Mycobacterium tuberculosis
Mycobacterium comprise several species : show branching filamentous form like fungal
mycelium (myces means fungus)
1. Mycobacterium tuberculosis complex that causes tuberculosis
2. Mycobacterium leprae that causes leprosy
3. Non tuberculous Mycobacterium species
Mycobacterium tuberculosis has been present in the human population since antiquity -
fragments of the spinal column from Egyptian mummies from 2400 B.C.
Mycobacterium tuberculosis complex
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They are antigenically and molecularly closely related to each other regarded by
some authors as variants of single species. Susceptible hosts may vary.
Unique property of Mycobacterium species is ACID FASTENESS that is resist
decolouration with even strong mineral acids
Due to
[Link] acid content
[Link] of cell wall
In india 40% of Indian population already infected with Mycobacterium
tuberculosis.10% will develop disease during their life time.
Those who have low immunity like HIV, Diadetes mellitus patients more likely
they develop Tuberculosis [Link] of the leading killer in economically reproductive
age group in india.
Symptoms of tuberculosis
PULMONARY TB
Cough lasting for more than 2 weeks
CONSTITUTIONAL SYMPTOMS:
▻ Fever – low grade at onset and high grade with progression of disease
develops in late afternoon
“night sweats”
▻ Loss of appetite, weight loss, malaise, weakness, unusual fatigue, headache
EXTRA PULMONARY
Symptom Depends on site of involvement
CONSTITUTIONAL SYMPTOMS:
Fever – low grade at onset and high grade with progression of
Disease develops in late afternoon
“Night sweats”
Loss of appetite, weight loss, malaise, weakness, unusual
fatigue, headache
NTP 1962 NATIONAL TB CONTROL PROGRAMME
RNTCP 1992 - REVISED NATIONAL TB CONTROL PROGRAMME
(DOTS STRATEGY- Directly Observed Treatment and Short Course Chemotherapy
strategy was followed and Alternate Day Drug Regimen and No FDC ( FIXED DOSE
COMBINATION)
Now called as NTEP 2020 - NATIONAL TB ELIMINATION PROGRAMME
Now No DOTS Strategy, Daily regimen and FDC is followed
Acid-fast Stain
Discovered by Paul Ehrlich and subsequently modified by Ziehl and Neelsen. Acid
fastness - due to presence of mycolic acid in the cell wall.
Ziehl-Neelsen Technique (Hot Method)
Smear = 3 × 2 cm in size from the yellow purulent portion of the sputum.
Smear - neither be too thick nor too thin - When placed over a printed matter, the
print should be readable through the smear.
The smear is air dried for 15–30 minutes and then heat fixed by passing over the flame
3–5 times for 3–4 seconds.
Step 1 (primary stain) - Carbol fuchsin (1%) for 5 minutes. Intermittent heating is
done by flaming until the vapours [Link] the slide. Heating helps in penetration of
dye.
Carbol fuchsin contains Basic fuchsin ,phenol, absolute alcohol, distilled water.
Step 2 (decolorization) - 25% sulfuric acid for 2–4 minutes. Then wash.
Step 3 (counter staining) - 0.1% methylene blue slide for 30 seconds. Then wash
Air dry and visualization under oil immersion.
Principle: Organisms that resist the decolouration with strong mineral acids
and retain the original primary stain (Carbol fuchsin) are called as Acid
fast
Interpretation
Mycobacterium tuberculosis appears as long slender, straight or slightly curved and
beaded, pink colored acid fast bacillus.
Cold method (Kinyoun’s method): Intermittent heating is not [Link] time is
allowed 10 minutes
Other alternative decolourizer is Acid-alcohol (Hcl plus Ethanol ). Malachite
green or picric acid can be used as counter stain alternatively. Concentration of sulfuric
acid - vary depending on the acid-fastness of the structure to be demonstrated.
For diagnosis two sputum samples are collected as per NTEP (One early
morning & One Spot sample or Two Spot one hour apart) . Hot method is more sensitive
than Cold method. Phenol act as mordant
Acid fast organisms:
. 1. Mycobacterium tuberculosis complex that causes tuberculosis
2. Mycobacterium leprae that causes leprosy
3. Non tuberculous Mycobacterium species (atypical Mycobacteria)
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[Link] spore
[Link] of cryptosporidium parvum
[Link] belli
Disadvantages
Detection limit of AFB staining is 10000 bacilli per ml of sputum
Cannot determine viability of bacilli
Advantages:
Rapid,
Cheap,
Peripheral laboratories,
Follow up treatment ( response to treatment can be assessed , positive grade to
negative indicates good response)
Infectiousness can be assessed (more the grade more infectious)
Mycobacterium tuberculosis complex is both acid and alcohol fast. Mycobacterium
smegmatis (commensal in urine) is acid fast only.
Acid fast organisms /structures Sulphuric acid (%) needed for
decolorization
1. Mycobacterium tuberculosis 25%
2. Mycobacterium leprae 5%
3. Nocardia 1%
4. Acid fast parasites such as 1%
Cryptosporidium , Cyclospora,
Cystoisosopra, Microsporidia
5. Bacterial spore 0.25-0.5%
GRADING OF SMEAR:
If the slide has: No. of fields to be examined Grading Result
1. No AFB in 100 0 Negative
100 fields
2. 1-9 AFB 100 Scanty* Positive
100 fields
3. 10-99 AFB 100 1+ positive
in 100 fields
4. 1-10 AFB 50 2+ positive
per field
5. More than 10 AFB 20 3+ Positive
per field
Uses of grading
1. More the grade more the infectious the patient
2. Treatment follow up 1. response to the treatment (positive grade to
negative indicates good response and
2. suspecting drug resistance - persistence positive smear
during follow up
Light Microscope
Advantage: Cheap
Rapid
Easy to perform
Peripheral Laboratories
Follow up treatment can be assessed
Infectiousness can be assessed
Disadvantages:
[Link] sensitive (Sputum sample need to contain 5000-10000 AFBml of
sputum.)
2. Young children, elderly & HIV infected persons may not produce
cavities & sputum positivity rate for AFB may be very low.
3. Cannot determine viability of bacilli
Detecting AFB by Fluorescence microscopy
The smear may be stained by auramine-O dye. In this method the TB bacilli are
stained yellow against dark background & easily visualized using fluorescent
microscope.
Advantages:
More sensitive
Rapid
Useful in large sample volume centers wherescreenig can be done
Disadvantages: Hazards of dye toxicity, and more expensive
Tuberculosis Diagnosis by
1. Clinical
2. Radiological (X –ray, CT scan etc;)
3. Microbiological
4. Pathological
MICROBIOLOGICAL
MICROSCOPE
[Link] MICROSCOPE
[Link] MICROSCOPE
CULTURE
SOLID: (Lowenstein Jensons Medium) a Gold standard, .
Need only 10 – 100 bacilli / 1 ml of sputum. MTB growth in LJ
media described as ROUGH TOUGH & BUFF COLONIES. Need 6 – 8
weeks for growing and further 4 weeks for Drug Susceptibility Testing.
LIQUID: BACTEC Mycobacteria Growth Indicator Tube (MGIT)
Mean positivity around 2 – 4 weeks
Detection and Drug Resistant can also be detected
MOLECULAR METHODS
1. CBNAAT (Catridge based nucleic acid amplication technique)
Detection limit is 5 genome copies/purified DNA OR
131 cfu/ml [Link] around time 2- 3 hours.
2. TRUENAT - Chip based nucleic acid amplication technique Micro PCR
technique. Portable .Point of care service.
3. LPA - Line probe assay . Turn around time 2- 3 Days
In Molecular Methods Drug Resıstant testing can also be done , detection of the genes
responsible for drug resistant
Eg; Rifampicin - rpoB gene
Isoniazid - inhA and katG genes
OTHER TESTS :(To detect Latent TB infection)
[Link] Skin Test also called as Mantoux test.
TST only indicate whether the individual is infected with MTB complex or not.
Intra dermal injection of .1ml of PPD -23 (Purified Protein Derivative) into the
flexor surface of forearm
Interpretation after 48 – 72 hours
≥ 10mm - Positive
6 -9 mm - Equivocal
≤ 5mm - Negative
Even the induration of 5 mm to be considered positive when tested on HIV
patients
FALSE POSITIVE
BCG VACCINATION
NTM INFECTION
FALSE NEGATIVE
IMMUNOCOMPROMISED STATUS
ANERGY INDUCED BY ACTIVE TB
DISADVANTAGE
Reproducibility
Inter & intra observer variation in measurement
Repeat testing interpretation is difficult due immunological recall
2. Interferron gamma release asssay
Treatment –
6 months duration. Fixed drug combination (HREZ). First line drugs
To prevent development of drug resistance
1. Isoniazid H
2. Rifampicin R First line drugs
3. Ethambutol E
4. Pyrazinamide Z
2 months HREZ and next 4 months HRE
For drug resistant bacilli second line drugs are used
Levoflox, Amikacin,Capreomycin,Ethionamide, Cyclosporine, Linezolid,
Clofazimine, High dose INH, P-amino salicylic acid,
Newer dugs are BEDAQUILINE AND DELAMANID
DRUG RESISTANT TB
[Link]- TB (Multiple drug resistant TB )
Resistant to Isoniazid and rifampicin drugs
Prevalance is 3.4 % in new and 18% in previously treated patients
Treatment duration is 24 months for conventional treatment and 9 months for
shorter MDR regimen
2. XDR TB (EXTENSIVELY DRUG RESISTANT TB )
MDR-TB plus fluoroquinolone plus one of the second line injectables
(amikacin, capreomycin , kanamycin)
INH + Rifa + Quinolones + one of the second line injectables are resistant
Treatment duration is 24 months
VACCINE FOR TB : BCG (Bacillus Calmette- Guerin )Vaccine at birth.
Live attenuated vaccine .
Intra dermal injection.
Helps in preventing serious extra pulmonary complication like
Meningitis ,Miliary TB. NOT THE PULMONARY TB
It also used as adjuvant therapy in Urinary blader cancer.( Intra
vesical immuno therapy)
CHEMOPROPHYLAXIS: INH Preventive Therapy for 6 months
[Link] less than 5 years who has house hold contact with Pulmonary TB patient
2. HIV positive individuals
3. .Children more than 5 years old and adults with house hold contact with
Pulmonary TB patient who have TB infection evidence like
Mx test positive or positive Interferron gamma release asssay
Chemoprophylaxis to be given after ruling out active TB disease.
