Mycobacteria

Why are the mycobacteria important?

The correct answer is: The mycobacteria are an important and varied group of bacteria. In
particular, they cause 3 million deaths each year and are a major cause of mortality in the
'developing' world. In the 'developed' world, the HIV epidemic has been associated with the
emergence of multi-drug resistant strains and in some countries, the increasing age of the
general population as well as immigration has led to a change in the incidence over the last 2-3
decades.
Which mycobacteria are of particular importance and how are they classified?
The four major species or groups of mycobacteria are:
1. Mycobacterium tuberculosis (often referred to as simply TB)
2. Mycobacterium bovis (causes some cases of TB, see below)
3. Atypical mycobacteria
4. Mycobacterium leprae - included in the Tropical Medicine course

What has been the epidemiology of tuberculosis since the 1990's in

'developed' countries?
Tuberculosis in 'developed countries' has been declining in incidence over the past 30-40 years.
However, recently some increase in cases has been reported from 'developed countries'
throughout the world. The reasons for this can be summarised as follows:
1. Development of multiple drug resistance in the tubercle bacilli.
2. Immunosuppression in AIDS patients and others who are immunosuppressed and others
e.g. cytotoxic drug therapy, etc.
3. Poor patient compliance in the continuation of their treatment. Patients are discharged
from hospital when non-infectious. They are, however, expected to continue their
therapy on leaving hospital for months.

4. Poor surveillance programmes - here patients may not be followed up for various
reasons resulting in poor compliance with treatment regimes leading possibly to spread
of TB and emergence of resistance.
5. Poverty, overcrowding and malnutrition.
6. Increasing immigration of foreign-born nationals from areas of high incidence.

What has been the epidemiology of tuberculosis since the 1990's in

'developed' countries?

In 'developing countries', it is estimated that a third of the population is infected with M.

tuberculosis. Eight million new cases occur each year, with up to three million deaths.
The changes and the reasons for them are similar to those listed for 'developed countries' but
in addition we must add inadequate drug therapy due to the cost of drugs and a lack of access
to good medical care such as for diagnosis. The combination of TB and HIV/AIDS has resulted
in very significant mortality and morbidity in some of the poorest parts of the world such as subSaharan Africa.
In Ireland, what has been the recent trend in TB notifications?

You answered:

The correct answer is: Over the last two decades, there has been a decline in overall numbers.
However, in the last 2-3 years the decline may have halted due to an increasingly elderly

patient population, better diagnosis and notifications, and increased immigration of foreign-born
nationals to Ireland from Eastern Europe and Africa.

Describe the morphology and microscopic appearance of mycobacteria

compared to other bacteria.
All mycobacteria differ from other bacteria in that they require a special stain usually the
Ziehl-Neelsen (ZN) to see them under the light microscope. A fluorescent auramine
stain is an alternative to the ZN stain.
Ordinary Gram stain will not penetrate the waxy capsular-like material surrounding the
cell wall.
They are alcohol and acid-fast slender bacilli, i.e. during the staining, the first dye applied
(Carbol-fuschin) cannot be subsequently removed by acids or alcohol.
When ZN stained and viewed under the microscope, the tubercle bacilli appear as red
slender bacilli against a blue background.
Special medium called Lwenstein-Jensen (LJ) or Middlebrook broth is required and
these bacilli take up to 8 weeks to grow. Modern automated methods, e.g. Bactec
reduces the time to a positive culture but these bacilli, unless most others, take weeks
and not days to grow.

What is the basis of the virulence of M. tuberculosis?

Pathogenicity is dependant on the ability of mycobacteria to remain alive and to multiply inside
phagocytic cells such as polymorphs and macrophages. Then they may escape by destroying
these cells and enter other cells and tissues. Humoral (antibody-mediated) immunity appears to
plays little part in the immune response to tuberculosis.
Recovery is associated with the development of cell-mediated immunity where macrophages
become "activated" and have the power to destroy these intracellular bacilli. Following infection,
the patient develops "delayed type" hypersensitivity.

What are the key pathological features?

These include:
Transient acute inflammatory reaction with polymorphs, which are unable to kill the
bacilli.

 Progressive infiltration of macrophages from the local tissues and the blood. The
macrophages are responsible for the destruction of the organism.
Macrophages phagocytose the bacilli. In a short time the appearance of the
macrophages change. The cytoplasm becomes pale and eosinophilic, and their nuclei
elongate and become vesicular. Their appearance bears a resemblance to epithelial
cells, hence the name "epithelioid cells."
Some macrophages fuse to become Langhans giant cells, i.e. nuclei disposed around
the periphery of the cell in a horse-shoe or ring arrangement.
Surrounding this mass of altered macrophages, there is a wide zone of small round cells,
mostly lymphocytes and fibroblasts.
Histologically, the necrosis is structureless and eosinophilic. Caseation would appear to
be caused by allergy to products of the bacillus - tuberculoprotein.
NB: Please consult Pathology lectures and notes also.
What is the source of M bovis and how is it transmitted?

You answered:

The correct answer is: The source is infected cattle and spread is generally by ingestion of milk
containing tubercle bacilli from tuberculous cows. Other animals e.g. dogs, cats and pigs may
become infected with M. bovis.

Describe the key features of primary or childhood-type pulmonary TB.

It is caused by Mycobacterium tuberculosis
Source: From a human, an infected "open" case of adult-type of pulmonary tuberculosis i.e.
where the lesion communicates with a bronchus and tubercle bacilli are coughed up, along the
respiratory tract.

Route of infection: By inhalation.

The classical lesion is the Ghon focus. This is a small wedge-shaped area situated in the
periphery of the lung, usually in the middle segments. This may then lead to:
1. Complete healing by calcification and fibrosis
OR
2. There may be spread to the hilar glands, which become enlarged and caseous. This primary
focus + enlarged hilar lymph glands is called the Primary Complex. This primary complex may
heal by calcification and fibrosis.
OR
3. There may be invasion into the bloodstream, which is called miliary tuberculosis, with
development of meningitis, and lesions in other body organs,
OR/AND
4. Hilar glands may be enlarged that they compress a bronchus causing segmental collapse.

Describe the key features of abdominal TB.

This is usually caused by Mycobacterium boviswhich is biologically very like M. tuberculosis,
both are often referred to as M. tuberculosis complex.
The infected cattle discharge the tubercle bacilli into the milk. Infection is acquired by drinking
raw (unpasteurised) milk from an infected cow.
Bacilli multiply in the Peyer's patches of the small intestine with involvement of the mesenteric
lymph nodes. The following may occur: (a) Recovery with healing mainly by calcification and fibrosis
OR
(b) Spread to overlying peritoneum with development of tuberculous peritonitis
OR
(c) Spread via the lymphatics to the thoracic duct and into the blood stream leading to military
infection. Here organs most commonly affected are bones, kidneys and brain. Patients if they die
usually do so from tuberculous meningitis.
The tonsils are commonly affected with characteristic spread to cervical lymph nodes. These
may calcify (recovery) or may soften and ulcerate through the skin (scofula).

How is abdominal (bovine) TB prevented?

This involves:
(a) Pasteurisation of milk. This process destroys most microbes, including TB and Brucella spp.,
but does not adversely affect the taste or nutritional value.
(b) Eradication of infected cattle. Large scale programmes are present in 'developed countries'
where all tuberculin-positive cattle are slaughtered. Despite this, there may be some infected
cattle still in many 'developed countries', e.g. U.S., U.K. and Ireland.
In 'developing countries', eradication programmes are not being carried out due to the expense
of slaughtering the infected cattle. Thus bovine tuberculosis in humans is still a problem in these
countries.

What is the sequence when the lungs are involved?

The focus is usually apical or sub-apical, usually in the right lung. This,
(a) heals by fibrosis,
OR
(b) softens producing a cavity which may be very large in severe cases.
The following may happen: 1. Haemoptysis due to erosion of blood vessel in the cavity
OR
2. Small extension to neighbouring parts of the lung via small bronchi,

OR
3. Widespread extension with massive caseation and death
However, usually the disease remains localised to the lungs, bloodstream invasion is unusual,
and lymph node involvement does not occur, unlike with primary TB.
Back
What happens when the intestine is involved?

This usually arises when the patient swallows his or her own infected sputum. Lesions arise
and ulcerate in to the wall of the ileum. The following may happen: 1. Local spread causing localised peritonitis.
2. Strictures.
3. Fistulae.
Involvement of local lymph nodes is minimal.
Involvement of local lymph nodes is minimal.

What does this mycobacterium cause?

This organism, which has never been grown in-vitro, causes leprosy, which affects the skin,
nerves and mucosa. It is very slow growing but is not as infectious as commonly believed. For
further details, please see Tropical Medicine course.

What are these mycobacteria, and why are they atypical?

You answered:

The correct answer is: These are mycobacteria, which have varying degrees of pathogenicity
for humans. The vast majority are found in the environment and when they do cause infections
in man, they are not readily transmitted from person to person, hence they are not considered
infectious like TB.

What kind of infections do they cause?

The majority of atypical mycobacteria are capable of producing pulmonary infections and
enlargement of lymph nodes (adenitis), particularly cervical adenitis. These infections may
present clinically in a similar manner to TB. The most clinically important atypical mycobacteria
are:
M. avium complex
M. kansasii

There are a large number of these atypicals but the majority are incapable of producing serious
disease.

What is the significance of Mycobacterium avium-intracellulare?

This is so-called because it causes disease in birds. This organism can cause serious disease in
immunosuppessed patients with disseminated disease in patients with AIDS.
It is always a primary infection
It is usually inhaled
Generalised disease can occur producing fever, weight loss and anaemia
Large numbers of bacilli are present in affected organs and in the blood and bone
marrow

How does M. kansasii differ from M. tuberculosis?

You answered:

The correct answer is: LJ slopes turn an orange colour after exposure to light for one hour.
(such atypical mycobacteria are called photochromogens
What category of infection does it cause?

The correct answer is: It usually produces a pulmonary infection resembling tuberculosis on Xray and is seen more commonly in middle-aged men with a history of chronic bronchitis. It is
common in tropical and sub-tropical countries and may invade the bloodstream in those who
are immunosuppressed and produce disseminated disease.

What approaches are there to making a diagnosis of TB, and briefly describe each one.

These involve:
Clinical suspicion
Skin tests
Blood tests
Microbiology laboratory investigations
Radiology
Tissue diagnosis or histology

(a) Clinical Suspicion

TB should be considered in any patient with a fever of undetermined origin or in a patient

unresponsive to conventional antibiotics. It should be suspected in patients at particular
risk such as alcoholics, HIV positive individual, etc.

(b) Skin Tests (Mantoux, Heaf, etc)

These are based on a delayed hypersensitivity reaction (Type IV) to protein derivative of
TB. They are less useful diagnostically in those countries where BCG vaccination (see
below) is routine. Intradermal injection of the purified protein derivative (PPD) of M.

tuberculosis produces a red indurated area 1 cm in diameter within 24 - 48 hours

(positive) in a patient who

(i) has had BCG vaccine

(ii) has had infection with M. tuberculosis or M. bovis in the past

(iii) is suffering at the time of the injection from infection with M. tuberculosis.

If negative on first testing, a stronger concentration of PPD is used, and this may produce
a positive reaction. If then negative, the patient has never been infected with
mycobacteria. Other possible but much less common explanations for a negative test
are:

(i) Very recent infection e.g. less than 4 weeks before

(ii) Disseminated miliary tuberculosis,

(iii) Immunosuppressed persons, the best example being AIDS where T cell activity is
greatly diminished.

(c) Blood test

Gamma-interferon tests use synthetic stimulating peptides based on two proteins of M.

tuberculosis and M. bovis that release interferon gamma by host T cells in patients with
infection. Two tests, the QuantiFERON TB and T-SPOT.TB, are currently available.
These tests are alternatives to skin tests, are used in diagnosing latent TB, and have a
high specificity even in populations exposed to the BCG vaccine.

Laboratory Diagnosis

Specimens will depend on site of tuberculous lesion within the body. Always send
multiple specimens. Swabs are unreliable; pus, tissue or fluids are far superior.

Pulmonary Tuberculosis: Sputum, 3 early morning or bronchoscopy samples

Meningitis: CSF

Renal: 3 consecutive early morning specimens of urine.

Investigations

Direct examination with ZN stain (see earlier figure).

Culture on LJ slope or automated monitoring systems (BACTEC), but takes up to 8

weeks or more, not days as with the culture of most other bacteria.

Figure Automated monitoring culture monitoring system (Humphreys & Irving)

PCR in certain areas is used to confirm a ZN +ve result due to M. tuberculosis and not
another species, or where microscopy is negative, e.g. TB meningitis.

Susceptibility tests on the tubercle bacilli isolated are then undertaken.

Diagnosis (other investigations)

Histology
All tissue from patients with undetermined pathology should be examined for granulomata,
especially caseating granulomata, and ZN stained to exclude TB. (See earlier figure)

What are the principles of TB prevention?

These include,
1. Improved social conditions (housing and nutrition), contact tracing and the effective therapy of
infected patients.
2. Bacille-Calmette-Guerin (BCG) vaccine

Consists of a live attenuated strain of M.. bovis attenuated by growing it on bile-potato medium.
Produces delayed-type hypersensitivity response similar to that caused by natural infection and
vaccinated person will become tuberculin-positive six weeks after vaccine. It is used in Ireland
but not in many other countries such as the USA, where therefore skin testing is more useful as a
diagnostic tool.
NB: BCG vaccine is never given to persons who are tuberculin test positive.

What are the general principles of TB therapy?

These include:
Combination therapy is the rule since resistance develops easily to any single drug, more
than one drug results in synergy and facilitates lower doses of each drug thus
minmising adverse drug events. At least 2 drugs should be used at any one time.
Isoniazid is the best drug available and should be included in any combination.
The minimum duration of therapy is six months but many patients require at least 9
months of treatment, especially if non-pulmonary disease.
Antibiotic susceptibility testing should always be performed (usually in a reference
laboratory) to guide treatment, especially if non-pulmonary disease.
The following are recommended regimens for the treatment of respiratory TB.
Rifampicin ]
Isoniazid ] for first twomonths
Pyrazinamide and/or ]
Ethambutol ]
followed by
Rifampicin ]
Isoniazid ] for further 4 months
This is the most popular regimen in the U.K. and Ireland

How do you assess the response to treatment?

A number of parameters should be considered:

Clinical improvement, e.g. disappearance of fever
Sputum usually becomes negative (by ZN), called sputum conversion, after two weeks
i.e. disease becomes non-communicable.
X-ray picture improves as cavities start to shrink after 4 weeks.
Follow up by regular sputum examinations and X-rays should be for up to 2 years

How do AIDS patients develop TB?

AIDS patients develop clinical evidence of tuberculosis due to two mechanisms:
(a) Reactivation of an old primary focus. Here, tubercle bacilli, which have survived in foci,
escape and multiply causing clinical tuberculosis. Clinically the disease may differ in many
respects to reactivation in normal non-immunosuppressed patients.
Disease in AIDS patients with low CD4 counts has been called "The new tuberculosis"
because there is a more generalised spread in the body e.g. frequent involvement of
extra-pulmonary sites such as lymph nodes and meninges.
X-ray of chest may not show the typical cavity of adult-type disease.
Tuberculin test may be negative due to inability of T cells to produce a delayed type
hypersensitivity response.

 While the disease presentation may be different these patients are highly infectious for
others e.g. those living with them or those caring for them.
As with other opportunist infections, the frequency of TB in HIV/AIDS has changed, with
the onset of improved anti-retroviral treatment
(b) Here the patients are infected with tubercle bacilli for the first time. Those unvaccinated will
develop primary childhood-type disease. In those who have been vaccinated with BCG, disease
may present with many of the features described in reactivated tuberculosis above.
In countries where primary tuberculosis is still common e.g. parts of Africa, reactivation of
disease and AIDS infection are common e.g. it is estimated that 2 - 4 million people in SubSaharan Africa are co-infected with tuberculosis and AIDS.
In the early 1990s, MDRTB emerged in New York city due to
poor compliance
slow response to treatment
inadequate public health measures such as isolation and contact tracing