Learning Objectives

1. Describe the morphology and antigens of Mycobacterium tuberculosis.

2. Describe Pathogenesis & Clinical features

3. Choose the appropriate lab diagnosis and interpret the results

4. Describe prevention and treatment

Introduction

1. Mycobacteria are slender bacilli that sometimes show branching filamentous forms resembling fungal mycelium (Myces meaning fungus).

2. They are difficult to stain but once stained, they resist decolorization with dilute mineral acids and are called Acid-fast bacilli (AFB).

3. They are aerobic, non-motile, non-capsulated, and non-sporing.

4. Growth is generally slow.

The genus includes obligate parasites (Mycobacterium tuberculosis complex), opportunistic pathogens (Atypical mycobacteria) and saprophytic mycobacteria (e.g. M. butyricum from butter, M. phlei from grass, M. stercoris from dung and M. smegmatis from smegma).

5. Mycobacterium Tuberculosis Complex includes 7 species that are responsible for mammalian tuberculosis viz. M. tuberculosis (human tubercle bacillus), M. bovis (bovine tubercle bacillus), M. microti (vole tubercle bacillus), M. africanum (intermediate form between M. tuberculosis and M. bovis), M. caprae, M. canettii (pathogen of cattle) and

M. pinniped (pathogen of seals).

6. Of these M. tuberculosis and M. bovis are typical tubercle bacilli and cause human lesions such as pulmonary tuberculosis.

Mycobacterium tuberculosis

1. The species contain 2 major types, classical and South Indian types of M. tuberculosis.

2. Classical type is virulent to a guinea pig but the South Indian type is attenuated in this animal. Also, the latter is prevalent in South India and in persons of Asian-ethnic origin living in other countries.

Antigenic Structure

1. The cell wall consists of lipids, proteins, and polysaccharides. The lipid content accounts for 60% of the cell wall weight.

2. Lipids of the cell wall particularly mycolic acid fraction are responsible for the acid-fastness of bacteria and the cellular tissue reactions of the body.

3. The cell wall is made up of four distinct layers:

1. Peptidoglycan (murein) layer, the innermost layer which maintains the shape and rigidity of the cell.

2. Arabinogalactan layer lies external to the peptidoglycan layer.

3. Mycolic acid layer is the principal constituent of the cell wall.

4. Mycosides (peptidoglycolipids or phenolic glycolipids) form the outermost layer of the cell wall.

ZN staining
Differentiating features of M. tuberculosis and M. bovis.

7. Susceptibility to Pyrazinamide:

i. M. tuberculosis is sensitive to 50 µg/ml pyrazinamide but other mycobacteria including

M. bovis are resistant.

However, if pyrazinamidase negative M.tuberculosis is detected, it will also be pyrazinamide resistant.

8. Susceptibility to Thiophen-2-Carboxylic Acid Hydrazide (T2H):

i. M. bovis is usually susceptible to 10 µg/ml of T2H.

ii. M. tuberculosis is usually not inhibited by this chemical, however, South Indian strains are susceptible.

9. Tween 80 Hydrolysis:

i. Certain mycobacteria possess an enzyme lipase and split Tween 80 into oleic acid and polyoxyethylated sorbitol which modifies the optical characteristics of the test solution from a yellow to pink. A pink colour indicates hydrolysis of Tween 80.

ii. M. kansasii and M. gordonae are positive, while M. bovis, M. africanum, M. avium

complex and M. scrofulaceum are negative. M. tuberculosis shows variable results.

Typing Methods:

1. Phenotypic:

i. Bacteriophage typing.

ii. Bacteriocin typing: M. tuberculosis can be typed by means of bacteriocins produced by rapidly growing mycobacteria. However, this method lacks discriminatory power.

2. Molecular typing

Mycobacteriophages

1. Some tubercle bacilli are infected with temperate phages. Many mycobacteria infected with

bacteriophages are not truly lysogenic, the phage genome appears as independent plasmid. There is no integration of phage genome into the host genome → Pseudolysogeny.

2. Tubercle bacilli have been classified into 4 phage types – A, B, C and I. Type I is intermediate between A and B.

3. Type A is worldwide in distribution and is the commonest type.

4. Infection due to type I is more frequent in India and neighbouring countries.

5. Type B occurs in Europe and North America.

6. Type C is rarely seen.

7. Phage 33D (isolated from an environmental mycobacterium) can lyse all variants of M. tuberculosis, but not BCG.

Molecular typing:

1. DNA fingerprinting is a more powerful epidemiological tool for differentiating between strains of tubercle bacilli.

2. IS6110 RFLP typing: Restriction endonuclease treatment yields nucleic acid fragments of varying length, the patterns of which are strain specific. RFLP using IS6110 as a probe is considered the gold standard of molecular typing.

3. Spoligotyping (Spacer oligotyping): This is based on polymorphism in the direct repeat (DR) locus. This is region present in all Mycobacterium tuberculosis complex in a unique

locus that contains well conserved sequences. This is more useful in strains that have no or very few copies of IS6110.

Pathogenesis

1. The infection is commonly acquired by inhalation of infected droplets coughed or sneezed into the air by a patient with pulmonary tuberculosis.

2. In bovine tuberculosis, infected cows develop lesions in the udder and bacilli are excreted in the milk which can then infect people who drink it raw.

3. Tubercle bacilli are engulfed by macrophages but they survive and multiply in macrophages.

4. These lyse the host cell, infect other macrophages, and sometimes disseminate to other parts of the lung and elsewhere in the body.

5. The cell mediated immunity (CMI) plays a major role to interact with these macrophages whereas humoral immunity appears irrelevant.

6. CD4+ helper T cells secrete interferon gamma, interleukin 2, tumor necrosis factor α, and others exerting different biological effects.

7. It may result in protective immunity or delayed type hypersensitivity (DTH) reaction.

8. Th-1 dependent cytokines activate macrophages to kill intracellular mycobacteria and thus result in protective immunity.

9. Th-2 cytokines induce delayed type hypersensitivity (DTH), tissue destruction and progressive disease.

10. Human tuberculosis is divisible into primary and secondary forms:

Primary Tuberculosis:

1. Inhaled tubercle bacilli are engulfed by alveolar macrophages in which they replicate to form a lesion called Ghon focus.

2. It is frequently found in the lower lobe or lower part of upper lobe.

3. Some bacilli are transported to the hilar lymph nodes.

4. The Ghon focus together with the enlarged hilar lymph nodes is called the primary complex.

5. In case of M. bovis which enters through mouth, the primary complexes involve the tonsil and cervical lymph nodes or the intestine, often the ileocaecal region, and the mesenteric lymph nodes.

Secondary (Post-primary) Tuberculosis:

1. It is caused by reactivation of the primary lesion (endogenous) or by exogenous infection.

2. Reactivation tuberculosis is likely to occur in immunocompromised individuals.

3. Granulomas of secondary tuberculosis most often occur in the apex of the lungs.

4. The necrotic element of the reaction causes tissue destruction and the formation of a large area of caseation termed tuberculomas.

5. Presence of caseous necrosis and cavities are two special features of secondary tuberculosis.

6. Cavities may rupture into blood vessels, spreading mycobacteria throughout the body, and breaking into airways, releasing the organisms in aerosols and sputum (open tuberculosis).

Immunity and Hypersensitivity

1. Two immunological responses develop simultaneously in the naturally infected host, one is antitubercular immunity and the other is tuberculin hypersensitivity.

2. Both responses are mediated by T-lymphocytes sensitised to the bacterial antigen.

3. Humoral immunity appears to be of no relevance in tuberculosis.

4. Immunity (resistance to infection) and hypersensitivity (allergy) are two different manifestations of the same mechanism in tuberculosis.

5. In non-immune host, the bacilli are readily phagocytosed and multiply inside the mononuclear cells. This intracellular parasitism is associated with the development of delayed hypersensitivity and of activated macrophages which have an increased ability to destroy the ingested bacilli.

6. After the first infection the host acquires some resistance against reinfection. In the immune host the sensitised T-lymphocytes (develop during primary infection) proliferate and release lymphokines which make the macrophages bactericidal.

7. Delayed hypersensitivity can be induced by infection with virulent or avirulent tubercle bacilli. For demonstrating delayed hypersensitivity, tuberculoprotein (tuberculin) injection is commonly employed

Koch’s Phenomenon

1. The response of a tuberculous animal to reinfection was best explained by Robert Koch.

2. When a healthy guinea pig is inoculated subcutaneously with virulent tubercle bacilli, the puncture site heals quickly and there is no immediate visible reaction.

3. After 10-14 days, a nodule appears at the site of injection which ulcerates and the ulcer persists till the animal dies of progressive tuberculosis.

4. The regional lymph nodes are enlarged and caseous.

5. If on the other hand, virulent tubercle bacilli are injected in a guinea pig which has received a prior injection of tubercle bacilli 4-6 weeks earlier, an indurated lesion appears at the site of injection in a day or so to form a shallow ulcer.

6. This ulcer heals rapidly without involvement of the regional lymph nodes or tissues → Koch’s phenomenon which is a combination of hypersensitivity and immunity.

Koch’s phenomenon is a combination of hypersensitivity and immunity and has got three components:

1.  A local reaction of induration and necrosis.

2.  A focal response in which there occurs acute congestion and even haemorrhage around the tuberculous foci in tissues.

A systemic response of fever that may sometimes be fatal

Tuberculin Skin Test

1. Tuberculin skin test is delayed or type IV hypersensitivity reaction.

2. A purified preparation of active tuberculoprotein was prepared by Seibert (1941) called Purified protein derivative (PPD).

3. The dosage of PPD is expressed in tuberculin unit (TU). 1TU is equal to 0.01 ml of Old tuberculin or 0.00002 mg of PPD-S. Another PPD is RT-23 with tween 80.

4. In India PPD RT-23 of strength 1 TU and 2TU are available.

5. 0.1 ml of PPD containing 5 IU of PPD-S is injected intradermally into flexor aspect of forearm (Mantoux test). A PPD-S dose of 1TU is used when extreme hypersensitivity is suspected.

6. In India 1TU of PPD RT-23 is recommended and not PPD-S.

7. In the Mantoux test the site of injection is examined after 48-72 hrs and interpreted as follows:

Positive test:

There is an induration of 10 mm diameter or more surrounded by erythema at the site of inoculation.

1. Positive test only confirms past infection with tubercle bacilli but does not indicate the presence of active stage of disease.

2. The test is helpful in children under 5 yrs for indication of active infection.

3. The test becomes positive 4-6 weeks after infection or BCG vaccination.

False negative:

The test may become negative in following conditions:

a. Miliary tuberculosis.

b. When anergy develops following overwhelming infection of measles, Hodgkin’s disease, sarcoidosis, lepromatous leprosy, malnutrition, administration of immunosuppressive agents and corticosteroids.

c. False positive:

This is observed in presence of related mycobacteria such as atypical mycobacteria.

Uses:

1. To measure prevalence of infection in a community.

2. To diagnose active infection in young children.

3. It is used as an indicator of successful BCG vaccination.

4. In recent years in vitro interferon-γ release assays (IGRA) have been introduced as a sensitive and more specific alternative to tuberculin skin test. This test is used in blood specimen which contains T-lymphocytes.

5. It uses ELISA to measure interferon-γ (IFN-γ) production by sensitised T-lymphocytes which are stimulated by M. tuberculosis antigens.

6. If an individual was previously infected with M. tuberculosis, exposure of sensitised T- lymphocytes to M. tuberculosis specific antigens results in IFN-γ production.

Laboratory Diagnosis:

Specimen:

a. Pulmonary tuberculosis: Sputum is the most common specimen. A morning specimen may be collected in a wide mouth container and a spot sample, at least 2–5 mL & preferably mucopurulent.

1. When sputum is not available, laryngeal swab or bronchial washings are collected. In children gastric washings may be examined as they tend to swallow sputum.

b. Extrapulmonary tuberculosis:

i. Meningitis: Cerebrospinal fluid (CSF) from tuberculous meningitis (TBM) often forms spider web clot on standing, examination of which may be more useful than of fluid.

ii. Renal tuberculosis: 3 consecutive days morning samples of urine are examined.

iii. Bone and joints tuberculosis: Aspirated fluid.

iv. Tissue: Biopsy of tissue.

Concentration of Specimens:

1. Concentration of a specimen is done to achieve:

a. Homogenisation of the specimen.

b. Decontamination i.e. to kill other bacteria present in the specimen.

c. Concentration i.e. to concentrate the bacilli in a small volume without inactivation.

2. Such concentrate is used for culture and animal inoculation besides smear preparation.

b. Extrapulmonary tuberculosis:

i. Meningitis: Cerebrospinal fluid (CSF) from tuberculous meningitis (TBM) often forms spider web clot on standing, examination of which may be more useful than of fluid.

ii. Renal tuberculosis: 3 consecutive days morning samples of urine are examined.

iii. Bone and joints tuberculosis: Aspirated fluid.

iv. Tissue: Biopsy of tissue.

Concentration of Specimens:

1. Concentration of a specimen is done to achieve:

a. Homogenisation of the specimen.

b. Decontamination i.e. to kill other bacteria present in the specimen.

c. Concentration i.e. to concentrate the bacilli in a small volume without inactivation.

2. Such concentrate is used for culture and animal inoculation besides smear preparation.

Petroff’s method:

3. It is a simple and widely used technique.

4. Sputum is mixed with equal volume of 4% sodium hydroxide and is incubated at 37℃ with frequent shaking for about 30 min. It is then centrifuged at 3000 rpm for 30 min.

5. The supernatant is discarded and the deposit is neutralised by adding 8% hydrochloric acid in presence of a drop of phenol red indicator.

The deposit is used for smear, culture and animal inoculation. Other methods:

6. Dilute acids (5% oxalic acid, 3% hydrochloric acid or 6% sulphuric acid), mucolytic agents

such as N-acetyl-L-cysteine (NALC) with 2% sodium hydroxide (NaOH) and pancreatin are used for concentration of specimens especially for automated culture systems as NALC liquefies sputum and NaOH kills normal flora.

7. In urine and CSF specimens centrifugation is done to concentrate the specimen. Centrifuged deposit is used for smear and culture examination.

Direct Microscopy:

1. Smear is made from the specimen on a new glass slide and stained by the Ziehl- Neelsen staining. It is examined under oil immersion lens.

2. The acid-fast bacilli (AFB) appear as bright red bacilli against a blue/green background.

3. To detect bacilli microscopically there should be at least 10,000 bacilli per ml of sputum.

4. A negative report should not be given till at least 300 fields have been examined.

Grading of Ziehl-Neelsen smear as per RNTCP guidelines

 1. Purulent part of the sputum is used for making a smear.

2. In case of urine specimen, care must be taken to exclude commensal M. smegmatis bacillus which is only acid-fast and not alcohol-fast, whereas, M. tuberculosis is both acid and alcohol fast.

3. If a large number of smears are to be examined, fluorescent microscopy is more convenient.

4. Smears are stained with fluorescent dyes such as auramine ‘O’ or auramine rhodamine and examined under ultraviolet light.

5. The bacilli appears as bright bacilli against dark background.

6. WHO has recommended use of LED (light emitting diode) fluorescence microscopy which are less expensive, able to run on batteries and can perform equally well without a dark room. Also it has more sensitivity than ZN staining.

7. Kinyoun’s modification of acid fast staining: This is a cold method where heating of the stain is not employed. Increasing concentration of phenol and increasing duration of staining is used instead.

Culture:

1. Culture is very sensitive method for detection of tubercle bacilli.

2. It may detect as few as 10 to 100 bacilli per ml.

a. Culture on LJ medium: The concentrated material is inoculated on two bottles of Lowenstein-Jensen (LJ) medium.

1. The culture media incubated at 37℃ in the dark and in the light.

2. Cultures are examined first after 4 days (for rapid growing mycobacteria, fungi and contaminant bacteria) and thereafter weekly till 8 weeks.

3. Tubercle bacilli usually grow in 2-8 weeks.

4. Longer incubation is necessary for strains originating from patients treated with antitubercular drugs.

5. In positive culture, characteristic colonies appear on culture medium. Smear is prepared from isolated colony and stained with ZN technique.

6. When acid-fast bacilli is slow growing, non-pigmented and niacin positive, it is regarded as M. tuberculosis. Confirmation is done by biochemical reactions.

b. In radiometric methods such as BACTEC 460, the growth may be detected in about a week by using 14C labelled substrates. Culture media contains 14C labelled palmitic acid. Mycobacteria metabolise the 14C-labelled substrates and release radioactively labelled 14CO2.

1. The instrument measures 14CO2 and reports in terms of ‘growth index’. A growth index of

≥ 10 is considered positive.

2. This method can also differentiate between M. tuberculosis and M. bovis. As M. bovis is susceptible to TCH, incorporation of TCH in the medium inhibits the growth of M. bovis but not that of M. tuberculosis.

3. This method has now been discontinued due to radioactivity.

c. Mycobacterial growth indicator tube (MGIT) is another rapid method for detection of mycobacterial growth.

3. It is a non-radiometric, automated method.

4. It consists of tubes containing liquid culture media, Middlebrook 7H9 medium supplemented with:

5. „OADC enrichment growth media (oleic acid, albumin, dextrose and catalase) - promote the growth M. tuberculosis and

6. „PANTA antibiotic mixture (polymyxin B, amphotericin B, nalidixic acid, trimethoprim, and azlocillin) to inhibit other organisms.

7. A fluorescent compound is embedded on the bottom of the tube which is sensitive to dissolved oxygen in the liquid medium. Thus, the dissolved oxygen in the uninoculated medium quenches any fluorescence from the compound.

8. When mycobacteria grow they deplete the dissolved oxygen in the liquid medium and allow the compound to fluoresce brightly which can be detected by observing the tube under UV light (Wood’s lamp).

9. The result are obtained in 8-14 days.

d. BacT/Alert 3D system is a non-radiometric, rapid and fully automated .

1. It uses liquid medium vials in which specimen is inoculated.

2. If mycobacteria are present in the specimen, CO2 is released by actively proliferating mycobacteria. The elevated level of CO2 lowers the pH in the medium which produces a colour change in sensor present in the liquid medium vial which is detected by the instrument.

Animal inoculation:

1. 0.5 ml of the concentrated specimen is inoculated intramuscularly into the thigh of 2 tuberculin negative healthy guinea pigs.

2. The animals are weighed prior to inoculation and thereafter at weekly interval.

3. They are tuberculin tested after 3-4 weeks.

4. There is progressive loss of weight and tuberculin test becomes positive in animals that develop tuberculosis. Animal is killed after 6 weeks.

Autopsy shows:

1. Caseous lesion at the site of inoculation.

2. Enlarged caseous inguinal lymph nodes. The infection may spread to other lymph nodes such as lumbar, portal, mediastinal and cervical lymph nodes.

3. Tubercles may be seen in spleen, lungs, liver or peritoneum.

4. Kidneys are unaffected.

5. The identity of the bacteria is then confirmed by demonstration of AFB from the lesions.

6. Infection with Y. pseudotuberculosis, Brucella, Salmonella and several fungi may produce lesions which macroscopically simulate tubercle.

7. M. tuberculosis is highly pathogenic to guinea pigs and hamsters and virtually non- pathogenic for rabbits, while M. bovis is highly pathogenic for both rabbits and guinea pigs.

Molecular Methods:

a. Polymerase chain reaction (PCR) is a rapid method in diagnosis of tuberculosis.

1. It is based on DNA amplification and has been used to detect M. tuberculosis directly in clinical specimens.

-Nested PCR targeting IS6110 gene.

-Cartridge-based nucleic acid amplification test (CBNAAT)

GeneXpert

1. CBNAAT system endorsed by WHO and RNTCP

2. ™Rapid: 2 hours

3. ™Principle: Real-time PCR technique detects

-MTB complex DNA and Rifampicin resistance (mutations of the rpoB gene)

4. No contamination: It employs single-use disposable cartridges

5. ™Procedure:.   The         entire      process  (sample processing,           nucleic   acid        extraction, amplification, and reporting of the result) is fully automated.

1. EPTB: WHO recommends GeneXpert as the initial test using CSF, lymph nodes and other tissues specimens.

2. ™Diagnostic utility: Detection limit 131 bacilli / mL of specimen.

Compared to culture:

-Detection of TB bacilli - 88% sensitive and 99% specific.

-Detection of rifampicin resistance: It is 95% sensitive and 98% specific

4. Disadvantages: very expensive, cannot further speciate.

b. The restricted fragment length polymorphism (RFLP) is used to type different strains for epidemiological purposes.

1. The principle of this technique is that restriction endonuclease treatment yields nucleic acid fragments of different lengths, the patterns of which are strain specific.

c. Line Probe Assay (LPA )

1. Probe-based detection of amplified DNA in the specimen.

2. ™Use of LPA in TB diagnostics: Identification of MTB complex

-Detection of resistance to antitubercular drugs – 1st & 2nd line

-Speciation of MTB complex and NTM

3. Limitation: Can be performed only on positive cultures or smear positive clinical specimens.

4. Commercial Kits - GenoType and INNO-LiPA

5. „Systems used to perform the assay - TwinCubator and GT-Blot

6. Principle: 2–3 days of turnaround time

i. DNA extraction from clinical specimens

ii. Multiplex PCR amplification with biotinylated primers

iii. Reverse hybridization: Chemical denaturation of amplicons  hybridization  streptavidin-conjugated alkaline phosphatase  detects hybrids by biotin-streptavidin moieties.

d. Ligase chain reaction (LCR) and Transcription mediated amplification (TMA) are other molecular methods for detection of M. tuberculosis. TMA is based on amplification of ribosomal RNA as compared to DNA amplification in PCR.

Chromatographic Methods:

1. The analysis of mycobacterial lipids by chromatographic methods, such as thin-layer chromatography, gas-liquid chromatography (GLC) and high performance liquid chromatography (HPLC), has been used to identify mycobacteria.

2. HPLC is very specific and rapid method for identification of species.

Serology:

1. Serology includes detection of antimycobacterial antibodies in patient serum.

2. Various methods such as ELISA, RIA, Latex agglutination assay have been employed.

3. Several antigens like BCG, antigens 5 & 6, 64 kDa, antigen 60 and 32 kDa protein have been tried for detection of antibody against them.

4. Diagnostic utility of these antibodies is equivocal and its use for diagnosis of active TB has been discouraged by WHO.

Upcoming Methods for TB Diagnosis

1. TrueNat (Molbios) - Chip Based Real Time PCR (TrueNat)

2. Automated battery operated device; can be used at primary health center level

3. Unit 1 (sample preparation device) - DNA extraction Then

4. Unit 2 (analyzer) - extracted DNA is added to the chip (coated with the probes) and loaded for amplification

5. Disadvantages: very expensive, cannot further speciate MTB complex and tests one sample at a time.

6. TB-LAMP (Loop-mediated isothermal amplification):

-Alternative to smear microscopy for identification (WHO)

-Does not detect drug resistance

7. ™Next generation GeneXpert (Xpert Ultra):

8. „Ultra cartridge with larger chamber for DNA amplification to accommodate larger amount of sputum

9. Two additional molecular targets to detect TB

10. „More sensitive and specific with detection limit of 16 bacilli/mL compared to 131 bacilli/mL of first generation GeneXpert

Sensitivity Testing

1. Drug-resistant mutants continuously arise at low rate in any mycobacterial population.

2. With the emergence of multidrug resistance in mycobacteria, it is essential to determine the sensitivity testing.

3. The isolated tubercle bacilli are tested in LJ media after incorporating different concentrations of antitubercular drugs in the media before inoculation.

Resistance Ratio Method:

1. This is the most important method. Bottles of LJ medium incorporating doubling concentrations of drug are inoculated with a test strain and a known sensitive strain (H37Rv strain of M. tuberculosis).

2. After 3 weeks of incubation at 37℃, culture media are examined for growth.

3. The medium with the lowest concentration of drug showing no more than 20 colonies is taken as the minimum inhibitory concentration (MIC).

4. The result is expressed as resistance ratio. Interpretation:-

5. Resistance ratio of 1 or 2 = sensitive strain.

6. Resistance ratio of 4 = Doubtful resistant strain, test should be repeated.

6. Resistance ratio of 8 = Unequivocal resistant strain.

Absolute Concentration Method:

1. The MIC of the drug against the test strain only is determined.

2. Known sensitive strain is not tested for MIC, therefore, this method is inferior to resistance ratio method.

Proportion Method

1. In this method, the number of colonies growing from a standard inoculum on a medium containing drug is compared with the number of colonies from same sized inoculum on a medium without drug.

2. When more than 1% of the mycobacteria grow in the presence of drug, it is regarded as a resistant strain.

Radiometric Method:

1. BACTEC radiometric method was most commonly used.

2. For each antituberculous drug tested, a standardized inoculum is inoculated into the liquid media, one containing drug and other without drug. The liquid medium contains 14C-labelled substrate.

3. The rate and amount of 14CO2 produced in the absence or presence of drug is measured by the special instrument and is then compared. This method has been discontinued due to radioactivity.

Non-Radiometric Method:

1. Mycobacterial growth indicator tube (MGIT) can also be used for sensitivity testing of M. tuberculosis.

BacT/Alert 3D System:

1. Liquid medium vials with and without antitubercular drugs are inoculated with growth of mycobacteria. Vials without drugs act as controls.

2. M. tuberculosis isolate is susceptible when the drug containing vial remains negative for growth or becomes positive later than the control vial.

3. The strain is resistant when the drug containing vial becomes positive prior to or on the same day as that of control vial.

Chemiluminescence:

1. Luciferase reporter mycobacteriphage (containing the firefly luciferase gene) has been used for susceptibility testing of M. tuberculosis.

2. Only viable mycobacteria can be infected with and replicate this mycobacteriophage; dead tubercle bacilli cannot.

3. The isolate of M. tuberculosis to be tested is grown in the presence and absence of drug and the reporter mycobacteriophage is added.

4. Following infection, a substrate of luciferase, luciferin is added.

5. If bacteria are viable, the luciferin is broken down and light is emitted which can be measured → known as Chemiluminescence.

6. If the isolate is resistant to drug, light will be emitted, while bacteria susceptible to the drug will not emit any light.

7. The amount of light emitted is directly proportional to the number of viable bacteria.

Epsilometer Test:

1. Epsilometer test (E-test) has also been applied for susceptibility testing of M. tuberculosis.

Mutation:

1. Demonstration of mutation in specific genes for different drugs is a useful indicator of drug resistance.

Treatment

2. The antitubercular drugs include bactericidal agents such as rifampicin (R), isoniazid (H), pyrazinamide (Z), streptomycin (S) and bacteriostatic agents include ethambutol (E), thiacetazone, ethionamide, paraaminosalicylic acid (PAS) and cycloserine.

.3. Short course regimens of 6-7 months are used.

4. A combination of 4 drugs (HRZE) given 3 times a week during an initial intensive phase for 2 months, followed by only 2 drugs (HR) 3 times a week during continuing phase of 4-5 months.

5. As resistant strains emerge readily by mutation and selection, combinations of two or more drugs are used.

6. The bacterial resistance may be primary (prior to start of treatment) or secondary which emerges during the course of treatment.

7. A serious consequence of unchecked drug resistance has been the emergence of multidrug resistance tuberculosis (MDR-TB).

8. MDR refers to resistance to rifampicin and isoniazid, with or without resistance to one or more other drugs.

9. MDR-TB is a global problem especially in HIV infected persons.

10. The directly observed treatment, short-course chemotherapy (DOTS) is being used to prevent deterioration of resistance problem by ensuring patient’s compliance.

11. When the patient does not show improvement, antibiotic sensitivity test is done to detect resistant strains.

12. Another serious condition extensively drug resistant tuberculosis (XDR-TB) has emerged recently.

13. XDR-TB is due to M. tuberculosis strains which are resistant to any fluoroquinolone and at least one of three injectable second line drugs (capreomycin, kanamycin and amikacin), in addition to isoniazid and rifampicin.

14. In recent years, two new terms extremely drug resistant tuberculosis (XXDR-TB) and totally drug resistant tuberculosis (TDR-TB) have been evolved.

15. These terms have been used for M. tuberculosis strains which are resistant to all first line drugs and second line drugs.

16. In 2007, 2 cases of TDR-TB were first reported from Italy. In 2009, Iran was the second country to report 15 cases of XXDR-TB or TDR-TB.

17. Recently in 2011, 12 such cases have been reported from Mumbai, India.

Prophylaxis

1. Protection from TB may be done by public health measures, BCG vaccination and by chemoprophylaxis. General measures such as nutrition and health education are also important.

Chemoprophylaxis

1. It is advocated only in some risk groups such as:

2. Individuals who are in unavoidable contact with a patient with open tuberculosis e.g. baby born to a mother with the disease.

3. Tuberculin positive but radiologically clear unvaccinated children.

4. Adults with the radiological evidence suggestive of inactive disease.

5. These individuals are given isoniazid alone with an assumption that the bacillary load is small and the chance of emergence of a drug-resistant mutant is remote.

6. RNTCP Guideline 2016 for Diagnostic Algorithm for Tuberculosis – Adults

7. For extrapulmonary TB (EPTB):

8. - EPTB specimens are paucibacillary  CBNAAT/MGIT are directly performed

Revised National Tuberculosis Control Programme (RNTCP) now renamed as National Tuberculosis Elimination Program (NTEP) – goal to end TB by 2025.

References