Mycobacterium leprae: Lepra – Molecular diagnosis (PCR)
Leprosy, also known as Hansen's disease, is a chronic infectious disease caused by Mycobacterium leprae, identified in the year 1873. Leprosy is an ancient disease that has afflicted humanity for many millennia and remains highly prevalent in economically disadvantaged countries. Each year between 200,000 and 250,000 new cases of leprosy are reported worldwide. Most occur in tropical and subtropical countries, including India, Brazil and the Central African Republic, which makes leprosy an unattended tropical disease. The large number of people emigrating from countries where this disease is endemic to European countries may allow an increase in the prevalence of it. In addition, more and more cases of drug-resistant and multidrug-resistant M. leprae are reported, which may pose a problem in preventing the onset and spread of the disease.
Leprosy is a chronic but curable granulomatous disease that mainly affects the skin, the nasal mucosa and the upper respiratory tract, the eyes, and the peripheral nervous system. It is known that the infection is transmitted directly from person to person, and the upper respiratory tract is considered the main route of entry and exit of the microorganism, but the mechanism has not been completely elucidated. Leprosy is not a very contagious disease. Susceptibility to infection is genetically determined, and 95% of all people appear to be resistant to the disease. People who develop leprosy usually incubate the infection for 3 to 5 years before manifesting the disease, a period that can last up to 30 years, making it difficult to detect the infection quickly. The earliest event is the presence of M. leprae in the skin, where it is selectively phagocytosed by macrophages and Schwann cells, invading in addition other non-typically phagocytic cells. Currently, it is accepted that the spread of the bacteria is carried out by blood. Depending on the immune response of the host, there are two main clinical manifestations opposite, which have been named according to histopathology as lepromatous and tuberculoid leprosy, and taking into account their bacillary load and number of lesions, such as multibacillary and paubacillary leprosy.
The tuberculoid or paubacillary form is characterized by the presence of one or few cutaneous lesions (less than five), with a low number of bacilli. Generally, patients have a concave, hypopigmented macula in the center, and well-defined edges. In patients with several lesions, its distribution is asymmetric. Histologically, the lesions observed consist of well-organized granulomas, with a high number of lymphocytes, epithelioid cells and, in some cases, giant cells. In these lesions, the bacilli are present in a small number and are often undetectable microscopically. Tuberculous lesions involve loss of sensation and thickening of the local peripheral nerves. The nerve condition occurs in the initial phase, and the superficial nerves that pass through the lesions have an increase in size. Larger peripheral nerves and those closest to the lesion may be palpable and visible, and the patient may have neuritic pain. The nerve condition can cause muscle atrophy, especially in the hands and feet, and often causes contractures. Trauma in these areas can lead to secondary infections and ulcers, and even cause the loss of phalanges.
Lepromatous or multibacillary leprosy is characterized by the presence of numerous systemic lesions (from five to hundreds), with a large number of bacteria, smaller than lesions of tubercular leprosy, and generally distributed symmetrically. The lesions can be constituted by macules, nodules, plaques or papules. The edges of the lesions are ill defined and the central part is elevated and indurated. Histologically, they are characterized by the existence of poorly organized or non-existent granulomas, a low number of lymphocytes, with the presence of macrophages, foam cells (called Virchow cells or leprosy cells), and abundant intracellular masses of bacilli that adopt a spherical morphology. The most frequent locations are the face, auricular pavilions, wrists, elbows, buttocks and knees. The lesions of lepromatous leprosy do not cause loss of sensation or thickening of the peripheral nerves. The most frequent symptoms that patients present are nasal congestion, epistaxis and respiratory distress and, occasionally, laryngitis and hoarseness. Also, depending on the location of the lesions, perforation of the septum and nasal deformity, keratitis and iridocyclitis, inguinal and inguinal adenopathies without pain, gynecomastia and, in men, testicular parenchyma affection causing sterility may occur.
Between these two opposing forms of leprosy, there are intermediate forms, called borderline or intermediate forms, which can be classified according to what resembles the tuberculoid or granulomatous form. In this case, the lesions increase in number and in heterogeneity. Unlike the leprosy and tuberculoid forms, the borderline forms of leprosy are unstable and can evolve into the lepromatous form in untreated patients or to the tuberculoid form in patients receiving treatment.
The most important pathological characteristic of leprosy, common to all forms of the disease, is the affectation and damage of the peripheral nervous system. M. leprae bacilli mainly invade the Schwann cells that coat the axons of the sensory and motor neurons in the peripheral nerves, causing the demyelination, de-differentiation and reprogramming of the cells. Axons deprotected by Schwann cells are also vulnerable to injury and can be destroyed by the host's inflammatory response to M. leprae. Generally, small myelinated and unmyelinated sensory neurons are affected first, and as the disease progresses, motor neurons and muscles can also be compromised. If the disease is not treated, M. leprae gradually causes the disfigurement of the extremities, the breakdown of tissues, and the development of physical disabilities. In addition, the peripheral nerves also serve as a safe haven for M. leprae, which is protected by the blood-nerve barrier of many host immune responses.
The causative agent of leprosy, Mycobacterium leprae, is an obligate intracellular bacillus, acid-fast, 0.25 x 7 μm, rod-shaped, slow-growing, and cannot be cultured in vitro. M. leprae is microscopically indistinguishable from other mycobacteria, and possesses a large amount of a specific phenolic glycolipid (PGL-1) in the outer capsule. It has the smallest genome among mycobacteria and almost half of its chromosome is occupied by non-functional pseudogenes. This loss of functionality is probably related to their inability to grow in artificial laboratory media, and influences the remarkably long generation time of the organism, of 12.5 days. In addition, most animals readily eliminate bacilli and cannot be experimentally infected with M. leprae. The only animals that naturally contract the infection are nine-banded armadillos, red squirrels and certain primates.
The impossibility of culture and replication in M. leprae animals has made it difficult to study the disease and diagnose. In general, the diagnosis of leprosy is based on the clinical findings and the detection of acid-fast bacilli in the material taken from the skin lesions, nasal mucus, or interstitial fluid or lymph (rich in macrophages containing the bacilli) of the ear lobe or elbow, if the patient does not present active lesions at the time of the examination. The main clinical and histological diagnostic problem is represented by the tuberculoid form of leprosy, due to the low number of bacilli present in the tissues. Among the detection techniques, smear microscopy is a technique of direct observation of microorganisms in smears of clinical samples after performing the Ziehl-Neelsen stain, which allows the detection of acid-fast bacilli. Direct detection of acid-fast bacilli has high specificity but low sensitivity, and approximately half of all leprosy patients have negative skin smears. In addition to smear microscopy, staining and observation of a skin biopsy taken from a skin lesion is another of the diagnostic possibilities for the identification of bacilli, as well as the histological involvement of the peripheral nerves. Among serological techniques, the detection of IgM antibodies against PGL-1 has some utility in patients with untreated lepromatous leprosy, as they are present in 95% of cases. However, only 60% of patients with tuberculoid leprosy are valuable. In addition, in endemic areas these antibodies can be detected in clinically healthy people. Finally, molecular techniques (PCR) stand out for their greater sensitivity, being able to detect up to one bacterium per sample, which is why they are very useful in cases of papular leprosy. The samples that can be used in this technique are very varied. However, the most useful samples are: samples of nasal septum mucosa, interstitial fluid of active lesions (or alternatively of the ear lobe or elbow), and tissue biopsies. One of the most common strategies is the molecular detection of M. leprae with specific primers targeting the pra gene, which encodes a 36 kDa protein antigen known as proline-rich antigen (pra).
Finally, given the suspicion of this disease, it must be taken into account that in humans, leprosy, in addition to M. leprae, can be caused by Mycobacterium lepromatosis, a mycobacterium with which it shares many common characteristics and causes lepromatous leprosy. and a specific form of the disease, diffuse lepromatous leprosy. However, while M. leprae causes the majority of cases of leprosy and is prevalent worldwide, M. lepromatosis is mainly endemic to Mexico and the Caribbean, although isolated cases have been reported from other countries.
Tests carried out in IVAMI:
- Molecular diagnosis (PCR) for M. leprae.
- Scraping mucosa of the nasal septum, sample of interstitial fluid of active lesions (or of the lobe of the ear or elbow if there are no active lesions at that moment), and tissue biopsies (fresh or paraffin).
Conservation and shipment of the sample:
- Refrigerated (preferred) for less than 2 days.
- Frozen: more than 2 days.
- Paraffin tissue biopsies can be stored and sent at room temperature or refrigerated. Fresh biopsies should be stored preferably frozen.
Delivery of results:
- Molecular diagnosis (PCR) for M. leprae: 24 to 48 hours.
Cost of the test:
- Molecular diagnosis (PCR) for M. leprae: Consult firstname.lastname@example.org