Mycobacterium leprae For MBBS (05-12-2017) By: Dr Puneet Kumar Gupta Assistant Professor, Microbiology

Disease of Historical importance World's oldest recorded disease Stigmatized disease Gerhard Henrick Armauer Hansen (1873-Norway)

The Bacterium

Armauer Hansen in 1868 Morphology : Straight rods. 1 - 8 x 0.2 - 0.5µm Single / groups. Intracellular. Acid fast bacilli with 5% H2 SO4. As agglomerates, bacilli being bound together by a lipid like substance (Glia) – called GLOBI

Parallel rows of bacilli in globi: CIGAR BUNDLE appearance – as in tissue section clumps of bacilli resemble cigarette ends GLOBI is seen in Virchow’s lepra cell or foamy cells (Large undifferentiated histiocytes)

Cultivation No artificial media / tissue culture available. Generation time: 12-13 days Mouse : Intradermally into Foot pads. Granulomatous lesions in 1- 6 months. Intact CMI : Limited replication. ↓CMI : Generalized leprosy. Armadillo: Highly susceptible. Chimpanzees, Mangabey monkey.

Important Experimental Animal

Most Important experimental Animal

Adaptation in artificial media: ICRC, Bombay 1962 Adaptation in artificial media: ICRC, Bombay 1962. – AFB from leprosy patients were isolated in human fetal spinal ganglion cell culture= ICRC bacillus (LJ adapted)

Warm humid environment 9 - 16 days. 46 days in Moist soil Warm humid environment 9 - 16 days. 46 days in Moist soil 2 hours in Sunlight 30 minutes U V rays Resistance

LEPROSY

Lepers are outcasts ?

Epidemiology Exclusively Human disease & only source is a patient Exact mode of transmission – not clear; probably via -Nasal secretions. (One nasal blow may release 8 x 108 bacilli) Entry via – respiratory tract or skin Asymptomatic infection not uncommon

Not very communicable – 5% spouses suffer Incubation period is 3-5 years. (2to 40 yrs) Continuous close contact. Rare in children < 5 Years. Confined to underdeveloped tropical countries & southern hemisphere currently India Prevalence 0.68/10000 population in 2012 32 states/UT achieved target of elimination Chhattisgarh, Dadar & Nagar Haveli Prevalence >1/10000

Annual Report 2015-16 from NLEP GOI (as on 1st April 2016) >1/10,000 Population (163 District out of 669) Total cases 86028

Annual Report 2015-16 from NLEP GOI >1-Chattisgarh, Dadar Nagar Haveli, Delhi, Odisha, Chandigarh, Lakshadeep

Classification of leprosy Table 27.10

IV. WHO classification. Based on bacterial load. 1. Paucibacillary IV. WHO classification Based on bacterial load. 1. Paucibacillary I, T T, BT 2. Multibacillary BB, BL, LL. Table 27.13 add

Clinically………………

Leprosy Slow, chronic & progressive Granulomatous disease of Peripheral nerves,skin and Muco- cutaneous tissues (Nasal mucosa). It affects Skin, liver, testes ,bones.

Source : Nasal or Skin Pathogenesis discharges from lesion. Portal of entry: Damaged skin -Inoculation. Nasal mucosa- Inhalation

Pathogenesis contd….: Infiltration of bacilli in cooler body tissues like skin (nose, outer ear), testicles & superficial nerve endings→ (maculae) visible lesions. A non-specific or Indeterminate skin lesion is the First sign of disease. Schwann cell is target cell. Neuritis leads to Anesthesia & muscle paralysis.

CMI severely depressed High infectivity Tuberculoid leprosy Lepromatous leprosy Extensive maculae, papules or nodules; destruction of skin. CMI severely depressed High infectivity Tuberculoid leprosy Lesions are large maculae on skin, superficial nerve endings. CMI is intact. Low infectivity Regression Progression

Lepromatous leprosy Generalized form with decreased CMI. “Lepromata” : Granulation tissue with plenty of vacuolated cells, from MN cells to Lepra cells. Ulceration Secondary infection & Mutilation of limbs. Skin lesions are extensive and bilaterally symmetrical.

Sites: Commonly face, ear lobules, hands and feet. Symmetrical thickening of peripheral nerves & anesthesia Bacilli invade mucosa of Nose , Mouth and Respiratory tract → shed in secretions. Bacteremia present. RE system, Eyes, testes, kidney & bone involved

Antibodies / other Abs are seen (exaggerated humoral response) Lepromin test is negative. CD8+ cells in plenty Antibodies / other Abs are seen (exaggerated humoral response) BFP= syphilis tests (STS) Infective form….more than other types – poor prognosis Lateral part of eyebrows are lost

Lepromatous leprosy Lepromatous leprosy

Complications : Acute exacerbations. Testicular atrophy, Gynaecomastia Diffuse thickening of face – (Leonine face). Necrosis of nasal bones, cartilage with loss of upper incisors. Corneal ulcers.

Localized form in individuals with intact CMI Localized form in individuals with intact CMI. Skin lesions : Few hypo or hyper pigmented macular patches (anesthetic) Sharply demarcated Seen on Face, trunk and limbs. Bacilli are scanty or absent. (paucibacillary) Infectivity is low. Tuberculoid leprosy

Diagnosed with Clinical + Histological evidences Diagnosed with Clinical + Histological evidences. Nerves : Peripheral Nerves to bigger nerves involved. Thickened, hard and tender. Deformities in hand & feet Lepromin test is positive. Auto antibodies production is rare. CD4+ cells.

Good prognosis

Peripheral neuropathy. V & VII th cranial nerve : Corneal ulcers Peripheral neuropathy. V & VII th cranial nerve : Corneal ulcers. Ulnar nerve : Claw hand. Lateral popliteal nerve : Foot drop. Posterior tibial & medial nerve: Trophic ulcers, Loss of digits. Complications

Dimorphous/Borderline type : Dimorphous/Borderline type : Lesions resembles both LL (bacteriology) & T T (Clinically). May turn to complete LL or T T type (depending on host resistance or chemo therpay)

Cirular, sharply demarcated Borderline lepromatous Borderline tuberculoid leprosy Lesions are Slightly asymmetrical with or without anesthesia. Cirular, sharply demarcated lesions. Raised erythematous border with anesthesia.

Indeterminate type:. Early stages : Maculoanesthetic patches Indeterminate type: Early stages : Maculoanesthetic patches. Lesions are not like T T or LL Spontaneous healing. Turn to either LL or T T type.

Indeterminate type

Immunity : High degree of innate immunity. Induces both AMI & CMI Immunity : High degree of innate immunity. Induces both AMI & CMI. Antibodies are not effective. LL Pts : Large number of CD8 cells. TT Pts : Predominantly CD4 cells. Genetic relation: T T : HLA – DR2 L L : HLA MTI, DQ1

Differential diagnosis of Leprosy Birth mark T. versicolor T.corporis

Pytiriasis alba Vitiligo Lichen planus

Fixed drug eruption Psoriasis Dermal leishmanoid Lupus vulgaris

Kaposi’s sarcoma Sarcoidosis

Lepra reactions: Acute inflammation of the disease due to Immunological reactions against bacilli. Medical emergency. Two types:

Jopling type 1: CMI response against bacilli Synonym: Reversal reaction Occurrence: Spontaneous, Chemotherapy Seen in BT, BB, BL. Due to influx of lymphocytes into lesions changed to T T morphology Lesions are painful, tender Erythema and swelling.

Jopling type 2 Synonym: Erythema Nodosum Leprosum (ENL) Due to vasculitis (Antigen – Antibody complex). Seen in LL & BL few months after starting the chemotherapy

Characterised by: Tender, inflamed subcutaneous nodules Fever Lymphadenopathy, arthralgia. (Ag from dead bacilli – Arthus type response) IgG, neutrophil & C in lesions

Lucio phenomenon: Cutaneous hemorrhagic infarct in LL cases.

Main features of lepra reactions. Type 1 Type 2 1.Immunological basis : CMI Vasculitis with Ag – Ab deposits. 2. Type of patient : BT,BB, BL BL, LL. 3. Systemic disturbances : Not seen . Present. 4. Hematological Not present Present disturbances: 5. T Helper response TH1 predominate TH2 6. Proteinuria Not seen. Frequently present. 6.Relation to therapy Seen in first Rare in first 6 months. 6 months

Lab diagnosis Microscopy Culture Serology (Ab detection) Molecular method Demonstration of CMI

Lab diagnosis Bacteriological Diagnosis is easy in LL types but difficult in TT. Specimen: smears collected from Nasal mucosa, Lesion, Skin (slit smear of ear lobule, forehead, Cheek, Chin, buttock) = 5-6 sites Sample from thickened nerve or Nodular lesion are collected for H/P

Nasal mucosa (nasal Blow or Nasal Scrapping) A blunt, narrow scalpel is introduced into the nose and internal septum Scraped sufficiently to remove a piece of mucous membrane, which is transferred to a slide and teased out into a uniform smear.

Taken from Edge of lesion Slit Skin smear: Taken from Edge of lesion Skin is pinched up tight to minimize bleeding a cut about 5 mm long made with a scalpel, deep enough to get into the infiltrated layers. Wipe off blood or lymph that may have exuded Scalpel blade is turned transversely to scrape the sides and bottom of the cut for tissue pulp smeared uniformly on a slide. About 5-6 different areas of the skin buttocks, forehead, chin, cheek and ears. Collection of skin smears

Smears are stained by the Ziehl-Neelsen technique using 5% sulphuric acid. Biopsy Nodular lesions and thickened nerves, and lymph node puncture may be necessary

Z-N staining: Decolorizer =5% H2SO4 Acid fast bacilli within the undifferentiated macrophages: L L Live bacilli : Solid, uniformly stained, parallel side, rounded ends, length five times width Dead bacilli :Fragmented and granular

Grading 1-10 / 100 oil immersion fields : 1+ 1 -10/10 “ “ : 2+ 1 -10/10 “ “ : 2+ 1 -10 / 1 “ “ : 3+ 10-100/ field : 4+ 100-1000 /field : 5+ >1000, clumps/globi in every field: 6+

Load of bacilli: 1. Bacteriological index (BI): total no of pluses (+) scored in all the smears divided by no of smears; Minimum 4 skin lesion, nasal scrap & both ear lobule must 2. Morphological index(% of uniformly stained bacilli) = Uniformly stained bacilli X 100 Total number of bacilli (= SFGB/Total x100)

Skin & Nerve biopsy.

Lymphohistiocytic infiltrate surrounding a nerve fiber

Culture: No Culture media Animal inoculation: Advantage: Disadvantage: Mouse foot pad: 9 banded Armadillo Advantage: 10 times more sensitive Drug resistance detection Evaluation of drug potency Check Viability Disadvantage: Time consuming (6-9 months) Ethical issue: animals used

LESIONS DEVELOPING FOLLOWING INOCULATION IN FOOT PAD OF MICE

Serological test : Antibodies against phenolic glycolipid Ag -ELISA (Antibody against PGL-I) Sensitivity LL: 95% TT: 60% -FLA-Abs-Fluorescent leprosy Ab Absorption Test Detect specific Ab irrespective of duration and stage of disease 92% sensitive & 100% specific Molecular diagnosis:

Detection of CMI (Lepromin test) : Detection of CMI (Lepromin test) : Skin test for delayed hypersensitivity to lepra bacilli. Antigens: 1. Boiled extract of Lepromatous tissue in isotonic saline. 2. Leprosins : Ultrasonicates of tissue – free bacilli from lesions. a). leprosins – H b). leprosins – A 3.Dharmender’s antigen. 4.Soluble antigen.

Two types of reactions on Intradermal injection 1 Two types of reactions on Intradermal injection 1. Early reaction of Fernandez : Erythema & Induration within 1 - 2 days Remains for 3 - 5 days. Poorly defined with little significance. 2. Late reaction of Mistuda. Erythematous, indurated , granulomatous nodular skin lesion. Seen in 1 - 2 weeks reaches to peak in 4 weeks. Indicates CMI status in leprosy patients.

Significance : 1. To classify the lesions of leprosy Significance : 1. To classify the lesions of leprosy. T T ( + ) L L ( - ) Borderline (+/-) 2. To assess prognosis & response to treatment. Positive: Good prognosis Negative: Bad prognosis 3. To assess the resistance of individuals to leprosy. 4. Identify candidate lepra bacilli

Treatment : Until 1982 : Dapsone only. Now MDT being given because of resistant strains. WHO recommended Multi drug therapy Paucibacillary case. (I, TT, BT) Rifampicin 600 mg/ month annually Dapsone 100mg / day till 2year

Multi bacillary case: (BB, BL, LL) Rifampicin 600mg / month Dapsone 100 mg / day annually Clofazimine 300 mg / month till 5 years + 50 mg / day (Ethionamide/prothionamide)

Vaccines: BCG, MAI complex vaccine. Mycobacterium w vaccine. Chemoprophylaxis: Dapsone ; in TT variety only

THANKS