Mycobacterium leprae: Introduction, Morphology, Pathogenicity, Lab Diag

Mycobacterium leprae: Introduction, Morphology, Pathogenicity, Lab Diagnosis, Treatment and Prevention

Introduction of Mycobacterium leprae

Mycobacterium leprae is the causative agent of a chronic infectious disease called Leprosy. Gerhard Armauer Hansen (1841-1912), who first discovered M. leprae in 1873, and leprosy is also known as “Hansen’s disease”. This bacterium has not been cultured so far in vitro even being the first bacterium to be identified as causing disease in humans. It belongs to the family Mycobatcteriaceae.

Classification of Mycobacterium leprae

Domain: Bacteria

Phylum: Actinobacteria

Class: Actinobacteria

Order: Actinomycetales

Suborder: Corynebacterineae

Family: Mycobacteriaceae

Genus: Mycobacterium

Species: M. leprae

Morphology of Mycobacterium leprae

Mycobaterium leprae is weak acid-fast, straight or slightly curved, 1-8 µm in length and 0.2-0.5 µm in width. The bacilli are seen singly or in groups, intracellularly or lying free outside the cells. The bacilli are bound together by a lipid-like substance called glia, which is present on the cell wall. The group of bacilli lying together are known as globi mass as shown above picture.

Additional properties of Mycobacterium leprae

Loss of acid fastness following pyridine extraction.
Ability to grow slowly in the footpad of mice.
Ability to oxidize 3,4 dihydroxyphenylalanine into pigmented product
Ability to invade peripheral nerve
Generation time: 12 days on average
Could not be cultured so far in-vitro

Pathogenicity of Mycobacterium leprae

It causes leprosy and the mechanism of leprosy completes in various steps as shown below-

Bacteria discharged from the nose
Inhaled by susceptible person
Taken up by alveolar marcophages
Disseminated through blood
Spread to nerve and skin
Organism proliferates especially in Schwann cells

Classification of Leprosy

Ridley-Jopling Classification
WHO classification

The World Health Organization (WHO) system distinguishes “paucibacillary” and “multibacillary” based upon the proliferation of bacteria (“pauci-” refers to a low quantity.)

Ridley-Jopling Classification of Leprosy (1966)

Indeterminate (I)
Tuberculoid (TT)
Borderline Tuberculoid (BT)
Mid Borderline (BB)
Borderline Lepromatous (BL)
Lepromatous (LL)

Most widely accepted
Based on clinical, bacteriological, immunological, and histopathological parameters, which divide leprosy into five recognizable groups
The various clinical manifestations are a result of variation in the host tissue response to lepra bacilli
The polar forms of tuberculoid leprosy (TT) and lepromatous leprosy (LL) correlate with good and poor immune status
Borderline forms (BB, BT, and BL) lie between these two poles and are immunologically unstable, tending to move towards one of the polar forms

WHO classification

Paucibacillary

Indeterminate – I
Tuberculoid – TT
Borderline Tuberculoid – BT

Multibacillary

Mid borderline – BB
Borderline Lepromatous – BL
Lepromatous – LL
All smear-positive cases

Suggestive signs of leprosy

Skin Patch: one or many, dry and with decreased or loss of hair
diffuse infiltration of the skin (red, thickened, shiny skin)
Skin nodules
Loss of sensation in the hands or feet
Weakness or paralysis of muscles supplied by a peripheral nerve (claw hand, foot drop, lagophthalmos)
Paraesthesia (tingling/burning sensation) in the face, hand, foot, or area of skin

3 Cardinal signs of Leprosy

Anesthesia of skin: Hypo-pigmented or Erythematous Skin Patch with loss of sensation
Enlargement of peripheral nerve
Presence of M. leprae in affected skin or nasal mucus

Laboratory Diagnosis of Leprosy

Specimen: It depends on the form of leprosy suspected by the treating physician; the preferred specimens may be. 1. Slit Skin smears from the earlobes, elbows, and knees2. Skin biopsy from edges of active patches3. Nerve biopsy from thickened nerves

Microscopy: Staining of Slit Skin Smear (SSS)

Ziehl-Neelsen (ZN) Method

Requirements for Staining

Staining Rack
Spirit lamp
Stain droppers
Stains:
1. 1% carbol fuchsin (freshly prepared)
2. 1% acid alcohol or 5% sulfuric acid
3. 1% methylene blue
Slide drying rack
Specimens: Four common sites for slit skin smears are -Right ear-lobe, Left ear-lobe, edge of active lesion (if no skin lesion right arm), and the edge of active lesion (if no skin lesion right thigh)

Procedure of Staining

Set staining rack over lab sink.
Align the slides on the staining rack.
Cover slides with 1% carbol fuchsin and heat gently for 15 minutes.
Wash slides with tap water.
Decolorize slides with 1% acid alcohol (HCl in 70 % Alcohol) for few seconds or 5% Sulphuric Acid solution for 10 seconds.
Wash slides once more with tap water.
Cover the slides with 1% methylene blue for 1 minute.
Wash slides once more with tap water.
Air dry slides on the drying rack.

Observation of Slit Skin Smear

Scan under 10X objective of a microscope.
Finally, observe under 100X objective (oil immersion).
Read slide systematically in a zigzag fashion.
Each smear is graded according to Ridley’s logarithmic scale as shown below.

Result Interpretaion of Slit Skin Smear

Grading: Ridley’s logarithmic scale

0 = Negative; no AFB in entire smear

1+ = 1-10 AFB in 100 microscopic field

2+ = 1-10 AFB in 10 microscopic field

3+ = 1-10 AFB in 1 average microscopic field

4+ = 10-100 AFB in 1 average field

5+ = 100-1000 AFB in on average field

6+ = >1000 AFB, AFB in 1 average field with clumps (globi)

Bacteriological Index (BI)

An estimation of the number of bacilli in the smear; semi-quantitative method (Ridley 1958)
BI is calculated by adding the index from the various sites and divided by the total number of sites examined
Maximum BI- site is also indicated inpatient report

Morphological Index (MI)

An estimation of solidly stained bacilli in a smear
Count 200 AFB of all morphology and note only solidly stained bacilli among them
- Expressed in Percentage.
- When bacilli are <200, expressed infraction
Characteristics of Solid bacilli:
- Uniformly stained organism
- Round ends
- Length is approximately 5 x width
- Parallel sides
MI can be used to assess the effectiveness of the treatment.
Fall of MI = sign of good response to treatment
Rise of MI= onset of drug-resistant of treatment

Culture: It is not cultivable either in artificial culture media or in tissue culture and therefore mouse footpad cultivation is common. Mycobacterium leprae suspected specimen is inoculated into a footpad of mice and kept at 20°C for 6-9 months. The nine-banded armadillo is also a natural host and reservoir of the Mycobacterium leprae.

Serology:

Antibodies to M. leprae demonstration specific PGL-1 antigens
Enzyme-linked immunosorbent assay (ELISA)
Latex agglutination test
The serology is a less effective diagnosis of the paucibacillary disease since serum antibodies are present in only 40–60% of such patients.
Fluorescent leprosy antibody absorption test (FLA-ABS): It is widely applicable to identify subclinical cases that detect M. leprae specific antibodies irrespective of duration and stage of the disease.

Molecular tests:

PCR is a highly sensitive assay than slit skin smear(microscopy) for Mycobacterium leprae detection.
It uses to identify DNA that encodes 65 kDa and 18 kDa M. leprae proteins and repetitive sequences in clinical specimens.

Miscellaneous Test

Skin test ( Lepromin test): It does not help in the diagnosis of diseases. It may give false-positive results.

Intradermal injection of Lepromin antigen can elicit the following reaction

Fernandez reaction ( after 48 hours)
Like Mantoux test

Interpretation

The patient might have infected with Leprosy bacillus some times in the past.
It can not be taken to indicate concurrent disease activity.
Mitsuda reaction ( after 3-4 weeks):
Associated with tuberculoid types of leprosy and indicates some degree of immunity.

Lepromin test is employed for the following purpose:

To classify the lesion of Leprosy patients. Lepramin test + ve in TT and -ve in LL type.
To assess prognosis and response of treatment. A +ve reaction after treatment indicates a good prognosis and -ve is a bad prognosis.

Conversion to lepromin test positivity during treatment is ign of the good response of treatment.

To assess the reaction to recruit the individuals to work with leprosy research. A negative one is susceptible.

Animal model is for leprosy research-

Mice (Swiss Albino Mice, nude mice)
Nine-banded armadillo

Applications of animal model

Testing of drug sensitivity
Assessing the value of new drug
Preparation of lepromin
Preparation of vaccine

Vaccine/s:

BCG: Different place different results

Uganda – good result

Burma- Bad result

Research underway:

Preparation of-

BCG + Killed purified M. leprae vaccine
Recombinant DNA vaccine

Treatment of Leprosy

The objective of treatment for the following purposes-

Early detection of the case
Provide appropriate treatment
Prevention of complications and disabilities

History of leprosy chemotherapy

1st medication with variable degree of response against Mycobacterium leprae: Chaulmogra oil
Cochrane & Mair: Dapsone monotherapy in 1940
Dapsone resistance recognized in the 1960 s
Rifampicin & clofazimine added in 1970 s
WHO recommended multidrug therapy (MDT) in 1982

Goals of MDT

The effective killing of Mycobacterium leprae in the shortest possible period
Prevention of the emergence of a resistant strain of M. leprae, in turn leading to the prevention of treatment failures and relapse

Advantages of MDT

Shorten the length of treatment
Reduce the problem of drug resistance
Decrease incidence & severity of reactions
Improve the patient compliance

Eligibility for MDT treatment

All newly diagnosed cases of leprosy
All those cases who did not complete treatment before (defaulters)
All relapse cases
Those cases who had taken monotherapy (Dapsone) treatment before

Anti-leprosy drugs are-

Dapsone
Rifampicin
Clofazimine
Moxifloxacin
Ofloxacin
Levofloxacin
Minocycline
Clarithromycin

MDT regimen for MB and PB leprosy is given below table.

Duration for MB Leprosy: 12 months (12 doses in 18 months)
Duration for PB Leprosy: 6 months (6 doses in 9 months)

Prevention of Leprosy

Following are the preventive steps in leprosy-

Health education
Vaccines (BCG has not sufficient protective effect in leprosy and thus new vaccine development is necessary.)
Chemoprophylaxis
Early diagnosis and treatment of leprosy may not only prevent deformities but also it will save a life.
Surveillance of contacts is very useful attempt to decrease the leprosy rate.

Keynotes

SSS has even been low sensitivity (10–50%, depending upon the expertise of laboratory workers), It is still the gold standard for all diagnostic techniques due to specificity of nearly 100%.
Molecular test, PCR assay has been increasingly used as an alternative for its diagnosis due to its higher sensitivity.
The sensitivity of the PCR is higher in the multibacillary patients due to have of a load of bacilli.
The difference between in SSS and Fite Faraco methods are as follow-SSS detects AFB in smear taken from dermis and Fite Faraco method detects AFB in tissue obtained by skin biopsy taken from clinically suspected lesions.
Four common sites for slit skin smears are -Right ear-lobeLeft ear-lobeEdge of active lesion (if no skin lesion right arm)Edge of the active lesion (if no skin lesion right thigh)
Follow up: Smear is always performed from previous smear site
If any new skin lesion also takes from the edge of the lesion.
SSS smear interpretation-Bacilli are red dots against a blue background whereas viable bacilli are seen as uniformly stained bacilli or solid bacilli having length 4 times greater than the breadth. Bacilli sides are parallel and the ends may be rounded, straight, or pointed. The dead bacilli ( Mycobacterium leprae) stain irregularly and appear as granular or fragmented. The bacilli may be seen singly, in small groups, or closely packed bunches called globi (as shown above image). Irregular blue-stained structures scattered among the bacilli are the cells of various structures in the skin.

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