Leprosy: Etiology, Pathogenesis, Lab Diagnosis

Leprosy is an age-old disease, associated with social stigma; historical records and literature show that people afflicted with leprosy have often been ostracized by their communities and families. Also known as Hansen’s disease, leprosy is a chronic infectious disease caused by an acid-fast bacillus, Mycobacterium leprae.

A man with leprosy (image by J. L. Losting.)

In 1873, Dr. Hansen discovered bacteria in leprosy lesions, which rule out that leprosy is a hereditary disease not a punishment from the gods. Leprosy mainly affects the skin, peripheral nerves, and mucosa of the upper respiratory tract (because their optimal temperature for growth is 30°C). It may affect many organs.

Features of M. Leprae and Leprosy

  1. Leprosy is not a highly infectious /very contagious infection.
  2. Infection is acquired by prolonged contact with patients with lepromatous leprosy (heavy shedders) who discharge M. leprae in large numbers in nasal secretions and from skin lesions.
  3. Route of Transmission: Skin and inhalation.
  4. M. leprae multiplies very slowly (with a doubling time of 14 days; slowest growing human bacterial pathogen).
    # Remember: antibiotic therapy must be continued for a long time, usually 1-2 years.
  5. Incubation period of the disease is about five years. Symptoms can take as long as 20 years to appear.
  6. Leprosy is curable with multidrug therapy (MDT). Three antibiotics (dapsone, rifampicin, and clofazimine) in combination are used for 6 months to 1 year based on the type of leprosy (paucibacillary or multibacillary).

Pathogenesis

M. leprae replicates intracellularly, typically within skin histiocytes, endothelial cells, and the schwann cells of nerves. Cell-mediated immunity (CMI) plays a major part in determining the response of the host to leprosy.

Pathogenesis of Leprosy

There are two distinct forms of leprosy-tuberculoid and lepromatous with several intermediate forms between the two extremes

  1. Tuberculoid leprosy: very few acid-fast bacilli in skin smear (paucibacillary disease): Cell-mediated immune (CMI) response is adequate and the lepromin test is positive.
  2. Lepromatous leprosy: large numbers of Mycobacterium leprae chiefly in masses within the lepra cells, often grouped together like bundles of cigars or arranged in a palisade (multibacillary disease). The cell-mediated immune  (CMI) response to the organism is poor and the lepromin test is negative.

Ridley and Jopling Classification

Ridley and Jopling (1966) have introduced a scale for classifying the spectrum of leprosy into five groups:

  1. Tuberculoid (TT)
  2. Borderline Tuberculoid (BT)
  3. Borderline (BB)
  4. Borderline Lepromatous (BL)
  5. Lepromatous (LL) 

WHO Classification of Leprosy

According to World Health Organization (WHO), leprosy is divided into two groups, paucibacillary and multibacillary.

Comparison of tuberculoid and  lepromatous leprosy

Feature
Tuberculoid
Lepromatous
Type of lesion
One or few lesions with little tissue destruction
Many lesions with marked tissue destruction
Number of acid-fast bacilli
Few
Many
Likelihood of transmission
Low
 High
Present
Reduced or latent
Lepromin skin test
Positive
Negative

Laboratory diagnosis

Sample

M. leprae in stained smear
M. leprae in stained smear

Ideally, at least six sites can be sampled, including earlobes, eyebrows, elbow, knees, nasal mucosa, and skin lesions. Skin biopsy from edges of active patches and nerve biopsy from thickened nerves can also be collected.

Taking a skin smear for the diagnosis of Leprosy

Traditionally smears used to be taken from four or even six sites, but two sites are now considered adequate in most cases. Skin smear is taken from one ear lobe and one lesion. Smear should be taken from the edges of the most active lesions (active lesions are raised and reddish in color). If there is no suitable skin lesion, a second smear can be taken from another ear lobe.

To take a skin smear, the following equipment will be needed

Equipment needed to take skin smear
  • Gloves
  • Swab and spirit
  • Scalpel handle and new blades
  • Dressing strips
  • Safe disposal for used blades
  • Spirit lamp
  • Slide box and new slides

Procedure

  • Wash your hands (1) and put on gloves.
  • Take a new, clean, unscratched microscope slide. Using a slide marker, write the patient identification (ID) number at the bottom of the slide (2). This number must be on the request form.
  • Clean the skin at the smear sites with a cotton wad drenched in alcohol. Let it dry.
  • Light the spirit burner.
  • Put a new blade on the scalpel handle. If you put the scalpel down, make sure the blade does not touch anything.
  • Pinch the skin firmly between your thumb and forefinger; maintain pressure to press out the blood.
  • Make an incision in the skin about 5 mm long and 2 mm deep (3). Keep on pinching to make sure the cut remains bloodless. If bleeding, wipe the blood away with cotton wad.
  • Turn the scalpel 90° and hold it at a right angle to the cut.
  • Scrape inside the cut once or twice with the side of the scalpel, to collect tissue fluid and pulp. There should be no blood in the specimen, as this may interfere with staining and reading.
  • Stop pinching the skin and absorb any bleeding with a wad of cotton. Spread the material scraped from the incision onto the slide, on the same side as the ID number. Spread it evenly with the flat of the scalpel, making a circle 8 mm in diameter (4).
  • Rub the scalpel with a cotton wad drenched in alcohol. Pass the blade through the flame of the spirit burner for 3 to 4 seconds. Let it cool without touching anything. Repeat the steps above for the second site. Spread this smear next to, but not touching, the first one.
  • Discard the scalpel blade safely. Dress the wounds and thank the patient.
  • Let the slide dry for 15 minutes at room temperature, but not in direct sunlight.
  • Fix the smears by passing the slide, with the smears upwards, slowly through the flame of a spirit burner, 3 times (5). Do not overheat. The slide should not be too hot to touch.
  • Put the slide in a slide box and send it to the laboratory with the skin smear request form.

Microscopy 

Slit-skin smear is stained with a modified Ziehl Neelsen stain (5% sulphuric acid or 1% v/v acid alcohol is used as a decolorizing agent). Find details about Ziehl-Neelsen stain procedure here. Stained slides are observed under a microscope (100x objective lens using immersion oil).

M. leprae is a slightly curved filament 3-10 m in length containing irregular arrangements of dense material sometimes in the shape of rods. Acid-fast bacilli appear as fine red rods against a blue background. They can be straight or curved, and the red color can be uniformly distributed (solid bacilli) or unevenly distributed (fragmented and granulated bacilli). Clumps of bacilli are called globi. Solid bacilli may suggest the presence of viable organisms and may be seen in new, untreated cases or in relapse cases. Bacilli which stain irregularly are probably dead and degenerating.

AFB (M. leprae) in skin smear
AFB in skin smear
  • In lepromatous leprosy: lipid-laden macrophages called ‘foam cells” containing many acid-fast bacilli are seen in the skin.
  • In tuberculoid leprosy: Very few acid-fast bacilli are seen and the appearance of typical granulomas is sufficient for diagnosis.

Based on the number of M. lepare and their morphology in the stained slides Bacteriological index (BI) and morphological index (MI) can be calculated. Bacteriological index (BI) is an expression of the extent of bacterial loads whereas morphological index (MI) is calculated by counting the numbers of solid-staining acid-fast rods (viable during sample collection).

BI and MI are useful in assessing the amount of infection, the viability of the organisms, and also the progress of the patient under treatment.

GradingBacteriological Index (BI)
0Absence of AFB in 100 fields
1+1-10 AFB/100 fields
2+1-10 AFB/10 fields
3+1-10 AFB/field, on average, in each field
4+10-100 AFB, on average, in each field
5+100-1000 AFB, on average, in each field
6+>1000 AFB, on average, in each field

According to WHO, a more accurate and reliable index of the bacillary content of a lesion is given by the logarithmic index of biopsies (LIB). These indices help to assess the state of patients at the beginning of the treatment and to assess progress.

Patients with lepromatous leprosy may give false-positive results in the nonspecific serologic tests for syphilis, such as VDRL and RPR.

Nine banded armadillo
Nine-banded armadillo

Culture

M. leprae has not yet been successfully cultured in vitro (either on artificial media or in cell culture) but it can be grown in the laboratory by injection into the footpads of mice or nine-banded armadillo. It is a slow-growing pathogen with a doubling time of 14 days.

Serology

A serologic test for IgM against phenolic glycolipid-1 is useful in the diagnosis of lepromatous leprosy but not useful in the diagnosis of tuberculoid leprosy.

Molecular diagnosis

Polymerase Chain Reaction (PCR) can be used as a means of diagnosis of leprosy and also as a tool for drug assessment.

Lepromin Skin Test

The lepromin skin test is not used to diagnose leprosy but to determine what type of leprosy a person has. Lepromin skin test is similar to tuberculin test.  An extract of M.leprae is injected intradermally and induration is observed 48 hours later in those in whom a cell-mediated immune response against the organism exists.

 The lepromin test is employed mostly for the following two purposes.

1.       To classify the lesions of leprosy patients.

2.       To assess the prognosis and response to treatment.

Acharya Tankeshwar

Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. As an asst. professor, I am teaching microbiology and immunology to medical and nursing students at PAHS, Nepal. I have been working as a microbiologist at Patan hospital for more than 10 years.

2 thoughts on “Leprosy: Etiology, Pathogenesis, Lab Diagnosis

  1. WHO DISCOVERED THAT INJECTION INTO THE ARMADILLO FOOT PAD COULD REPLICATE AN INFECTION WITH ASSOCIATED NEUROPATHY? WHY EVEN MENTION THE MICE SINCE THEY DONOT DEVELOP A DISEASE STATE WHEN INJECTED.?

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