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Diagnostic

 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  
 
 

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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.

Of the several distinct components of the TB Control program, case finding


remains the corner stone for the effective control. However, there are no
definite guidelines available as of today as to how to use optimally the
number of diagnostic tests ranging from simple The vast majority of people
who have TB germs in their bodies do not have an active case of the disease.
Even if the disease is active, the disease is quite advanced. Simple AFB
microscopy to complex molecular biological techniques has become available
over a period, to establish or rule out diagnosis of tuberculosis in a given
patient. Although, detection and cure of smear positive TB remains foremost
priority of control program, diagnosis and management of smear negative TB
cases can’t be overlooked.

Figure 1. Prevalence of tuberculosis in 2009 (Reports from WHO)

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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.

Figure 2. Mycobacterium tuberculosis

3. Transmission  and  Pathogenesis


People suffering from active pulmonary TB coughs, sneeze, speak, or spit;
they expel infectious droplets (aerosolized) 0.5 to 5 µm in diameter. Each one
of these droplets contain the bacilli and may transmit the disease, since the
infectious dose of tuberculosis is very low and inhaling less than ten bacteria
may cause an infection. Pathogenesis of tuberculosis is basically dependent
upon the interplay between immune response and presence & multiplication
of bacilli in the host tissue (Des Prez et al, 1985). In healthy individuals (if
infected by the bacilli) progression of disease may hault due to strong cell
mediated immune response, but pathogens may remain latent. Reactivation
of the active disease may occur depending upon genetic influences,
nutritional status, or immune response. Infection with tuberculosis passes
through several stages. In most cases, host defenses either clear infection or

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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.

TB Prevention and Control in the United States:

The basic strategies for the prevention and control of TB in USA include:

1. Early detection and treatment of patients with active TB disease.


The most important strategy for minimizing the risk for M.
tuberculosis transmission is the early detection and effective
treatment of persons who have infectious TB.

2. Preventive therapy for infected persons. Identifying and treating


persons infected with M. tuberculosis can prevent the progression
of latent infection to active infectious disease.

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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:

1. Political or Management commitment.


2. TB diagnosis through sputum microscopy (x-ray is only a
secondary diagnostic tool).
3. Availability of complete and quality anti-TB medications.
4. Supervised treatment (a responsible person making sure that the
patient takes the anti-TB medication everyday).
5. Recording and reporting of cases and outcomes.

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.

6.1 Microbiological  techniques  


6.1.1 AFB  microscopy
Use of microscopy in rapid detection of tuberculosis is of great value
especially in detection of active disease. This technique involves microscopic

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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.

Other methods are silver impregnation and cold techniques. Flourochrome


method is advantageous over ZN staining as

1. Fluorochrome staining is found to be more efficient (45%) in


comparison to ZN staining (29%) in detecting cases associated
with HIV seropositivity, especially paucibacillary cases.
2. Samples can be scanned at lower magnification as bacilli
fluorescence bright yellow against a dark background.
3. Negative smears can be scanned in 2-3 minutes rather than 15
minutes by Ziehl-Neelson method.
4. Non tuberculous Mycobacteria are as likely as M. tuberculosis to
be detected by fluorescent microscopy in specimens from patients
from areas endemic for NTM lung disease and at low risk for
AIDS.

Figure 3. Photomicrograph of Mycobacterium tuberculosis using acid-fast stain

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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.

6.1.2 Bacterial  culture

The definitive diagnosis of tuberculosis continues to depend on culture of


M.tuberculosis from secretions or tissue from infected host together with
compatible clinical picture of the disease. Sputum smear and culture still
remains “Gold standard” for the diagnosis of tuberculosis and valuable test for
screening suspects of PTB.

Various medias used for culturing includes Middlebrook 7H10/7H11/7H12,


Lowenstein-Jensen Petragnani, ATS medium etc. In developing countries,
Löwenstein Jensen (LJ) medium is the most commonly used me- dium for
culture of Mtb (recommended by WHO). Recently blood agar media has been
used for culturing the mycobacteria (Drancourt M. et al, 2002).

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.

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Figure 4. Mycobacterial grown in agar based media

6.1.3 BACTEC  (BACTEC,  Becton  Dickinson)  method

This commercial technique can be used to overcome the drawback of poor


sensitivity of microscopic technique & time consumption of bacterial culture.
This rapid radiometric culture system has been accepted for the culture
isolation of mycobacteria using an enriched Middlebrook 7H12 containing
14C labeled palmitic acid.

The method involves detection of mycobacteria based on their metabolism


rather than their visible growth. Growth of mycobacteria is detected by
quantitatively measuring of the 14 CO 2, liberated by the metabolism of 14C-
labeled substrate present in the medium. Growth is defined in terms of
growth index.

The main advantage of BACTEC culture system over the conventional LJ


culture technique is early detection of smear positive specimens. This method
shortens the time of detection from 4 weeks (in case of bacterial culture) to 7-
14 days (Middlebrook et al, 1997; Roberts et at, 1983). BACTEC 460 system
can differentiate between TB complex from mycobacteria other than
tuberculosis (MOTT) bacilli. It can differentiate between live/dead and
sensitive/resistant bacilli. The disadvantages include potential contamination
between culture bottles, aerosolization while monitoring culture bottles,
problems related to use and disposal of radioactive material, labour required
during loading/unloading of bottles. BD diagnostics has the launch of the BD
BACTEC™ MGIT™ 320 mycobacteria culture system a new, smaller capacity
system to quickly and accurately detect tuberculosis (TB) by offering range of

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solutions for TB diagnosis from specimen collection to final test result.

Panta-plus kit: This kit is modified Mitchinsons combination of


antimicrobial drugs i.e. Polymixin B, Amphotericin 8.5, Nalidixic acid,
Trimethoprim, and Azocillin. This mixture is specially formulated in
BACTEC system to isolate mycobacteria. It suppresses the growth of residual
normal microbial flora, which may have survived during the digestion &
decontamination process of specimen. Growth observed is due to
mycobacteria.

NAP TB differentiation test: Susceptibility to p-nitro-α-amino-β-


hydroxypropiophenon (NAP) & p-nitrobenzoic acid used in BACTEC TB
system to differentiate M. tuberculosis complex from other mycobacteria. The
growth of tuberculosis complex is inhibited by NAP, which is determined by
decrease or no increase in 14CO2 output while other bacteria grow in presence
of NAP & show an increase in 14CO2 output.

MGIT (Mycobacteria growth indicator tube): New method developed


by Becton Dickinson microbiology system for the detection of mycobacteria. It
contains a modified, non-radioactive 7H9 broth in conjunction with a
fluorescence quenching-based oxygen sensor it allows rapid antimicrobial
susceptibility test (AST), and thus, overcomes the limitations of radiometry
(BACTEC).
Sensitivity & time is comparable to BACTEC 460. The fluorescent compound
is contained in a silicon plug at the bottom of tube, which contains
mycobacterial growth medium. The dissolved O2 in the medium quenches any
fluorescence from compound. However, inoculation of medium with sample
containing mycobacteria & their subsequent growth, utilizes the O2 & thus
compound fluoresce. The fluorescence is detected by UV-transilluminator.
Detection of isolates is rapid as multiple tubes can be inspected
simultaneously, no instrumentation is required & resistant forms can be
identified. Limiting factors include inefficiency in detection of deep-seated or
occult lesion, lower rate of recovery was observed in smear negative clinical
specimens & cost ineffectiveness.

6.2 Histopathological  evidence  in  biopsy  specimens

Tissue biopsy is done to observe the histopathological changes in the cells


infected by M. tuberculosis and diagnosis of TB.

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6.3 Chromatographic  techniques

6.3.1 High  performance  liquid  chromatography  (HPLC)

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.

Denaturing HPLC is now being used to rapidly identify rifampicin resistant


Mycobacterium tuberculosis in low as well as high-prevalence areas. It
provides definitive species identification for more than 50 mycobacterial
species and mutation in less than 3 hrs. Highly sensitive, accurate (98.6%) &
automated HPLC that utilizes fluorescence detection of mycolic acid to
identify MTB & MAI complex directly from fluorescence stained smear-
positive sputum specimens & young BACTEC cultures. It can detect XDR as
well as MDR TB. It can be carried out in 2 hrs but it requires standarization
of the instrument, large number of organisms/ml of specimen is required to
carry out the experiment and is costly.

Figure 5. HPLC instrument

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Sherlock mycobacteria identification system (SMIS)
It uses computerized software to identify mycobacterial species on the basis
of mycolic acid pattern generated by HPLC.

6.3.2 Gas  chromatography

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

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6.4 Immunological  techniques

Immunological techniques help in the diagnosis of tuberculosis in patients


with smear negative & extrapulmonary tuberculosis. These techniques are
based on immunologic perspective of TB bacilli & host response. Antigenic
diversity of Mycobacterium tuberculosis and Mycobacterium bovis is detected
by means of monoclonal antibodies (Coates, A.R.M et al, 1981).
Immunological tests for the detection of M. tuberculosis antigens and
antibodies to the antigens have been explored to identify individuals at risk
of developing TB or with latent TB infection. These methods measure specific
cellular or humoral responses of the host to detect presence of infection or
disease. They do not require a specimen from the site of infection.

6.4.1 Mantoux  test

It is an interdermal test using 0.1ml (5TU) of PPD (Purified Protein


Derivative) (Nash et al, 1980). It is also called Mantoux screening test,
Tuberculin Sensitivity Test, Pirquet test, or PPD test for Purified Protein
Derivative. Purified protein derivative (PPD) tuberculin is a precipitate of
non-species-specific molecules obtained from filtrates of sterilized,
concentrated cultures. The induration of more than 15mm is indication of
positivity to infection. This test is based on the fact that mycobacterial
infection produces delayed hypersensitivity to certain products of organism
contained in culture extracts called “Tuberculin”. Seibert et al, 1994, studied
chemistry of tuberculin and Sokol et al, 1975 did the measurement of delayed
skin test responses.

A false positive result may be caused by nontuberculous mycobacteria or


previous administration of BCG vaccine. False negative result comes in those
that are immunologically compromised.

6.4.2 Enzyme  Linked  Immunosorbent  Assay  (ELISA)

It is a serodiagnostic method for diagnosis of tuberculosis. It is an antigen-


antibody based assay. It was first used by Nassuau et al, 1976 for the
detection of IgG antibody to antigen of M.tuberculosis. Grange et al, 1980
used ELISA to evaluate antibodies to mycobacterium in IgG, IgA and IgM
classes. There are three methods of enzyme immunoassay:

1. Double antibody sandwich ELISA.


2. Indirect ELISA.
3. Competitive ELISA.

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Various advantages of ELISA ARE:

1. It cab be utilized to detect various antibodies against M. tuberculosis.


2. ELISA can be performed using various antigen preparation of M.
tuberculosis.
3. It can be performed in any body fluid i.e., serum, CSF, and sputum.
4. It can be repeated in shorter intervals without developing booster
response as in Mantoux test & doesn’t require patient to attend, for
reading of values.
5. Paramedical person can collect serum and small amount of serum is
sufficient for analysis (Tandon et al, 1980).
Disadvantages of this method are that the localization of exact site of lesion
particularly for extra-pulmonary tuberculosis is difficult. Decreased
sensitivity hampers serologic test & problem of cross-contamination with
other mycobacteria occurs. High antibody titer using ELISA can’t distinguish
between infection with atypical & typical mycobacteria or active disease. No
standardization so far, is done for the diagnostic utility of ELISA to be
exploited.

           

     
Figure 6. ELISA instrument

6.4.3 Radioimmunoassay  (RIA)

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

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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.

6.4.4 Agglutination  tests

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.

Agglutination tests was made more sensitive by Jagganath et al by the use of


Staphylococcus aureus bearing protein A (SAPA), which binds to
immunoglobulin SAPA was added to the system just prior to the addition of
sensitized erythrocytes. Sensitivity increased 2 to 8 times more as compared
to passive haemagglutination system. Major advantages of this method are
that the result can be obtained in one day, material used for the test can be
stored at room temperature, technically easier to perform and it is an
economical method and can be applied on mass scale. Large-scale validation
of a latex agglutination test has been done for diagnosis of tuberculosis.

6.4.5 Immuno-­‐precipitation  tests

An Immuno-precipitation test basically involves:

1. Gel matrix precipitation.


2. Immunoelectrophoresis.

In gels, immunodiffusion with antigens and antibodies may occur in one or


two dimensions. These methods are among the least sensitive method for the
antibody detection but permits direct comparison of precipitation bands, to
establish identity or non-identity. To detect antigen of the clinical samples
precipitation should be in large amount and radiolabeling of the samples with
gamma emitting isotope was done to detect it with appropriate photographic
emulsions. Immunoelectrophoresis includes conventional, rocket IE and 2-D
IE (crossed IE).
This method is a useful tool but excessive number of precipitation bands
makes it difficult to relate antibodies in a patient’s serum from reference.

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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).

6.5 Molecular  biology  technique  

Molecular biology plays important role in study of genetic material especially


with the introduction of PCR. DNA recombinant technology has helped in
identifying immunodominant antigens, promiscuous T-cell epitopes of
diagnostic and vaccine potential tuberculosis. Many molecular tests for
detecting, identifying and testing antibiotic susceptibility have been reported
since several years. These methods do not require a culture and can
significantly reduce diagnosis time from a few weeks to a few days. There is a
tremendous need for molecular tests that can sensitively detect the presence
of M. tuberculosis in clinical samples, as well as its possible resistance to
antibiotics

6.5.1 Polymerase  chain  reaction  (PCR)

Diagnostic PCR is a rapid technique of DNA amplification that uses specific


DNA sequences to serve as markers for the presence of microorganisms
(Eisenstein et al, 1990; Cousins et al, 1990; Markham, 1993). It is highly
specific and sensitive method (Noordhoek et al, 1994). It is helpful in
detection of Mycobacterium tuberculosis in HIV infected as well as
extrapulmonary cases (Torrea et al, 2006).
Various clinical samples with various sets of primers to amplify
mycobacterial DNA have been used. This assay uses primer specific for the
conserved regions in target genes to produce amplified products from
mycobacteria. Biotinylated primers are used to label amplicon. Most
commonly used genetic marker in mycobacteria is IS6110, a highly specific
insertion element present in genome. Amplification of specific DNA
sequences is done using Taq polymerase enzyme. Ribosomal RNA (rRNA)
sequences are also of choice asamplification targets as these sequences are
found in abundance (Boddinghaus et al, 1990).

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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.

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Polymerase Chain Reaction Commercial kits for PCR

1. Amplicor: Amplicor assay is developed by Roche diagnostic


System. It is the first benchtop system to fully automate the
amplification and detection steps of the Polymerase Chain
Reaction (PCR) testing process on a single instrument. It
combines five instruments into one (thermal cycler, automatic
pipettor, incubator, washer and reader). It is a PCR kit used to
amplify a target within the 16sRNA of the M.TB. Studies have
proved that Amplicor PCR is more specific (98%) than PCR using
IS6110 as a target (79%).

2. Amplified Mycobacterium Tuberculosis Direct Test


(AMTD): AMTD is developed by Gen-Probe. It is a direct
specimen assay for the identification of M.TB from respiratory
samples and can be completedin 4-5 hrs. In this method RNA is
amplified and the product is detected with a specific
chemiluminescent probe. The MTD test is intended for use only
with specimens from patients showing signs and symptoms
consistent with active pulmonary tuberculosis and can be used
as an adjunctive test for evaluating either AFB smear positive
or negative sediments prepared using NALC-NaOH digestion-
decontamination of respiratory specimens

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.

6.5.2 DNA  chips

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

6.5.3 Luciferase  Reporter  Mycobacteriophage  (LRM’s)  test

The utility of luciferase reporter mycobacteriophages (LRPs) for detection,


identification, and antibiotic susceptibility testing of Mycobacterium
tuberculosis has been evaluated in a clinical microbiology laboratory. This is a
rapid system based on firefly luciferase to measure the drug susceptibility of
mycobacterium. This test uses mycobacteriophage, a virus that infects M.
tuberculosis & has cloned gene for production of luciferase reporter enzyme.
The luciferase mycobacteriophage (LRM) when mixed with culture of
bacterial cell, results in production of light in presence of ATP. If the drug
kills the bacteria no light is produced, revealing sensitivity of organism to
drug.

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.

6.5.4 Nucleic  acid  probes

Nucleic acid probe is a DNA or RNA fragment, labeled by a radioisotope,


biotin, etc., that is complementary to a sequence in another nucleic acid
(fragment) and that will, by hydrogen binding to the latter, locate or identify
it and be detected; a diagnostic technique based on the fact that every species
of microbe possesses some unique nucleic acid sequences which differentiate
it from all others. Commercially marketed probes for M tuberculosis and M
avium are also available.

It is based on specific DNA sequence present in mycobacteria. The sensitivity


of this method is equivalent to smear examination by Ziehl-Neelson staining
and it is highly specific for identification of M.tuberculosis, MAC, M. avium
(Drake et al). These probes are being used in for rapid confirmation of the
identity of mycobacterial isolates. When used along with other techniques
(such as BACTEC, Septi-Chek, MGIT) detecting growth of bacilli, it is very
useful in rapidly confirming the diagnosis as identity can be established

  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.

6.5.5 Restriction  fragment  length  polymorphism  (RFLP)

Restriction Fragment Length Polymorphism (RFLP) is a difference in


homologous DNA sequences that can be detected by the presence of
fragments of different lengths after digestion of the DNA samples in question
with specific restriction endonucleases. RFLP, as a molecular marker, is
specific to a single clone/restriction enzyme combination DNA fingerprinting

It is a new technique helping in strain identification of the microorganisms. It


relies on repetitive DNA elements in the chromosome of the bacteria i.e.
IS6110, a 1361 base pair insertion sequence, specific for the Mycobacterium
tuberculosis. In this technique, DNA is extracted from the culture, cleaved by
restriction endonuclease & DNA samples are separated by electrophoresis
(Southern blotting), hybridized & detected by labeled DNA. The DNA from
each mycobacterial isolate is depicted as series of bands on x-ray film to
create fingerprints. Banding pattern reflecting number & position of IS6110
within chromosome is obtained. Molecular epidemiology of tuberculosis can
be known by this method. Distinction of sensitive/resistant strains &
reactivation/or reinfection with new strains can be done.

6.6 Radiological  and  imaging  methods

Radiology is the branch or study of medicine that utilizes imaging


technologies like x-rays, CT scans, and MRIs to diagnose and treat diseases.
No other field of medicine has played such a big role as in that of chest
medicine excepting orthopedic surgery.

6.6.1 X-­‐ray

It is especially useful in pulmonary tuberculosis (Leung et al, 1999), in which


postero-anterior view of chest is taken (Hlawatsch et al, 2000). In
disseminated TB (miliary TB), a pattern of many tiny nodules throughout the
lung fields is common. It is efficacious in the detection of active pulmonary
tuberculosis in patients with acquired immunodeficiency syndrome (AIDS). It
is a very rapid method. Different features at each stage of pulmonary
tuberculosis are helpful in its diagnosis such as in primary stage
lymphadenopathy, pleural effusion, miliary disease, or lobar or segmental

  19  
atelectasis can be distinctly seen, while postprimary tuberculosis, the earliest
radiologic finding is the development of patchy, ill-defined segmental
consolidation.

Figure 9. Chest radiograph of patient having TB (indicated by arrows)

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.

Abreugraphy (also called Chest photofluorography, or mass miniature


radiography) is a technique for mass screening of tuberculosis using a
miniature (50 to 100 mm) photograph of the screen of x-ray fluoroscopy of the
thorax, first developed in 1935. It has been used to detect asymptomatic
tuberculosis.

6.6.2 Computed  tomography  (CT)

CT is a powerful nondestructive evaluation (NDE) technique for producing 2-


D and 3-D cross-sectional images of an object from flat X-ray images. A basic
problem in imaging with x-rays (or other penetrating radiation) is that a two-
dimensional image is obtained of a three-dimensional object, which was
solved in the early 1970s with the introduction of CT.
Various advantages are:

  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.

Figure 10. Computed Tomography scanner

Diadavantages of CT are that it can’t distinguish cavities formed by atypical


mycobacterium and similar appearance of lesion is observed in other
granulomatous such as cysticercosis, fungal, amoebic and non-specific
infections. Repeated imaging leads to risk of exposure of patient to harmful
radiations same as Χ-ray.

6.6.3 Magnetic  resonance  imaging  (MRI)

Magnetic resonance imaging (MRI) is a noninvasive imaging methodology,


which helps in diagnosis and treatment of various diseases. It is primarily a
medical imaging technique that is used in radiology to visualize detailed
internal structure and limited function of the body. To perform MRI, patient
is placed in the external magnetic field of the MRI magnet. It can
demonstrate the lesion in brain stem, temporal lobes, posterior fossa, spinal

  21  
tuberculosis (Kisore et al, 2001) and the regions blind to CT.

Figure 11. MRI scanner

MRI can distinguish between tuberculomas and myelomas (Chung et al,


2000) and diagnose intracranial tuberculomas (Gupta et al, 1988). MRI is
useful in the evaluation of peritonitis and adnexal masses. It is helpful in
detecting pott’s disease. High-resolution computer tomography (HRCT) scan
and 99mtechnetium-methylene diphosphate (99mTc- MDP) bone scintigraphy
are more sensitive and specific than the chest X-ray for the detection of
pulmonary calcification. MRI can demonstrate different pathological stages of
hepatic tuberculoma, providing reliable clues to a correct diagnosis.

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

Diagnostic sonography (ultrasonography), is an ultrasound-based diagnostic


imaging technique used to visualize subcutaneous body structures including
tendons, muscles, joints, vessels and internal organs for possible pathology.
This method is useful in picking up early pleural pathology especially
minimal pleural effusion. It is useful in detection of genitourinary TB. It can
accurately demonstrate small quantities of ascitic fluid and is an effective
method for detection of peritoneal disease. Ultrasound has the advantage of
being less expensive, widely available, and easy to perform in comparison to
CT.
Sonography is not as sensitive as intravenous urogram or CT scanning
because of problems in identifying calyceal, pelvic, or ureteric abnormalities.

  22  
6.6.5 Fiberoptic  bronchoscopy  (FOB)

Fiberoptic bronchoscopy is a procedure that allows a clinician to examine the


breathing passages (airways) of the lungs that can be used for diagnostic as
well as therapeutic purposes. Role of FOB in diagnosis of pulmonary
tuberculosis is very useful in patients with smear negative AFB microscopy.

1. Bronchoalveolar lavage: It is called liquid biopsy of lungs, useful in


diagnosis of pulmonary tuberculosis.
2. Bronchoscopic biopsies: It involves direct biopsy of endobronchial
lesion, transbronchial biopsy or transbronchial needle aspiration.
3. Direct Biopsy. Transbronchial Biopsy (TBLB). Transbonchial
needle aspiration (TBNA).

6.6.6 Laser  therapy

Laser therapy has been tried for the management of tuberculosis. It is


supposed to be effective in multicavitary disease with heavy bacterial load.
Laser has two advantages. First, it has capacity to kill bacteria rapidly
thereby decreasing bacillary load. Second, laser improves the penetration of
antitubercular drugs in the walled off lesion and help in early cavitary
closure.

It is of proven benefit in cases with tracheal and bronchial stenosis,


lymphadenopathy and sinuses. In patients with resistant tuberculosis
undergoing surgical treatment various types of laser have been given.
Surgical (CO2 and YAG) and therapeutic lasers (Helium-Neon, Ultraviolet
and semiconductive) are now used. This method is unable to localize the
lesion and detect deep-seated lesion.

6.7 Radionuclidic  emission  based  imaging  technique

It is used as nuclear medicine technique. This modality has gained universal


acceptance as one of the most powerful discipline in non-invasive diagnosis.

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.

Radiopharmaceuticals used for detection of tuberculosis are:

1. Tc-99m tetrafosmin (Degermenci, B. et al, 1998) and Tc-99m MIBI


(onsel, C. et al, 1998), 99mTc-hexakis methoxy isobutyl isonitrile
(Mehrossadat Alavi, 2008) are used in the diagnosis of pulmonary TB.

2. 99mTc-INH and 99mTc-EMB has been used successfully in diagnosis of


pulmonary or bone tuberculosis (Singh, N. et al, 2009, Singh, N., 2010).

Figure 12. Localization of 99mTc-INH in patient with bone TB in ankle

3. Technetium-99m hexakis-2-methoxyisobutyl isonitrile is used in the


detection of neoplastic lung lesions (Santini, Mario et al, 2008).
4. 18F-FDG PET to detect infection or the inflammatory response has been
established in numerous disease processes including tuberculosis. Dual-
Phase 18F-FDG PET has been used in the diagnosis of pulmonary
nodules in a patient with tuberculosis.
5. Occult abscesses in tuberculosis patients were detected with 111In-
labeled leukocytes (WR Martin et al, 1979).
6. Extrapulmonary, peritonitis, and military TB, can be detected with

  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.

Table 1 Comparison of various diagnostic methods to detect TB

Type of test Test Advantages Limitations

AFC counts Specific Not sensitive


Microbial Tests Culture Gold standard Time consuming
BACTEC test Specific Easy sampling
Chromatographic GLC CNS specific Non-specific
Techniques HPLC CNS specific Not sensitive
Immunological ELISA Easy sampling Non-specific
Tests Mantoux test Easy sampling Non-specific
Molecular
PCR Sensitive Doubtful results
Biological tests
X-ray Sensitive Non-specific
Radiographic and
Imaging CT Sensitive Non-specific
MRI Sensitive Cost ineffective

  25  
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