Mycobacterium
The mycobacteria are a group of non-motile rods that are defined on the basis of a distinctive staining property. They are relatively impermeable to various basic dyes but once stained, they retain the dyes with tenacity. They resist decolourisation with acidified organic solvents and are therefore called acid-fast. Mycobacteria range from widespread innocuous saprobic inhabitants of the soil and water to organisms responsible for tuberculosis and leprosy. In both of these chronic diseases, intracellular infection and delayed hypersensitivity play an important role. The mycobacteria are considered to be transitional forms between eubacteria and actinomycetes, some of the latter, in the genus Nocardia are acid-fast.
The leprosy bacillus, discovered by Hansen in 1879, was the first bacterium shown to be associated with human disease. It is strikingly adapted to man: it is difficult to transfer to any other host and has not been grown on any artificial media. In contrast, the agents of human tuberculosis (M. tuberculosis and M. bovis) are readily cultivable on simple media and are pathogenic for various lower animals, especially guinea pigs and mice. With the decreasing prevalence of tuberculosis, it has been recognised that similar but generally milder infections are caused by a variety of mycobacterial species “atypical mycobacteria”, previously ignored because they do not cause disease in test animals. These organisms have reservoirs in the environment or in lower animals.
Mycobacterium tuberculosis infects 1.7 million people world-wide, about 1/3 of the world’s population and causes 3 million deaths each year, the most for any single infectious disease. After inhalation of the tubercle bacilli, the initial lesion appears as an area of nonspecific pneumonitis. Delayed hypersensitivity develops in 2 to 4 weeks resulting in granulomatous inflammation and the characteristic tubercles. The pulmonary focus and the granulomatous lesion in the hilar lymph node are known as the primary complex. The next stage in the inflammatory process consists of caseation necrosis. Primary tuberculosis in children usually stabilises and heals. The caseous lesions heal by fibrosis and calcification. The healed primary complex is referred to as the Ghon focus. In a small minority of individuals, the infection is not brought under control and the primary lesions become larger, coalesce, and liquefy. When this material is released, a cavity is formed in the lung.
Most tuberculosis in adults is secondary to reactivation of long-dormant foci remaining from the primary infection. The foci are usually located in the posterior apical portions of the lung. By the time disease is recognised, liquefaction of the caseous lesion has occurred and the resulting cavity provides a favourable site for the rapid proliferation of bacilli. These may then be transmitted to other individuals via droplet nuclei produced by aerosols of infected sputum. Almost every organ in the body may be the site of extrapulmonary tuberculosis. The genito-urinary, bones, joints, pleura, and peritoneum are most commonly involved. Extrapulmonary manifestations may also occur as a result of reactivation of dormant lesions seeded during the primary infection. HIV patients are particularly prone to reactivation of pulmonary TB, the extent of which depends on the amount of immunosuppression. HIV-positive individuals may also acquire new infection from others in their environment.
Laboratory Diagnosis
A provisional diagnosis of tuberculosis is usually made by demonstrating acid-fast bacilli in stained smears of sputum or of gastric washings. For rapid staining of smears, some laboratories employ fluorescence microscopy. Whether the smear is positive or not, the material should be cultured since fewer organisms can be detected, the human tubercle bacilli can be differentiated from other acid-fast bacilli, and testing for anti-microbial sensitivity can be carried out. Solid culture media are preferred for primary isolation and they often contain egg yolk to promote growth. These media often contain malachite green or other antibiotics to inhibit the growth of other organisms. A positive culture usually grows in 2 to 4 weeks. A selective liquid medium with a radiolabelled carbon substrate allows automated detection of growth several days sooner than with conventional culture.
Delayed type hypersensitivity to tuberculin is highly specific for tubercle bacilli and this is the basis of the tuberculin test. A positive test reveals previous mycobacterial infection but it does not establish the presence of active disease. Reactivity appears about 1 month after infection and persists for many years. The tuberculin test (or Mantoux test) is a useful diagnostic test and epidemiological tool. Conversion from negative to positive provides good evidence for a recent infection which should set in motion investigation of contacts and possible source cases. However, 5 to 10% of patients with nondisseminated tuberculosis have negative initial tests, and the frequency is higher in miliary and extrapulmonary disease.
Treatment
Before effective drugs were available, half of patients with active pulmonary tuberculosis died within 2 years, and only a quarter were cured. Sanatorium treatment was only moderately effective for noncavitary but hardly at all for advanced cavitary disease. With the advent of chemotherapy, protracted bed rest and lengthy isolation became unnecessary. Initially, a 4-drug regimen should be tried for 6 months, usually isoniacid, rifampin, pyrazinamide, and ethambutol.
Isoniacid (INH) is the cornerstone of therapy and should be included in all regimens unless a high degree of INH resistance exists and the regimen includes rifampin. Rifampin is the second major antituberculous agent. The most important complication of rifampin is hepatitis. Pyrazinamide is an essential component of 6-month regimens. Its beneficial effect is mostly limited to the first 2-3 months of treatment. Ethambutol is a component of most regimens. Streptomycin was the first major antituberculous drugs but was promptly replaced by INH as the cornerstone of therapy.
Second line agents are less efficacious and/or more toxic than the first-line drugs. These include ethionamide, prothionamde, cycloserine, kanamycin, capreomycin, thiacetazone, para-aminoslicylic acid and other agents.
M. bovis causes tuberculosis in cattle and is also highly virulent for man. In the past man usually get infected through unpasteurised or other diary products produced from tuberculous cows. After ingestion, the organism penetrates the mucosa of the oropharynx and intestine, giving rise to early lesions in the cervical or mesenteric lymph nodes. Subsequent dissemination from these sites principally involves the bone and joints. Infection of the vertebra has resulted in the hunchbacks of previous generations. When inhaled e.g. by diary farmers, the organism can also cause pulmonary tuberculosis indistinguishable from that caused by M. tuberculosis. Bovine tuberculosis had been virtually eliminated by the pasteurisation of milk and the virtual eradication of tuberculosis in cattle.
Besides mycobacterium tuberculosis and bovis, various other acid-fast bacilli have been cultured from respiratory secretions of apparently tuberculous individuals. Because they possessed little or no virulence for guinea pigs or rabbits, they were long considered as saprobes. It was only until the 1950s that the role of these “atypical mycobacteria” were recognised when such organisms were repeatedly recovered from the sputum or infected tissue in the absence of classic mycobacteria. Moreover, as the incidence of classical tuberculosis decreases, the importance of these atypical mycobacteria increases. In certain parts of the world, as much as 20% of newly diagnosed mycobacterial pulmonary disease is caused by these organisms. The proportion is even higher in soft-tissue and disseminated disease.
1. Mycobacterium Avium Complex
The M. avium complex includes M. avium and intracellulare. M. avium causes tuberculosis in chickens, other birds, and swine. There are many documented cases caused by M. avium, mostly in farmers and men with silicosis. M. intracellure is not usually pathogenic for birds or animals. M. intracellure is a frequent cause of disseminated infection in patients with AIDS. In contrast to the more virulent M. tuberculosis, the identification of MAC in an isolated sputum culture does not constitute definite evidence of disease because MAC can colonise healthy persons. Because the presence of MAC in the sputum does not constitute a public health hazard and MAC pulmonary disease is not rapidly progressive, it is important to obtain evidence to establish MAC disease before embarking on a prolonged course of therapy. The diagnosis of disseminated infection can be made by the identification of MAC from a sterile site. MAC is generally resistant to the first-line antituberculous agents.
2. Mycobacterium Scrofulaceum
M. Scrofulaceum is a common cause of lymphadenitis in children aged 1 to 3 years. Lymphadenitis usually involves a single node or a cluster of nodes in the submandibular area. Characteristically, the nodes enlarge slowly over a period of weeks. There are very few local or systemic symptoms. Untreated, the infection will usually point to the surface, rupture, form a draining sinus and eventually calcify. Infection in other tissues occurs occasionally. A very few cases resembling progressive primary tuberculosis have been encountered in children. In children, metastatic bone disease may be prominent. Colonies are usually yellow-orange even when grown in the dark (scotochromogenic). They are usually resistant to antituberculosis drugs in vitro.
3. Mycobacterium Kansasii
M. kansasii and M. avium-intracellulare account for most of the human mycobacterial disease attributable to acid-fast organisms other than tubercle bacilli. M. kansasii is photochromogenic; the overnight change of the colonies to yellow is followed by the formation of red crystals of B-carotene on exposure to light for several more days. Most strains are sensitive to rifampin and to several other drugs. M. kansasii has been identified as an agent of disease in nearly all parts of the world. It characteristically produces a chronic lung infection that closely resembles pulmonary tuberculosis. Symptoms are usually milder than tuberculosis. M. kansasii may infect extrapulmonary tissues often as a result of inoculation or haematogenous dissemination. M. kansasii is among the most predictably sensitive of the mycobacterial species.
4. Mycobacterium Ulcerans
M. ulcerans, found mainly in Africa and Australia, will grow only below 33oC. It causes chronic deep cutaneous ulcers in man. It usually produces lesions in the cooler parts of the body. It has a unique drug sensitivity pattern;- resistance to INH and ethambutol and susceptibility to streptomycin and rifampin. Human disease responds poorly to drug treatment and extensive excision followed by skin grafting is often necessary.
5. Mycobacterium Marinum
M. marinum also grows best below 33oC. It causes a tuberculosis-like disease in fish and a chronic skin lesion known as “swimming pool granuloma” in humans. Infection is acquired by injury of a limb around a home aquarium or marine environment and can lead to a series of ascending subcutaneous abscesses. M. marinum resembles M. kansasii in being photochromogenic. M. marinum varies in susceptibility to antimicrobial agents.
6. Mycobacterium Fortuitum Complex
Most of the fast-growing disease-associated mycobacteria are members of this complex, which may be divided into two accepted species: M. fortuitum and M. chelonae. They abound as saprobes in soil and water. A wide variety of clinical symptoms may be encountered. Sporadic infections have involved almost every tissue and organ systems, and outbreaks have followed cardiothoracic surgery, peritoneal dialysis, and haemodialysis. The spectrum of diseases caused by these organisms includes soft-tissue abscesses, sternal wound infections after cardiac surgery, prosthetic valve endocarditis, disseminated and localised infection in haemodialysis and peritoneal dialysis patients, pulmonary disease, traumatic wound infection, and disseminated disease often with cutaneous lesions.
The most predictably effective therapy for infections due to rapidly growing mycobacteria is surgical removal of all involved tissues. The rapidly growing mycobacteira vary widely in their susceptibility to chemotherapeutic agents.
7. Mycobacterium Xenopi
M. xenopi was first isolated in 1959 from a South African toad. It had been reported to cause pulmonary infection in several parts of the world, most prominently in England. Contaminated hot water tanks may serve as reservoir for infection, especially in institutions that care for patients with chronic lung disease. Most infections resemble pulmonary tuberculosis. It is scotochromogenic and grows optimally at 42oC
8. Mycobacterium Szulgai
M. szulgai is a distinct species with a potential ability to produce chronic lung disease, as well as infection of lymph nodes and bursae. Lung disease generally resembles that of M. tuberculosis.
9. Other species
A group of acid-fast bacilli with little or no pathogenicity for man or animals may be found in human specimens and mistaken for established pathogens. These species include M. gordonae, M. gastri, M. terrae-triviale, and M. flavescens. However under some circumstances, these species may cause disease in man resulting in local, generalised, and pulmonary infections.
M. simiae is a photochromogen which has been isolated from monkeys and from tap water. It is an infrequent cause of pulmonary infections. Cases have occurred in monkey handlers and those with no association with monkeys. M. Malmoense and M. haemophilum are nonphotochromogens which are infrequently associated with human disease. M. Malmoense cause mainly pulmonary disease and M. haemophilum mainly skin disease.
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)
Pathogenesis
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.
Epidemiology
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.
Staining
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.
Culture
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.
Guinea pig
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.
Specimens
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
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
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.
Newer methods
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.
Tuberculin test
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
Optimal
Growth Catalase
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
scrof,, fort.
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.