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DIAGNOSIS OF TUBERCULOSIS
Principles and PracticeDr.T.V.Rao.MD.
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Robert Koch Discovers Mycobacterium
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A Global Emergency
The Tuberculosis in the beginning of the 21st Century declared as Global Emergency
(WHO)Dr.T.V.Rao MD 3
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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 DiseaseDr.T.V.Rao MD 4
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Tuberculosis is a Global Problem
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Tuberculosis - Important communicable disease spread by
Respiratory route
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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.
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Tuberculosis in the era ofHIV / 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 MD 8
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Why we fail to Diagnose Tuberculosis.
Lack of health infrastructure. Control is plagued with lack of Accurate, Robust, and Rapid Diagnostic methods, Technologies.
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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 10
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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.
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When to suspect Tuberculosis
Cough longer than 3 weeks.Fever for 1 month, or both.Blood stained sputum.Nigh sweats, weight lossAge between 14 and 70 years
( Correlates National Tuberculosis Programme ).
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DIAGNOSTIC METHODS
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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.
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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.
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X - ray examination of chest most easily available Investigation.
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Microbiological Investigations are
essential for definitive Diagnosis of Tuberculosis.
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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.
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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.
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Observe to identify Sputum from Saliva.
SPUTUM
Specimens appear mucoid even, blood stained.
Contains many
Polymorphonutrophils.
SALIVA
Appears clear, watery, and frothy.
Contains many squamous epithelial cells
Absence of Polymorphoneutrophils.
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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.
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Microscopy and Tuberculosis
Microscopy with Ziehl – Neelsen’s staining
A century old procedure
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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 MD 23
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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.
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Smear showing Acid Fast Bacilli.
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What is Smear PositivityWHO
All patients who have submitted two Specimens
and found to be positive for identification of AFB
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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.
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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.
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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
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Growing role ofFluorescent 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.
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Acid Fast Bacilli as seen under Fluorescent Microscope
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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.
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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 MD 33
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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.
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Methods of Culturing.
Culturing on Lowenstein Jenson’s culture medium remain the affordable ,economical method in developing world.
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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.
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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.
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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.
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Growth of Acid fast bacilli on L J Medium.
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ADVANCES IN CULTURING
TECHNIQUES.There are emerging Modern Media
with accurate detection, are replacing the Egg and Agar based
medium.
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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
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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.
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Molecular Methods in Diagnosis of Tuberculosis
Several methods are available, mainly used as
Research tools
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Real Time PCR replacing older Methods
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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.
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Rapid Diagnostic Methods in Tuberculosis
Past decade has seen several emerging technologiesHow far practicable ?
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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.
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Emerging TechnologyMODS
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.
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Non Specific Tests
Tuberculin test( Mantoux Test )
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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.
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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 MD 51
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Dealing with Tuberculosis In HIV / AIDS patients.
Diagnosing Tuberculosis in HIV infected is a priority and
improve quality of Life
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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.
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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.
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Extra Pulmonary Tuberculosis
Poses several challenges, Yet no optimal, specific
diagnostic methodsDr.T.V.Rao MD 55
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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.
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Identification of Atypical Mycobacterium
A growing concern on infections with less known,
uncommon Mycobacterium in immunosuppressed, an
emerging infectious disease.Dr.T.V.Rao MD 57
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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.
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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.
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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).
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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
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Microscopy in TuberculosisTODAY
In spite of several scientific, and molecular advances Microscopy in Tuberculosis continues to be back bone in Diagnosis. Dr.T.V.Rao MD 63
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