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.

To confirm the diagnosis of leprosy and classify the disease, different types of specimens may be collected from the patient depending on the clinical presentation and the availability of laboratory facilities. The most common specimens are:

• Skin biopsies: These are small pieces of skin tissue that are taken from the edges of active lesions, such as patches, nodules, or papules. Skin biopsies are used for histopathological examination and molecular diagnosis of leprosy. They can also be used for culturing M. leprae in animal models, such as mouse foot pads or armadillos .
• Nasal discharges: These are samples of mucus or fluid that are obtained from the nose of the patient by gently blowing or swabbing. Nasal discharges are used for microscopy and molecular diagnosis of leprosy, as they may contain M. leprae bacilli, especially in multibacillary cases .
• Scrapings from the nasal mucosa: These are samples of cells and tissue that are scraped from the inner lining of the nose, particularly from the inferior turbinate folds of the nasal septum. Scrapings from the nasal mucosa are used for microscopy and molecular diagnosis of leprosy, as they may also contain M. leprae bacilli .
• Slit-skin smears: These are samples of tissue fluid and cells that are prepared by making superficial incisions in the skin and scraping out some material. Slit-skin smears are usually collected from the earlobes, elbows, and knees, as these are sites where M. leprae tend to accumulate. Slit-skin smears are used for microscopy and calculation of bacillary and morphological indices .

The specimens should be collected by trained personnel using sterile instruments and following standard precautions to prevent infection and contamination. The specimens should be labeled properly and transported to the laboratory as soon as possible.

One of the most common and reliable methods for laboratory diagnosis of leprosy is microscopy. Microscopy involves examining stained smears of specimens under a microscope to detect and identify acid-fast bacilli (AFB), which are characteristic of Mycobacterium leprae, the causative agent of leprosy.

The most widely used staining technique for AFB is the Ziehl-Neelsen (ZN) stain, which was first introduced by Paul Ehrlich and later modified by Franz Ziehl and Friedrich Neelsen. The ZN stain uses carbol fuchsin, a basic dye, to stain the AFB red, and acid alcohol, a decolorizing agent, to remove the dye from non-AFB. A counterstain, such as methylene blue, is then applied to stain the background and non-AFB blue.

The ZN stain exploits the acid-fastness of AFB, which means that they retain the dye even after exposure to acid alcohol. This property is due to the presence of mycolic acids, long-chain fatty acids that form a waxy layer on the cell wall of AFB. The mycolic acids make the cell wall impermeable to most stains and solvents, but also allow the dye to penetrate and bind strongly when heat or phenol is applied.

The ZN stain can be performed on various types of specimens collected from leprosy patients, such as skin biopsies, nasal discharges, scrapings from the nasal mucosa and slit-skin smears. Slit-skin smears are prepared by making superficial incisions in the skin and scraping out some tissue fluid and cells. The smears are collected from different sites depending on the type and extent of leprosy lesions. For example, in cases of patches, the samples are obtained from the edge of the lesion rather than from the center.

The stained smears are examined under an oil immersion objective (100x magnification) for the presence and number of AFB. The AFB appear as red rods, either single or in groups, against a blue background. The AFB may also form globi, which are clusters of bacilli bound together by a lipid-like substance called glia. The globi are often found inside foamy macrophages called Virchow`s lepra cells or foamy cells.

To quantify the bacterial load in the smears, two indices are calculated: the bacillary index (BI) and the morphological index (MI). The BI is an expression of the number of AFB per microscopic field. It is obtained by counting six to eight stained smears under the oil immersion lens and totaling the number of pluses (+). Each plus corresponds to a range of AFB per field as follows:

The MI is an expression of the percentage of viable AFB in the smears. It is based on the assumption that uniformly stained bacilli with carbol fuchsin are alive, while irregularly stained bacilli are dead or degenerated. The MI is obtained by dividing the number of solid-staining bacilli by the total number of bacilli counted in at least 100 fields and multiplying by 100.

The BI and MI are useful for classifying leprosy patients into paucibacillary (PB) or multibacillary (MB) forms, monitoring treatment response and detecting relapse or drug resistance. Generally, PB patients have a BI of less than 2+ and a high MI (>50%), while MB patients have a BI of more than 2+ and a low MI (<50%).

The lepromin test is a skin test that is used to study the host immunity to M. leprae, the bacterium that causes leprosy. The test is not used to diagnose leprosy, but to determine the type and severity of the disease. The test involves injecting a small amount of killed M. leprae under the skin and observing the reaction after 48 hours and 21 days.

The lepromin test can elicit two types of reactions:

• The Fernandez reaction is a delayed-type hypersensitivity reaction that appears within 48 hours of the injection. It is characterized by a localized area of inflammation, swelling, and redness measuring 10 mm or more in diameter. This reaction indicates that the person has been exposed to M. leprae in the past and has developed cellular immunity against it. The Fernandez reaction is usually positive in people with tuberculoid or borderline tuberculoid leprosy, which are milder forms of the disease with fewer bacteria and less nerve damage.
• The Mitsuda reaction is a granulomatous reaction that appears after 21 days of the injection. It is characterized by a nodule or an ulcer at the injection site. This reaction indicates that the person has a strong cellular immunity against M. leprae and can mount a granulomatous response to contain the infection. The Mitsuda reaction is usually positive in people with tuberculoid or borderline tuberculoid leprosy, and negative in people with lepromatous or borderline lepromatous leprosy, which are more severe forms of the disease with more bacteria and more nerve damage.

The lepromin test can help to classify the type of leprosy and guide the treatment and prognosis of the disease. However, it has some limitations, such as:

• It is not useful for diagnosing leprosy, as it does not detect active infection or indicate prior contact with M. leprae.
• It is not reliable for detecting indeterminate or early cases of leprosy, as the immune response may not be fully developed yet.
• It may give false negative results in people with immunosuppression, malnutrition, or other infections that impair cellular immunity.
• It may give false positive results in people who have been vaccinated with bacillus Calmette-Guérin (BCG) or exposed to other environmental mycobacteria that cross-react with M. leprae.
• It may cause adverse reactions such as pain, itching, scarring, or infection at the injection site.

Therefore, the lepromin test should be interpreted with caution and in conjunction with other clinical and laboratory findings.

M. leprae is not cultivable either in artificial culture media or in tissue culture systems. 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.

The mouse foot pad technique was first described by Shepard in 1960 and it represents the first truly useful and reproducible animal model of M. leprae infection. Its employment has enabled research into basic questions with respect to the microbiology of M. leprae, and the epidemiology, treatment and control of leprosy.

The technique involves making a small incision on the plantar surface of the hind foot of a mouse and injecting about 0.03 ml of a suspension of M. leprae obtained from human or armadillo tissue. The inoculated mice are housed in cages with wire mesh floors to prevent ulceration of the foot pads. The foot pads are examined periodically for swelling and harvested at various intervals for counting the bacilli by microscopy.

The mouse foot pad technique is useful for several applications, such as:

• Determining the viability of M. leprae isolated from patients or other sources
• Testing the susceptibility of M. leprae to various drugs or drug combinations
• Studying the immunological response of mice to M. leprae infection
• Comparing the virulence and infectivity of different strains of M. leprae
• Producing large quantities of M. leprae for biochemical and molecular studies

However, the mouse foot pad technique also has some limitations and drawbacks, such as:

• It is labor-intensive and time-consuming, and requires a large number of animals
• It is expensive in terms of the costs of animal purchase and maintenance
• It is imprecise and insensitive, compared with the techniques employed in working with cultivable micro-organisms
• It may be contaminated by other acid-fast nocardioform bacteria that can grow in mouse foot pads
• It may not reflect the true situation of human leprosy, as mice do not develop leprosy-like lesions or immune reactions

Therefore, until a more simple and sensitive technique for demonstrating the viability of M. leprae is developed, the mouse foot pad system remains an essential tool for leprosy research, but it also needs to be complemented by other methods such as serology and molecular diagnosis.

Serodiagnosis of leprosy is based on the detection of antibodies to M. leprae-specific antigens in the serum of patients. These antibodies are mainly of the IgM and IgG classes, and their levels correlate with the bacterial load and the clinical spectrum of the disease. Serodiagnosis can be useful for screening, monitoring, and prognosis of leprosy, especially in multibacillary cases.

Several methods have been developed to measure serum antibodies to M. leprae antigens, such as enzyme-linked immunosorbent assay (ELISA), latex agglutination test, Mycobacterium leprae particle agglutination (MLPA) test, and fluorescent leprosy antibody absorption (FLA-ABS) test . These methods use different antigens derived from M. leprae, such as whole sonicate, purified protein derivatives, recombinant proteins, or synthetic peptides.

One of the most widely used antigens for serodiagnosis of leprosy is the phenolic glycolipid-I (PGL-I), which is a unique and specific component of the cell wall of M. leprae. PGL-I can be detected by ELISA or by a simple dipstick test that can be performed in the field. PGL-I antibodies are more prevalent and higher in lepromatous patients than in tuberculoid patients, and they decrease after effective treatment.

Another antigen that has been used for serodiagnosis of leprosy is the 35-kDa protein, which is a major secretory protein of M. leprae. This antigen can be detected by ELISA using a monoclonal antibody (MLO4) that recognizes a specific epitope on the 35-kDa protein. The 35-kDa protein antibodies are also more common and higher in lepromatous patients than in tuberculoid patients, and they show a good correlation with the bacillary index.

Serodiagnosis of leprosy has some limitations, such as cross-reactivity with other mycobacteria or non-mycobacterial antigens, variability in antibody levels among individuals or populations, and lack of sensitivity in paucibacillary cases . Therefore, serodiagnosis should not be used as a sole criterion for diagnosis of leprosy, but rather as an adjunct to clinical and microbiological methods. Serodiagnosis can also provide valuable information for epidemiological surveillance, assessment of treatment efficacy, and identification of relapse or reinfection cases .

Polymerase chain reaction (PCR) is a technique that amplifies specific DNA sequences from a sample. PCR can be used to detect and identify M. leprae DNA in various clinical specimens, such as skin biopsies, nasal swabs, blood, urine, and saliva. PCR has several advantages over conventional microscopy and culture methods for leprosy diagnosis:

• PCR is more sensitive and specific, meaning it can detect low numbers of M. leprae and distinguish them from other bacteria.
• PCR can be performed on different types of samples, including those that are difficult to obtain or process by other methods.
• PCR can provide information on the viability, strain type, and drug resistance of M. leprae.

There are different types of PCR techniques that have been used for leprosy diagnosis, such as conventional PCR, quantitative PCR (qPCR), real-time PCR (RT-PCR), nested PCR, multiplex PCR, and loop-mediated isothermal amplification (LAMP). These techniques vary in their speed, accuracy, cost, and equipment requirements. Some of the most commonly used PCR targets for leprosy diagnosis are:

• RLEP: A repetitive element present in multiple copies in the M. leprae genome. It is highly specific and sensitive for M. leprae detection.
• 16S rRNA: A gene that encodes a component of the bacterial ribosome. It is conserved among bacteria but has some variations that allow identification of different species.
• 65 kDa and 18 kDa: Genes that encode heat-shock proteins of M. leprae. They are immunogenic and can elicit antibody responses in infected patients.

PCR can be useful for diagnosing leprosy in cases where clinical or histopathological features are atypical or inconclusive, or where acid-fast bacilli are not detected by microscopy. PCR can also be used to monitor treatment response, diagnose relapse, or determine the need for chemotherapy. However, PCR also has some limitations and challenges for leprosy diagnosis:

• PCR requires specialized equipment and trained personnel, which may not be available or affordable in resource-limited settings.
• PCR can be affected by sample quality, contamination, inhibitors, or false positives or negatives.
• PCR does not indicate the viability or infectivity of M. leprae, which may have implications for transmission and treatment outcomes.
• PCR results may not correlate well with clinical manifestations or disease classification, which depend on host immune responses.

Therefore, PCR should be used as an adjunct tool to support clinical and histopathological diagnosis of leprosy, rather than as a standalone test. PCR should also be interpreted with caution and in conjunction with other relevant information.

Leprosy is a curable disease that can be treated with a combination of antibiotics. The World Health Organization (WHO) provides free treatment for all people with leprosy. The treatment depends on the type and severity of the disease, and usually lasts between 6 months to 2 years. The main goals of treatment are to:

• Stop the infection and prevent transmission
• Reduce morbidity and prevent complications
• Eradicate the disease and prevent relapse
• Promote physical, social and psychological rehabilitation

The treatment options for leprosy include:

• Chemotherapy: This involves using multiple drugs to kill the bacteria that cause leprosy. The WHO recommends different regimens for paucibacillary (PB) and multibacillary (MB) leprosy. The drugs used are:

Chemotherapy can cure the infection and prevent further damage, but it cannot reverse the nerve damage or physical deformities that may have occurred before the diagnosis. Therefore, it is important to diagnose and treat leprosy as early as possible.

• Immunotherapy: This involves using agents that can enhance the immune system`s response to the bacteria. Immunotherapy can help improve the clinical outcome and reduce the risk of reactions and relapses. Some of the agents used are:

Immunotherapy can be used in combination with chemotherapy or as an alternative in cases where chemotherapy is not feasible or effective.

• Surgery: This involves performing procedures to correct deformities, restore function, relieve pain and improve appearance. Surgery can be done for:

Surgery can be done at any stage of the disease, but it is more effective if done before irreversible changes occur.

• Rehabilitation: This involves providing physical, social and psychological support to people affected by leprosy. Rehabilitation can include:

Rehabilitation can help people with leprosy cope with their condition, improve their quality of life and regain their dignity.

Leprosy is a treatable disease that requires a comprehensive approach that includes chemotherapy, immunotherapy, surgery and rehabilitation. With early diagnosis and proper treatment, people with leprosy can achieve a complete cure and live a normal life.

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