False-positives mean that the results that
were reported positive were truly negative
Patients are treated unnecessarily
Treatment may be continued longer than
necessary
Medications will be wasted
Dr.T.V.Rao
23 MD

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 MD

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QUALITY CONTROL TEACHES MANY

MATTERS

Dr.T.V.Rao MD

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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 MD

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Acid Fast Bacilli as seen under Fluorescent Microscope

Dr.T.V.Rao MD

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Light emitting diodes (LED)

The recent development of light
emitting diodes (LED), with the
appropriate fluorescent light
output for FM and low power
consumption, has led to the
development of simple, robust
LED FM microscopes, requiring
minimal mains or battery power
and no dark room requirement.
The WHO has recommended
rolling it out as an alternative to
LMs in resource-limited settings,
Dr.T.V.Rao MD

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Culturing
Most useful in

Surveillance,
Drug sensitivity testing patterns.
Identify treatment failures.
Useful in Patients presenting with respiratory symptoms, Xrays suggestive, but smear negative. Can prove culture
positive.
Cultures remain suggestive and helpful in early treatment
periods, failed drug regimes.
Dr.T.V.Rao MD

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Methods of Culturing.
Culturing on
Lowenstein Jensons
culture medium
remain the affordable
,economical method
in developing world.
Dr.T.V.Rao MD

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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 MD

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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 MD

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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.
Dr.T.V.Rao MD

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MGIT Technology
BDs MGIT Technology is a
commercial liquid culture system
and is the leading rapid culture
method in the developed world.
MGIT stands for Mycobacteria
Growth Indicator Tube. It is a
system that determines whether
or not bacteria will grow.
Typically, it is used to test for the
existence of TB and/or the
effectiveness of various TB drugs
on various strains of TB bacteria
Dr.T.V.Rao MD

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Molecular Methods in
Diagnosis of Tuberculosis
Several methods are available, mainly used as
Research tools

Real Time PCR replacing older Methods

Dr.T.V.Rao MD

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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 MD

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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 MD

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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 MD

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DETECTION OF ANTIBODIES
Although the detection of antibodies against
MTB in the blood is a relatively simple and costeffective method, recent meta-analyses and
systematic reviews concluded that commercial
serological tests provided inconsistent results. As
the overall test performance and data quality of
these assays were poor, the WHO currently
recommends against their use for the diagnosis
of pulmonary and extra pulmonary TB.
Dr.T.V.Rao MD

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( Mantoux Test )

Dr.T.V.Rao MD

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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 MD

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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. But of No Use
Dr.T.V.Rao MD

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WHO monitoring of Xpert MTB/RIF roll-out

Xpert MTB/RIF is an
automated, cartridgebased nucleic
amplification assay for
the simultaneous
detection of TB and
rifampicin resistance
directly from sputum in
under two hours.
Dr.T.V.Rao MD

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GeneXpert platform
The technology is based on the
GeneXpert platform and was
developed as a partnership with
support from the US National
Institutes of Health. WHO
recommended use of the
technology in December 2010
and is monitoring the global rollout of the technology to
promote coordination.
Dr.T.V.Rao MD

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GeneXpert MTB/RIF
The Xpert MTB/RIF is a cartridge-based, automated
diagnostic test that can identify Mycobacterium
tuberculosis (MTB) and resistance to rifampicin
(RIF). It was co-developed by Cepheid, Inc. and
Foundation for Innovative New Diagnostics, with
additional financial support from the US National
Institutes of Health (NIH) and technical support
from the University of Medicine and Dentistry of
New Jersey
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How the test works

The Xpert MTB/RIF detects DNA sequences
specific for Mycobacterium tuberculosis and
rifampicin resistance by polymerase chain
reaction It is based on the Cepheid
GeneXpert system, a platform for rapid and
simple-to-use nucleic acid amplification
tests (NAAT).
Dr.T.V.Rao MD

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How the test works

The Xpert MTB/RIF purifies, concentrates,
amplifies (by real-time PCR) and identifies
targeted nucleic acid sequences in the
Mycobacterium tuberculosis genome, and
provides results from unprocessed sputum
samples in 90 minutes, with minimal biohazard
and very little technical training required to
operate
Dr.T.V.Rao MD

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the Xpert MTB/RIF assay is:

99.7% sensitive and 98.5%
specific when compared to
culture for smear positive
sputum.
76.1% sensitive and 98.8%
specific when compared to
culture for smear negative
sputum3

Dr.T.V.Rao MD

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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 MD

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Do not neglect Fundamentals in

Microbiology

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REAL PCR NEEDED TODAY IN

MICROBIOLOGY

MICROBIOLOGISTS NEED
P Patience
C commitment to work
R Responsibility
or else we will be ignored
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WHAT ARE ESSENTAILS TO START LABORATORY

A separate Ventilated Room with poor exhaust
Minimal Laboratory Equipment as Bacteriology culture laboratory
Biosafety Cabinet grade 3 ?
Two technicians having basic experience in handling Biohazard
Specimens
Documentation facilities

A GREAT DETERMINATION
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We can train and learn many things at this

Internet site
Essentials for the Mycobacteriology Laboratory:
Promoting Quality Practices
The Association of Public Health Laboratories
works to strengthen laboratories serving the
public's health in the US and globally. - See
more at:
http://www.aphl.org/AboutAPHL/Pages/default.
aspx#sthash.A9SaRJiV.dpuf
Dr.T.V.Rao MD

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NEVER FORGET
India spends least on patients with just 28$
among the BRICS Countries
Many believe India has Million Missing patients
India has close to 1,00,000 cases of Multidrug
resistant TB which is hard to diagnose and treat
Most Important Undocumented and untreated
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Dr.T.V.Rao MD

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I THANK YOU AND MICROSCPE

Dr.T.V.Rao MD

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