Leprae of Mycobacterium Infections
Although M. leprae has never been cultivated, it has long been recognised as the aetiological agent of human leprosy as it is readily demonstrated in stained smears of exudates of persons with leprosy. M. leprae is indistinguishable in morphology and staining properties from M. tuberculosis and leprosy has many clinical features in common with tuberculosis. The failure to culture the organism has hampered its investigation and made it difficult to test strains for drug sensitivity. The organism can be propagated in the foot pads of mice which is a relatively cool environment. It can also cause disseminated disease in the armadillo which is used to raise antigen for skin testing i.e. the lepromin test. The lepromin test may give a positive result in tuberculous individuals and occasionally in healthy individuals. It is mainly used in determining the prognosis of disease (see below)
M, leprae causes granulomatous lesions resembling those of tuberculosis, with epitheloid and giant cells but without caseation. The organisms are predominantly intracellular and can proliferate within macrophages, like tubercle bacilli. Leprosy is distinguished by its chronic slow process and by its mutilating and disfiguring lesions. These lesions may or may not be characteristic as to be diagnostic for leprosy. The organism has a predilection for skin and nerves. In the cutaneous form of the disease, large firm nodules are distributed widely and on the face they create a characteristic leonine appearance. In the neural form, segments of peripheral nerves are involved, more or less as random, leading to localised patches of anaesthesia. The loss of sensation in the fingers and toes increases the frequency of minor trauma, leading to secondary infection and mutilating injuries
In either form of the disease, three phases may be distinguished:
(1) Lepromatous (progressive) type – the lesions contain many leprae cells, which are macrophages with a characteristic foamy cytoplasm, in which acid-fast bacilli are abundant. The lepromin test is usually negative. The disease is progressive and the prognosis is poor.
(2) Intermediate type – bacilli are seen in areas of necrosis but rare elsewhere, the lepromin test is positive, and the long-term outlook is fair.
(3) Tuberculoid (healing phase) – the lesions contain few leprae cells and bacilli, fibrosis is prominent, and the lepromin test is usually positive.
The organism may be widely distributed in other tissues such as liver and spleen without any ill effect. Deaths of leprous patients are not caused by leprosy itself but by intercurrent infections with other organisms such a tuberculosis.
Diagnosis and treatment
Diagnosis is accomplished by demonstrating acid-fast bacilli in scrapings or fluid from ulcerated lesions. The lepromin skin test is useful in determining whether or not the patient is in the lepromatous stage and thus the prognosis. Serological tests are becoming available. Therapy with dapsone or related compounds usually produces a gradual improvement over several years and is continued for a long period after clinical remission. However, the emergence of resistance lead to the inclusion of rifampin and clofazime to the treatment regimen. It is recommended that all three drugs should be given for a period of at least 2 years. Treatment results may be evaluated by counting the acid-fast bacilli in serial biopsies of skin scrapings.
Leprosy is apparently transmitted through contact and is not highly contagious and patients so not need to be isolated. The incubation period ranges form a few months to 30 years or more. Apparently, M. leprae can lie dormant in tissues for long periods. The prophylactic use of BCG vaccine or dapsone has not been successful. There are thought to be 10 million lepers world-wide and the disease is found mainly in Asia and Africa.
Laboratory techniques for mycobacteria
The commoner species of mycobacteria are classified into two groups: (1) the typical tubercle bacilli i.e. M, tuberculosis and M. bovis, and (2) the atypical mycobacteria. Mycobacterium are difficult to stain but once stained, they resist decolourisation with acid and alcohol. Three main methods are used to detect tubercle bacilli in sputum and other materials.
Typically, the human tubercle bacilli are slender, straight or slightly curved rods. They are non-motile, non-sporing and uncapsulated. They remain uncoloured with simple stains but show acid-fast staining with warm carbol fuchsin followed by 20% H2SO4 or by 3%HCl in 95% ethanol (Ziehl-Neelsen method). Bovine tubercle bacilli tend to be shorter and thicker than the human type.
The tubercle bacillus is an obligate aerobe and grows at temperatures from 30-41oC, optimally at 35-37 oC. Lowenstein-Jensen media with glycerol or sodium pyruvate is generally used for isolation. The specimen is incubated on slopes of L-J medium at 37 oC. Growth is slow so that colonies only appear after 2-3 weeks. The slopes should be incubated for a total of 6-8 weeks before discarding. M. tuberculosis colonies on L-J media are rough, buff to yellowish in colour, and tough when picked off. Dispersed uniform growth can be obtained by subculturing two or three times in Dubos and Davis liquid medium continuing Tween 80. The addition of glcerol to L-J medium improves the growth of M. tuberculosis, but not that of M. bovis. Sodium pyruvate, on the other hand increases the growth of M. bovis and some strains of drug-resistant M. tuberculosis.
Tubercle bacilli are killed by heat at 60oC for 15-20 min. They survive many weeks in moist conditions in the dark. They are killed rapidly by sunlight. They are relatively resistant to chemical disinfectants. The tubercle bacilli are catalase positive and do not produce acid in sugar-containing media. Biochemical tests are generally not used in their confirmation. Several phage groups of M. tuberculosis had been identified and it is now possible to use phage typing to demonstrate case to case spread of infection.
The guinea pig is highly susceptible to infection by both M. tuberculosis and M. bovis. After subcautaneous injection of an infected specimen, a local swelling appears within a few days which proceeds to caseate and finally ulcerate. The organism spreads to the lymph nodes, spleen, liver, and peritoneum. The animal dies within 6-15 weeks. Because modern methods of culture are so efficient, inoculation of sputum into animals is rarely indicated.
1. Sputum – sputum should be examined microscopically firstly in a direct smear made from the untreated specimen and then again in a smear made from the centrifuged, concentrated deposit that has been treated and homogenised.
2. BAL, Laryngeal swab and gastric lavage – where sputum is absent, material for culture may be obtained from a BAL, laryngeal swab, or gastric lavage.
3. Urine – special caution must be exercised in interpreting the finding of mycobacteria in urine. Samples may be contaminated by M. smegmatis from the urethral orifice.
4. Pleural and peritoneal fluid – as tubercle bacilli are usually scanty, a large specimen e.g. 50-100 ml should be obtained.
5. CSF – as large as possible a specimen should be obtained.
6. Pus – examine with direct smears and Z-N stain.
7. Tissue – tissue should be homogenised.
Sensitivity tests are done on slopes of L-J glycerol medium each containing a series of concentrations of a particular antibiotic. After incubation for 3 weeks at 37oC, the slopes are examined for growth and the lowest concentration of antibiotic showing no more than 0-20 colonies is taken as the end-point, or nominal “MIC”. The conventional methods of sensitivity testing involve a delay of 3 weeks before results are available. Rapid methods are available which measure the evolution of 14CO2 from a radio-labelled substrate in the medium e.g. Bactec.
Identification tests should be carried out at the same time as sensitivity tests. The following five tests are routinely carried out for the purpose of identification:-
1. L-J slope with para-nitrobenzoic acid (PNB) 500 mg/l – all tubercle bacilli are sensitive to PNB and thus should fail to grow.
2. L-J slope with thiophen-2-carboxylic acid hydrazide (TCH) – of the strains which are sensitive to PNB, only strains of M. tuberculosis are resistant to TCH and can grow on the TCH medium.
3. Dubos and Davis medium for the niacin test – M. tuberculosis produce niacin and so differs from M. bovis and BCG which do not.
4. L-J slope for incubation in an incubator with light for pigment production – atypical tuberculoid bacteria can be divided in (1) photochromogens, (2) scotochromogens, and (3) non-chromogens. (see below)
5. Sauton agar with 0.2% picric acid – rapidly growing mycobacteria can be distinguished from slowly growing mycobacteria by their ability to grow on Sauton agar containing 0.2% picric acid. Typically, rapid growing mycobacteria will show good growth within 5 days at 37oC
Photochromogens are slowly growing mycobacteria that form yellow or orange pigment when their cultures are exposed to light, but not when they are kept in the dark. In the standard test for photochromogenesis, two L-J slopes are seeded, one is wrapped in aluminium foil to exclude light from it and both are incubated for 14 days at 37oC in a light incubator. Scotochromogens are slowly growing mycobacteria that form pigment in both light and dark. Non-chromogens are those that do not form pigment at all, of which the most important members are those of the MAC complex.
HPLC is now routinely used in large laboratories for the identification of mycobacteria, as is the case of nucleic acid probes on culture. The role of PCR in the routine diagnosis of mycobacteria infection has yet to be fully established.
Antibodies to mycobacteria in patient’s serum can be demonstrated by various serological techniques such as HAI and EIA. However, the presence or absence of antibodies, or their titres when present, has shown little correlation with the clinical state of the patient. The principal immunological response in tuberculosis is the development of cell-mediated immunity. It is demonstrated by the delayed hypersensitivity reaction which follows the intradermal inoculation of tuberculin. A positive test in a person who has not been vaccinated with BCG indicates that tuberculous infection has taken place in the recent or distant past, but is not necessarily a sign of active disease. In the absence of BCG vaccination, a positive reaction is valuable confirmatory evidence. The test is also useful in screening children who have been in contact with an open case of disease. However, the test may be negative in advanced or miliary infection. Two methods of testing are currently in use: Mantoux test and heaf test.
Tuberculin test – this is the standard method by which all other methods are compared. A test dose of 0.1 ml of Purified Protein Derivative (PPD) containing 5 Tuberculin Units is injected intracutaneously into the skin of the forearm. The development of an area of palpable firm induration greater than 10 mm in diameter is recorded as positive. The extent of the accompanying erythema is irrelevant. If the reaction is completely negative, the test may be repeated by giving an injection of 100 Tuberculin Units.
Heaf test – this test is done with a multiple puncture apparatus with 6 needles that prick tuberculin 1-2 mm deep into the skin. A drop of undiluted PPD is spread onto the area of the skin selected for inoculation, the instrument is pressed against this are of skin and the needles are released. The site is inspected 72 h later. When a reaction comprising the presence of erythema and oedema or induration around at least 4 of the punctures is regarded as positive. Because of its ease of performance, the Heaf test is principally used in epidemiological surveys and as a test for immunity before BCG vaccination. For diagnostic purposes, the more accurate Mantoux test should be used. A fresh apparatus must be used for each person tested in order to prevent the spread of HIV and HBV infections.
Some Characteristics of Nontuberculous Mycobacteria Commonly Encountered in Human Material
Clinical Temperature Tween Iron Growth
Species Significance* (oC) Pigment 25‑38oC 68oC Nitrate Hydrolysis Urease Uptake Rate
M. avium‑intracellulare + 37 ‑ or S Weak + - - - Slow
M kansasii + 37 P Strong + + + + Slow
M xenopi + 42 S Weak + ‑ - - Slow
M. scrofulaceum + 37 S Strong + ‑ - + Slow
M. simiae + 37 P(weak) Strong + ‑ - + Slow
M szulgai + 37 S/P Strong + + ± + Slow
M. gordonae ± 30‑37 S Strong + ‑ + - Slow
M flavescens ± 30‑37 S Strong + + + + - Intermediate
M. terrae‑trivale ± 30‑37 - Strong + + + - Slow
M. gastri - 30‑37 - Weak ‑ ‑ + + Slow
M marinum + 30 p Weak + ‑ + + Intermediate
M. fortuitum + 30‑37 - Strong + + ± + + Fast
M. chelonae + 30‑37 - Strong + ‑ - + - Fast
M. smegmatis - 25‑45 - Strong + + + + + Fast
+ May be pathogenic for hosts without general immune suppression
± documented pathogen only for hosts with abnormal local and/or general defense mechanisms
P = photochromogenic, S = scotochromogenic,
S/P Scotochromogenic when grown at 37oC but variably photochronnogenic at 25oC.
TABLE 35‑2. Nontuberculous Mycobacterial Diseases of Man
Disease Common Associated Species Other Associated Species
Chronic cavitary lung disease in adults MAI, M. kans. M. xenopi, szulgai, simiae, malmoense
Local lymphadenitis in children MAI, M. scrof M. kans., M. fort.
Arthritis, tenosynovitis, and osteomyelitis, MAI, M. kans. M. fort., M. terrae, M. marinum,
including hand infection M. xenopi
Bursitis M. kans. M. szulgai
Skin nodules and abscesses M. marinum, M. haemophilum, M. fort. M. kans., MAI, M. fort., M. szulgai
Buruli or Bairnsdale ulcer M, ulcerans
Disseminated disease MAI, M. kans. M. scrof., M. fort.
Leprosy M leprae
Abbreviations: MAJ, M. avium‑intracellulare, M. fort., M. fortuitum‑chelonao,. M. scrof, M. scrofulaceum, M. kans., M. kansasn.