Professional Documents
Culture Documents
Methods
for
Tuberculosis
Namrata
Singh,
PhD
Table
of
Contents
1.
Introduction
2
2.
Causative
Agent
3
3.
Transmission
and
Pathogenesis
3
4.
TB
Vaccine
4
5.
TB
DOTS
Program
5
6.
Diagnosis
5
6.1
Microbiological
Techniques
5
6.1.1
AFB
Microscopy
5
6.1.2
Bacterial
Culture
6
6.1.3
BACTEC
8
6.2
Histopathological
evidence
in
biopsy
specimens
9
6.3
Chromatographical
Techniques
10
6.3.1
High
Performance
Liquid
Chromatography
10
6.3.2
Gas
Chromatography
11
6.4
Immunological
Techniques
12
6.4.1
Mantoux
Test
12
6.4.2
Enzyme
Linked
Immunosorbent
Assay
12
6.4.3
Radioimmunoassay
13
6.4.4
Agglutination
Test
14
6.4.5
Immunoprecipitation
Test
14
6.4.6.
Fluorescent
Procedure
15
6.5
Molecular
Biology
Techniques
15
6.5.1
Polymerase
Chain
Reaction
15
6.5.2
DNA
Chip
17
6.5.3
LRM
Test
18
6.5.4
Nucleic
acid
probe
18
6.5.5
Restriction
Fragment
Length
Polymorphism
19
6.6
Radiological
Techniques
19
6.6.1
X-‐ray
19
6.6.2
Computed
Tomography
20
6.6.3
Magnetic
Resonanace
Imaging
21
6.6.4
Ultrasonography
22
6.6.5
Fibero-‐optic
Bronchoscopy
23
6.6.6
Laser
Therapy
23
6.7
Radionuclidic
emission
based
imaging
technique
27
7.
References
27
1
1. Introduction
Tuberculosis is a dreaded infectious, contagious disease and is one of the
major health hazards even with sophisticated research and various
diagnostic modalities for its detection and cure. Approximately, one third of
the world’s population has been infected with Mycobacterium tuberculosis
and there are six to eight million new cases of disease and two to three
million deaths each year. TB most commonly affects the lungs but also can
involve almost any organ of the body. Although manifestations of tuberculosis
are usually limited to the chest, the disease can affect any organ system and
in patients infected with human immunodeficiency virus usually involves
multiple extra-pulmonary sites including the skeleton, genitourinary tract,
and central nervous system.
2
2. Causative
agent
The causative agent of tuberculosis is Mycobacterium tuberculosis (MTB). It
is a rod shaped, non-motile, slow growing, aerobic, gram positive, facultative
intracellular parasite. Robert Koch first discovered MTB in 1882. Cole et al,
deciphered the genome of the H37Rv strain (virulent strain), which was
published in 1998. The size of the genome is 4 million base pairs, with 3959
genes. 40% of these genes have characterized functions. The genome contains
250 genes involved in fatty acid metabolism, with 39 of these involved in the
polyketide metabolism generating the wax coat. They have certain proteins
that impair growth and functions of macrophages.
3
drive it into a chronic, latent stage that is asymptomatic and potentially long
lasting. Antigen-specific immune response provides indirect read out of
bacterial metabolic changes during infection differing in active, inactive and
latent TB.
4. TB
Vaccine
(BCG)
BCG, or bacille Calmette-Guérin, is a vaccine for TB disease. In developing
countries, BCG vaccine as part of their TB control programmes, especially for
infants. This was the first vaccine for TB and developed at the Pasteur
institute. BCG vaccination is for persons who have a reaction of <5 mm
induration after skin testing with 5TU of PPD tuberculin. BCG vaccines are
live vaccines derived from a strain of Mycobacterium bovis that was
attenuated by Calmette and Guérin. BCG provides some protection against
severe forms of pediatric TB, but cannot be relied against adult pulmonary
TB. All currently used vaccines are derived from the original M. bovis strain.
These strains differ in their characteristics when grown in culture and in
their ability to induce an immune response to tuberculin. These variations
may be caused by genetic changes that occurred in the bacterial strains
during the passage of time and by differences in production techniques.
Methods and routes of vaccine administration, and by the environment and
characteristics of the populations in which BCG vaccines have been studied
affect the protective efficacy of BCG. BCG vaccination often results in local
adverse effects, serious or long- term complications are rare. If BCG is
accidentally given to an immunocompromised patient (with HIV infection,
leukemia or lymphoma) and immunosuppressed (whose immunologic
responses have been suppressed by steroids, alkylating agents, anti-
metabolites, or radiation), it can cause disseminated or life-threatening
infection. The first recombinant tuberculosis vaccine rBCG30, entered clinical
trials in the US in 2004. A very promising TB vaccine, MVA85A, is currently
in phase II trials in South Africa by a group led by Oxford University.
The basic strategies for the prevention and control of TB in USA include:
4
3. Prevention of institutional transmission. Effective TB infection-
control programs are implemented to prevent the transmission of
M. tuberculosis. TB is a recognized risk in health-care settings and
is a particular concern in places, where HIV-infected people work,
volunteer, visit, or receive care.
5. TB
DOTS
Program
The TB-DOTS program, which stands for Tuberculosis Directly Observed
Short-course, has five components:
The TB-DOTS program complies with the World Health Organization (WHO)
standards as a prescribed, cost-effective strategy to detect, treat and cure TB.
6. Diagnosis
There are two basic approaches for the diagnosis of tuberculosis. The direct
approach includes detection of mycobacteria or its products and the indirect
approach includes measurements of humoral and cellular responses of the
host against tuberculosis. Certain non-conventional diagnostic approaches
proposed included the search for biochemical markers, detection of
immunological responses and early detection other than colony counting
(Palomino, J.C., 2005). Tuberculosis is diagnosed by various conventional
diagnostic modalities, depending upon correlation of clinical findings,
radiological & bacteriological investigation and these modalities were
demonstrated by Holm et al, 1947. The key to the diagnosis of tuberculosis is
a high index of suspicion. The diagnostic methods used for detection of MTB
are microbiological, histopathological, immunological, chromatographical,
molecular biological, radiological & nuclear medicine based imaging.
5
examination of acid-fast bacilli (AFB) (Gordin et al, 1990; Hann et al, 1996).
In developing countries, microscopy of sputum is by far the fastest, cheapest
and most reliable method for the diagnosis of pulmonary tuberculosis. Direct
microscopic examination of sputum specimens from patients suspected of
pulmonary tuberculosis is routinely done in many hospitals. The sample
taken from patients are generally sputum but others can be used are
cerebrospinal fluid (CSF), pus, biopsy samples. The bacilli can be stained
with basic fuchsin dye or preferably with flourochrome (auramine-
rhodamine).
Since 1982, the mycobacteriology laboratory has been using the Ziehl-Neelsen
fuchsin stain (Z-N) for the detection of acid-fast bacilli (AFB) in the sputum.
Franz Ziehl and Frederick Neelson discovered ZN stain.
6
The main advantages of smear microscopy are that it is an inexpensive, simple
method and is easy to perform, read and detect transmitters of tubercle bacilli.
Certain disadvantages associated by this method are poor sensitivity
(technique sensitivity may vary depending on smearing, staining, and smear
reading), large number of organisms required/ml of specimen to study, false
positive results due to acid fast food particles, false negative results due to
improper collection, low prevalence which questions the validity of this
technique.
To culture for tuberculosis, portions of the sputum are placed in tubes of broth
that promote the growth of the organism; growth and identification may take
two to four weeks. The samples obtained are decontaminated by 2% NaOH & n-
acetyl cysteine to remove the pyrogens from the upper respiratory tract.
Centrifugation is done to remove the fat contents affecting the sedimentation of
the mycobacterium. Growth rate and pigmentation properties are used to
differentiate mycobacterial species.
The major advantages are include culturing of mycobacteria for its detection is
several times more sensitive than microscopy & few viable organisms can be
detected in the sample. The disadvantages in this method are that it is slow
(results come only after 2-6 weeks) and the treatment of patients is delayed
due to atypical presentation & slow confirmation by culture probably because
of long generation time of 18-24 hrs.
7
Figure 4. Mycobacterial grown in agar based media
8
solutions for TB diagnosis from specimen collection to final test result.
9
6.3 Chromatographic
techniques
HPLC uses a liquid mobile phase at high pressure to carry sample through
the column packed with particulate material or stationary phase, where the
separation of two components takes place (Butler and Kilburn, 1988; Butler
et al, 1991). HPLC is used to detect species-specific (till genus level) mycolic
acid produced by mycobacteria ((Butler, W.R et al, 1986). Mycolic acids
extracted from saponified mycobacterial cells are converted to the p-
bromophenacyl esters, and the unique mycolic acid pattern associated with
each species is detected by chromatographic separation of the esters.
10
Sherlock mycobacteria identification system (SMIS)
It uses computerized software to identify mycobacterial species on the basis
of mycolic acid pattern generated by HPLC.
GLC uses gas (hydrogen) in the mobile phase and liquid in the stationary
phase. The analysis of the microbial short-chain fatty acids (methyl esters) by
GLC is based on the comparison of retention time of the tested sample to the
retention times of the known standards. Certain mycobacteria (including
MAC), contain bound wax estermycolates, which yield long-chain secondary
alcohols, particularly, 2-eicosanol, when subjected to hydrolysis. This can be
detected by HPLC. It is used to study short-chain fatty acids & cleavage
products of mycolic acid by selected ion monitoring of CSF from mycobacteria
but differentiation within M.tb complex is not possible. The presence of
tuberculostearic acid in spinal fluid or serum of patient has been detected
using GLC & mass spectrophotometry (Mardh et al, 1948; Brooks et al, 1987;
French et al, 1987). This is a rapid, sensitive & specific technique for
detection of tuberculosis meningitis (French et al, 1987). This method is very
important tool in better determination of species of non-tuberculous
mycobacteria, MAC, M. mal- moense, and M. tuberculosis causing pulmonary
TB.
The major drawbacks of this method are that it requires expensive &
analytically complex equipment. Single false positive result occurs probably
by intrathecal treatment with amikacin. Difficulty in standardization as
specialized expertise is required. Negative result is obtained in patients
suffering from systemic lupus erythromatosus.
Figure 6. GC instrument
11
6.4 Immunological
techniques
12
Various advantages of ELISA ARE:
Figure 6. ELISA instrument
S.A Berson & R.S Yalow, developed this method in 1950’s for the
determination of insulin in human serum. It is a very sensitive technique
used to measure concentrations of antigens.
RIA is highly specific, easy, cost effective, objective and sensitive method. It is
13
useful in the diagnosis of sputum negative samples but repeated labeling of
the samples is required as half-life of I123 is 60 days and I131 is 8 days
(radionuclide used for the assay), radiation hazards and problems related to
radiowaste disposal are the loopholes of this method. It requires special
precautions and licensing, since radioactive substances are used.
Middlebrook and Dubois found that carbohydrates from the tubercle bacillus
passively gets absorbed to untreated RBC of sheep and those antibodies from
TB patients caused agglutination. The kaolin agglutination test (KAT) has
been applied in the diagnosis of pulmonary tuberculosis patients in Kenya,
East Africa.
14
6.4.6 Fluorescent
procedures
Nassau and Merrick presented encouraging result with the use of fluorescent
antibodies test using sera of 248 bacteriologically proved cases of TB. The test
consists of fixation of smears of M. tuberculosis, reaction of test serum upon
addition of fluorescence coupled goat anti-human IgG and measurement of
fluorescence. This test is referred to as modified soluble antigen fluorescent
antibodies (SAFA). Chaparas et al. introduced it as serodiagnostic test of
tuberculosis. Comparative studies of fluorescent antibody tests was done for
tuberculosis and paratuberculosis with antigens coupled to insoluble spheres
or taken up by macrophages, indicating that it might have good prospects for
routine examination for antibodies against species of Mycobacterium
(Goudswaard, J. et al).
15
Figure 7. Schematic Representation of the PCR cycle.
Steps involved are:
1. Denaturing at 96°C.
2. Annealing at 68°C.
3. Elongation at 72°C (P=Polymerase enzyme).
The first cycle is complete. The two resulting DNA strands make up the
template DNA for the next cycle, thus doubling the amount of DNA
duplicated for each new cycle.
Advantages of this method are that it is highly specific and sensitive method,
which helps in detection of resistant forms. PCR amplification followed by
single strand complimentary polymorphism and directs DNA sequencing
helped in identifying rifampicin & streptomycin resistance. It is helpful in
diagnosis of cases of pleural effusion, neurotuberculosis, oculat TB,
tubercular infection in skin and lymph nodes, bone, kidney, genitals and GIT.
The disadvantages associated with this method are difficulty in preparation
of sample for PCR amplification & there is possibility of contamination of
sample with extraneous, undesirable genetic material.
16
Polymerase Chain Reaction Commercial kits for PCR
Direct comparison was done for both types of kits. Studies showed both
nucleic acid amplifications are rapid, sensitive and specific for the detection
of M.TB in respiratory specimens.
It is a microchip that holds DNA probes that form half of the DNA double
helix and can recognize DNA from samples being tested. A technology still
under development that appears promising involves oligonucleotide arrays or
DNA chips (molecular biology coupled with computer technology), which have
been designed to determine the specific nucleotide sequences diversity of
rpoB and 16s rRNA genes for species identification. DNA chip is combined
with an image analysis system for TB detection (assay of IS 6110 gene). This
method detects M. tuberculosis complex rapidly in respiratory specimens,
readily adapts to routine work and provides a flexible choice to meet different
cost-effectiveness and automation needs in TB-endemic countries.
17
Figure 8. DNA microchip
This method is rapid (48-72 hrs), sensitive & specific. It can be used to
distinguish between sensitive & resistant strains. LRP’s are comparable in
sensitivity, specificity and speed to the MGIT 960 and BACTEC 460. This
method is cumbersome, so it is not preferred.
18
within 1 or 2 days with gene probes as compared to much longer time
required with classical biochemical tests. Recently, ribosomal RNA gene
region has been extensively explored for designing systems for ribosomal
DNA fingerprinting and for development of probes/ as well as gene
amplification assays for various types of mycobacterial species including M
tuberculosis, M leprae, M avium, M gardonae.
6.6.1 X-‐ray
19
atelectasis can be distinctly seen, while postprimary tuberculosis, the earliest
radiologic finding is the development of patchy, ill-defined segmental
consolidation.
Major disadvantages are that there are chances of getting normal image in
infected person, non-specificity & exposure of patients to harmful radiation.
The main limitations of the chest radiography as a survey tool pertain to the
judgement of the activity of the lesion and the objective interpretation of
radiological abnormalities, in particular the etiology of the shadows. The
enlarging lymph glands may constrict bronchi and cause lobar or segmental
collapse, but bronchial ulceration from (usually post-primary tuberculosis) is
also fairly common and explains the occurrence of non-bovine tuberculosis
elsewhere in individuals with a normal chest x-ray. Further, the
interpretation of abnormal shadows seen in the x-ray is influenced by the
experience of the x-ray reader and the overall impression about the
prevalence of disease in the community.
20
1. CT can give important information in case of pulmonary tuberculosis
and chest with normal radiograph (Davidson et al).
2. High resolution CT predicts the disease activity in sputum smear-
negative pulmonary tuberculosis (Lai et al, 1997).
3. It is a sensitive method for detection of cavities, tuberculous
spondilitis/arthritis, renal, intracranial, ureteric, and bladder
tuberculosis and is superior to conventional radiography.
4. CT shows a ring enhanced hypodense soft tissue mass surrounding the
sternum, with marked cortical thickening (Allali et al, 2005).
5. Involvement of the liver and spleen in miliary tuberculosis may appear
on CT as tiny low-density foci widely scattered throughout the organ.
21
tuberculosis (Kisore et al, 2001) and the regions blind to CT.
The limiting factor of this technique is cost ineffectiveness & its dependence
upon anatomical changes in the lesion. Fistulae or sinus tract formation may
complicate bladder tuberculosis although these complications are rare and
are demonstrated better on CT and MRI scans.
6.6.4 Ultrasonography
22
6.6.5 Fiberoptic
bronchoscopy
(FOB)
It is highly specific and sensitive method, which not only detects but also
locates the pathological lesion and also gives idea of the size of lesion.
Instrumentation and radiopharmaceuticals make the nuclear medicine
modality useful in the diagnostic field. For the diagnosis of the disease the
radiopharmaceutical is injected to the patient and then image is taken under
gamma camera. Radiopharmaceuticals are chemical compound containing
radioisotopes within its structure and are used in the field of nuclear
medicine as tracers in the diagnosis and treatment of several diseases. 85% of
23
radiopharmaceuticals are used for the diagnostic purposes and 95% of them
are technetium-99m labeled.
24
gallium-67 scan and computed tomography.
7. Radiolabeled monoclonal antibodies are used in detection of
tuberculosis.
8. 99mTc-ciprofloxacin has done the diagnosis of deep-seated tubercular
infections (Britton, K.E. et al, (2002).
9. Tuberculosis meningitis and apoptosis is detected by labeling antigens
and antibodies with iodine.
10. Radiolabeled leucocytes are used in diagnosis of pulmonary TB.
25
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26
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29