Laboratory diagnosis of Leprosy caused by Mycobacterium leprae


Leprosy, also known as Hansen`s disease, is an infectious disease that damages the skin and nervous system. The condition can be cured with early diagnosis and treatment. Leprosy is caused by two types of bacteria: Mycobacterium leprae and Mycobacterium lepromatosis. The exact route of transmission is not known, but it is likely to spread through respiratory droplets of cough or sneezing of an infected person.

Leprosy can be classified into two groups based on the number of bacteria present in the skin lesions: paucibacillary (PB) and multibacillary (MB). PB leprosy has fewer than five skin lesions and no detectable bacteria in the skin smears. MB leprosy has more than five skin lesions and positive skin smears for bacteria. The classification of leprosy is important for determining the appropriate treatment regimen and the risk of complications.

The diagnosis of leprosy is based on the characteristic signs and symptoms of the disease, such as light-colored or red skin patches, reduced sensation of touch, numbness, weakness in the hands and feet, pain in the joints, disfiguring skin sores, weight loss, eye damage, hair loss, etc. The diagnosis is confirmed by laboratory tests that can identify the bacteria or their antigens in the specimens collected from the affected sites.

The laboratory diagnosis of leprosy involves the following methods:

  • Specimen collection: Skin biopsies, nasal discharges, scrapings from the nasal mucosa and slit-skin smears are collected from the patients. Skin smears are prepared by making superficial incisions in the skin, scraping out some tissue fluid and cells. Skin smears are collected from the leprous lesions, such as nodules, thick papules, and areas of infiltration. In cases of patches, the samples are obtained from the edge of the lesion rather than from the center. Nasal smears are collected by scraping material from the mucous membrane of the internal nasal septum. Skin and nerve biopsy are collected from active edge of the patches and thickened nerve for histological confirmation.
  • Microscopy: The specimens are stained by Ziehl-Neelsen technique using 5% sulfuric acid for decolorization. Under oil immersion objective, red acid-fast bacilli are seen, arranged singly or in groups (cigar like bundles), bound together by lipid-like substance, called glia to form globi. The globi are present inside the foamy macrophages called Virchow’s lepra cells or foamy cells. The presence of acid-fast bacilli confirms the diagnosis of leprosy. The bacillary index (BI) is an expression of the extent of bacterial load. It is calculated by counting six to eight stained smears under the oil immersion lens. The morphological index (MI) is an expression of the percentage of uniformly stained bacilli in the tissues that are believed to be viable.
  • Lepromin test: The lepromin test is used to study host immunity to M. leprae. The test is an intradermal skin test performed by using lepromin antigen, which is a suspension of killed M. leprae obtained from infected human or armadillo tissue. The test elicits two types of reaction: The Fernandez reaction that appears in sensitized subjects 48 hours after skin testing as a localized area of inflammation with congestion and edema measuring 10 mm and more in diameter; and the Mitsuda reaction that appears after 3–4 weeks after testing with lepromin as a nodule at the site of inoculation that may undergo necrosis followed by ulceration. The Fernandez reaction indicates past infection with M. leprae while the Mitsuda reaction indicates host ability to give a granulomatous response to antigens of M. leprae.
  • Mouse Foot Pad Cultivation: M. leprae is not cultivable either in artificial culture media or in tissue culture. The only certain way to cultivate M. leprae is by inoculating the specimens into foot pad of mice and keeping at 20°C for 6-9 months. Other animals such as nine banded armadillo can also be used which is a natural host and reservoir of the pathogen.
  • Serodiagnosis: Serodiagnosis of leprosy is based on detection of antibodies to M. leprae specific PGL-1 antigens. Enzyme linked immunosorbent assay (ELISA) and latex agglutination test are used to detect serum antibodies. The serology is useful primarily in patients with untreated lepromatous leprosy, as most of patients have higher levels of serum antibodies. The serology, however, is less useful for diagnosis of paucibacillary disease, because serum antibodies are present in only 40–60% of such patients.
  • Molecular Diagnosis: Polymerase chain reaction (PCR) for identifying DNA that encodes 65 kDa and 18 kDa M. leprae proteins and repetitive sequences of M. leprae is used to detect and identify M. leprae in clinical specimens. PCR is used to monitor treatment, diagnose relapses, or determine the need for chemotherapy. The technique is most useful in cases of leprosy showing atypical clinical or histopathological features but positive for acid-fast bacilli. It is not useful for diagnosis of cases when acid-fast bacilli are not detected by light microscopy.