Mycobacterium tuberculosis: mode of transmission, pathogenesis, clinical diseases, lab diagnosis and treatment

Mycobacteria

• The name mycobacterium is derived from the word “mold” meaning fungus like bacterium.
• Mycobacterium comprises acid-fast bacilli ie.  Resistant to decolorization by weak mineral acids because they have an unusual cell wall that contains mycolic acid acid instead of NAM and has a very thick high lipid content (60-70%).
•  Because of their lipid-rich waxy cell wall, they are difficult to stain with basic aniline dyes. But once stained, they resist decolorization by acidified alcohol (3% hydrochloric acid) after prolonged application of a basic fuschin dye or with heating of this dye following its application. They are therefore called Acid-fast Bacilli (AFB). The lipid rich cell wall also makes the bacteria resist the commonly used disinfectants and laboratory staining reagents.
• Another important feature of these organisms is that they grow more slowly than other human pathogenic bacteria because of their hydrophobic cell surface.
• Because of this hydrophobicity, organisms tend to clump so that nutrients are not easily allowed into the cell.
• Growth is slow or very slow with colonies becoming visible in 2 to 60 days at optimum temperature.

Classification of Mycobacteria:

• Currently there are more than 100 recognized species in the genus mycobacterium.
• Mycobacteria can be divided into three major groups on the basis of fundamental differences in epidemiology and association with disease. These are:
• M. tuberculosis complex
• Non tuberculosis mycobacteria (NTM)        
• Lepra bacilli

Mycobacterium tuberculosis complex

• The term complex is frequently used in the clinical microbiology laboratory to describe two or more species whose distinction is complicated and of little or no medical importance.
• The mycobacterial species that occurs in human and belong to M. tuberculosis complex include M. tuberculosis, M. bovis, M. bovis BCG and M. africanum: all species are capable of causing tuberculosis.

Mycobacterium tuberculosis

•       M. tuberculosis was first isolated by Robert Koch on 24^th March, 1882.
• It is the causative agent of tuberculosis in Human.

General Morphology of M. tuberculosis:

• Shape and size: M. tuberculosis is a straight or slightly curved rod measuring about 0.5-0.6 µm in breadth and 3-4 µm in length.
• They occur singly or in pairs or as small clumps. The size depends on the condition of growth and long filamentous, club sized and branching forms may sometimes be seen.
• They are non-motile, non-sporing, non-capsulated and are acid fast bacilli due to the presence of mycolic acids in their cell wall.
• They are weakly grown positive, with Ziehl-Neelsen stain, M. tuberculosis stains bright red and appear slender, straight or slightly curved rod with beaded or barred appearance. With fluorescent stains mycobacteria can be demonstrated by yellow-orange fluorescence.

Habitat

• The main reservoirs of M. tuberculosis are infected humans.
• M. tuberculosis primarily the respiratory tract of the infected human host.
• The organism can also occur as a pathogen in animal.

Mode of transmission of TB:

• Humans acquire M. tuberculosis infections aerosolized dropletes.
• Persons with pulmonary tuberculosis (PTB) when they cough, sneeze or speak, dropletes containing bacteria are released and people nearby who get infected.
• A single cough can produce 3000-5000 tiny droplet nuclei. Theoretically, although a single tuberculosis bacilli may cause disease in practice 5-200 inhaled bacilli are essential for infection.
• Two factors determine an individual risk of exposure:
• Concentration of droplets nuclei- contaminated air.
• The length of time breathing that air.
• Occasionally infection occurs by ingestion of infected milk (M. bovis)

Virulence factors:

• The factors determining the virulence of M. tuberculosis are poorly understand. M. tuberculosis do not produce any toxins.
•  The following are some virulence factors however, their existence as virulence factors are still doubtful.

1. Cord factor:

• It is a glycolipid derivation of mycolic acid present on outer surface of M. tuberculosis.
• It is responsible for-
• Inhibition of migration of polymorphonuclear leucocytes and elicits granuloma formation

2. Immunogenic and can induce protective immunity in animals

• Cause bacilli to grow in culture in serpentine cords (parallel arrangements of bacilli form)

3. Sulfolipids:

• They prevent fusion of phagosome and lysosome inside the macrophages, thereby allowing the bacteria to multiply within the macrophages.
• Antibacterial resistance to antibiotics is acquired mostly by mutation
• The main pathology in the infected tissue caused by mycobacterial infection is primarily due to responses of the host to M. tuberculosis infection rather than any virulence factor produced by it.

Pathogenesis of tuberculosis:

• Tuberculosis may be primary or post primary depending on the time of infection and the type of host immune response.

1. Primary tuberculosis:

• It represents the initial infection caused by M. tuberculosis is an infected host.
• On inhalation of the aerosolized bacteria (infectious droplets), most of the larger droplets become lodged in the upper respiratory tract (nose and throat), where infection is unlikely to develop. However, the smaller droplet nuclei may reach the small air sacs of the lungs (the alveoli), where infection begins.
• The bacilli in the lungs are endocytosed by alveolar macrophages by binding lipoarabinomannan on the bacterial cell wall through mannose receptors and binding opsonized mycobacteria through complement receptors.
• At this stage, macrophages are naive and unable to kill mycobacteria which multiply the host cell, infect more macrophages.
•  Some bacilli are transported by macrophages to the hilar lymph nodes. They are attracted to the site by the presence of bacilli, cellular components and chemotactic factors such as complement of the serum.
• An antibody mediated immune (AMI) will not aid in the control of a MTB infection because MTB is intracellular and if extracellular, it is resistant to complement killing due to high lipid concentration in its cell wall. Thus a CMI response is necessary to control infection.
• Ghon’s focus formation: Lysis of macrophages and MTB results in the formation of granuloma or tubercle. The center of the tubercle is characterized by “caseation necrosis” meaning it takes a semi-solid or “cheesy consistency. MTB cannot multiply within these tubercles because of the low pH and anoxic environment. MTB can however, persist within these tubercles for extended periods. The granuloma is also known as Ghon’s focus commonly located in the in the lower part of the upper lobe. The Ghon focuses together with enlarged hilar lymph node constitutes the Ghon complex or primary complex. In minority of the cases, the lesson heals spontaneously in 2-6 month leaving behind a calcified nodule.

2. Active tuberculosis:

• It may develop as a progression of primary infection.
• In a few, particularly in children with unpaired immunity or other risk factors, the primary lesson/tubercle may enlarge.
• The growing tubercle may invade a bronchus and thus MTB infection can spread to other part of the lungs.
• Similarly the tubercle may invade an artery or other blood supply line. The hematogenous spread of MTB result in extrapulmenary tuberculosis also known as milliary (disseminated) tuberculosis.
• The secondary lessons caused by milliaryTB can occur at almost any anatomical location but usually involve the genitouninary system, bones, joints, lymph nodes and peritoneum.

**Note:  Growing tubercle- although many activated macrophages can be found surrounding, the tubercles many other macrophages present remain unactivated or poorly activated. MTB uses these macrophages to replicate and hence the tubercle grows.

• With the passage of time, MTB are either killed or they remain dormant within the macrophages or they are actively multiplying leading to progressive 1° infection as described above.
•  The first two conditions are called the latent TB infection.
• The chest x-ray in both of these conditions looks similar. However, when clearly viewed by microscope, in case of dead case, the macrophage remains healthy. However, in the dormant case, the live MTB can be observed within the macrophages.

3. Secondary tuberculosis:

• It is caused due to reactivation of the latent infection or exogenous infection and differs from primary type in many respects.
• It affects mainly the upper lobes of the lungs, the lesson undergoing necrosis and tissue destruction, leading to cavitation.
• Lymph node involvement is unusual.
• The necrotic materials break out into the airways, leading to expectoration of bacteria-laden sputum, which is the main source of infection to other healthy individuals come in contact.
• In the immunodeficient patients cavity formation is unusual. Instead there is widespread dissemination of lessons in the lungs and other organs.
• 2° tuberculosis develops months or years later due to poor health, malnutrition or defective immune responses.

Clinical symptoms of Tuberculosis:

     The clinical manifestation of TB depends on the site of infection.

Pulmonary TB:

• The main symptoms of pulmonary tuberculosis in adults are a chronic cough with the production of mucoid or mucopunulent sputum which may contain blood (hemoptysis).
• In the later stages of the disease there is usually loss of weight, fever, night sweats, tiredness, chest pain and anemia.   
• Rheumatoid factor is occasionally detected in the serum.
• Complications of PTB includes:
• Dry pleurisy or pleural effusion, lung collapse, acute miliary TB and occasisonally tuberculosis meningitis.
• In children PTB can occurs weight loss, enlargement of lymph glands that may cause bronchial obstruction and emphysema.

Extrapulomonary tuberculosis:

• It usually occurs as a result of spread of the bacilli through blood circulation, during the initial stage of multiplication at lungs depending on the site of infection, extrapulmonary TB can be:

Renal and urogentital TB:

• Tubercle bacilli reach the kidney and genital tract by way of the blood circulation.
• The typical symptoms include dysuria, increased frequency of urination and flank pain.
• There is usually recurring fever.
• TB of genital tract in male cause epidymitis or growth in the scrotal area.
• Complications: Hydronephrosis and autonephrectiomy.

Tuberculosis meningitis:

• It occurs more frequently in infants and young children as a complication of primary tuberculosis.
• The conditions may persist as headache, which is either intermittent or persistent.
• The condition is usually rapidly fatal under an early stage.

Gastrointestinal tuberculosis

• The clinical manifestation of the condition depends on the site affected in the gastrointestinal tract.
• For eg. Infection of stomach or duodenum manifest as ‘‘abdominal pain mimicking peptic ulcer disease’’, whereas infection of the large intestine manifest as pain in abdomen, diarrhea etc.
• Complications- intestinal perforation, abstruction and malabsorption.

Skeletal tuberculosis

• Spine is involved resulting in Potts disease.
• Complications: paraplegia

Tuberculosis lymphadenitris or scrofula

• Involves the neck along the sternocleidomastoid muscle. The condition is usually unilateral with little or no pain.
• Other condition include miliary tuberculosis, tuberculosis of the skin and tuberculosis of middle ear and ocular structure.

Laboratory diagnosis of M. tuberculosis:

Specimens:

• Samples depends on the site of infection
• PTB:- sputum,
• Tubercular meningitis:- CSF
• Genitourinary TB:- urine.
• Others- largyngeal swabs, gastric lavage, pleural fluid, pus sample, nasopharyngeal aspirates.

1. Microscopy

• Ziehl-Neelsen staining
• Auramine Rhodamine Fluorochrome stain
• Kinyoun stain

2. Culture (Gold standard method)

• M. tuberculosis is an obligate aerobe and characteristically grows very slowly in media. Optimum temperature is 37°C and the bacteria do not grow below 25°C or above 40 °C. Optimum pH is 6.4 – 7.0 pigment is not produced.
• It is ideal to inoculate the concentrated specimen into one egg-based and one agar-based.
• Cultures are incubated at 35 °C in the dark in an atmosphere of 5 % and 10 % CO2 and high humidity. Tubed media is incubated in a slanted position with screw caps loose for at least 1 week to allow for evaporation of excess third and the entry of CO2. Cultures are examined weekly for growth. Most isolates appear between 3 and 6 weeks, a few appear after 7 or 8 weeks of incubation. After 8 weeks of incubation negatives cultures are reported and the cultures are discarded.
• On LT medium (Lowenstein Jensen) at 35-37°C, M. tuberculosis produces raised dry, cream (buff) colored colonies. Visible colonies are usually produces 2-3 weeks after incubation but cultures should be incubated for up to 6 weeks before being discarded.
• On liquid medium, MTB produces growth that appears first at the bottom and then grips upto the slides and produces a surface pellicle that may extend along the slides above the medium. In glycerol broth, they form a whitish winkled pellicle on the surface and a granular deposit at the bottom. Virulent strains usually form a long serpentine cords in the liquid media. The liquid media are not used for routine culture of the bacilli.

Some culture media used to cultivate M. tuberculosis:

• Agar based medium :Middlebrook 7H10 medium, Middlebrook 7 H11 medium, Middlebrook 7H10 7H11  medium (biphate medium)
• Egg based medium: Lowerstein Jensen (LJ)medium and Ogawa medium
• Blood based – Tarshis medium
• Liquid medium : Dubo’s solution, Middlebrook 7H9, Bactec 12B medium (worldwide use nowadays)

3, Biochemical test of M. tuberculosis

• Niacin: positive
• Catalase: positive
• Nitrate reduction: positive
• Urease: slightly positive
• Arylsulfatase test: positive
• Neutral red best: negative
• Amidase test: positive
• Pyrazinmidase test: positive
• Tween hydrolysis test: negative
• Growth on medium containing paranitrobenzoic acid- negative
• Rate of growth- slow grower
• Sensitive to 50 mg/ml pyrazininamide

4. Animal inoculation

• The concentrated material is inoculated intramuscularly into the thigh of two healthy guinea pigs, about 12 weeks.
• The animals are weighed before inoculation and at intervals thereafter. Progressive loss of wt. is an indication of infection.
• Infected animals show a positive tuberculin test. One of the animal is killed after four weeks and autopsied.
• Autopsy shows;
• Caseous lesions at the site of inoculation
• Enlarged caseous inguinal lymph node
• Tubercles may be seen in lungs
• Spleen-enlarged with irregular necrotic foci.
• Peritoneum may show few tubercles
• Kidney unaffected

5. Rapid and automated methods

• The conventional methods are very slow and time consuming and require 6-8 weeks of isolation. Hence, recently more rapid and automated methods are being used for diagnosis of TB.
• Those methods are:
• Modified cultural techniques
• Bactect 460 TB, rapid radiometric culture system
• Septi-check AFB system
• Mycobacterial growth indicator tubes (mGIT)
• ESP culture system
• Bacteriophage based test to detect M. tuberculosis in sputum.

Non-conventional method of identification

• Phenotypic method
• Technique based on lipid analysis
• Technique based on isoenzyme analysis and protein electrophoresis
• Technique based on immunological relatedness of enzymes

Genetic method

• DNA probes
• RNA based probes
• PCR etc.

7. Serodiagnosis

• Immunodiagnosis methods based on the detection of antibodies to various mycobacterial antigens have not come into widespread clinical use compared with other infectious disease.

8. Mantoux test ( tuberculin skin test):

• Mantoux test is a Tuberculin skin test used for the diagnosis of TB.
• In this test, 0.1 ml or 5 tuberculin units of PPD (purified protein derivative) is injected intradermally into the volar aspect of the forearm using a 27-G needle.
• PPD should be injected between the layers of the skin and not subcutaneously. The results is read after 48-72 hours.

Results :

• Positive: Induration of 10 mm or more at the site of infection surrounded by erythema. The positive test only confirms past infection by tubercle bacilli but does not present active stage of disease. Histological examination shows a dense infiltrate of mononuclear cells around capillaries and skin appendages.
• Negative: Induration of less than 5 mm
• Equivocal: Induration between 6-9 mm.
• False positive: observed in presence of atypical Mycobacteria.
• In case of negative test, with the PPD of 5TU, then the test may be repeated using PPD of 10 or 100 TU.

Treatment of tuberculosis:

• Anti-tuberculosis drugs destroys the tubercle bacilli, thereby preventing further complications of early primary disease and progression of disease.
• Anti-tuberculosis drugs are divided as-
• First line drugs:
  • Isoniazid, Rifampicin, Ethambutol, Streptomycin (injection) and Pyrazinamide
• Second line drugs:
  • Used for the cases of TB where first line drugs are ineffective.
  • Includes ciprofloxacin, cycloserine, ethionmide, kanamycin, ofloxacin, levofloxacin, capreomycin and others.

Mycobacterium tuberculosis: mode of transmission, pathogenesis, clinical diseases, lab diagnosis and treatment