Diagnostic  Methods  for  Tuberculosis    Namrata  Singh,  PhD  

 

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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.

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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

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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

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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:

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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

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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.

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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

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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

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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 Culture Gold standard Time consuming Microbial Tests BACTEC test Specific Easy sampling GLC CNS specific Non-specific Chromatographic

Techniques HPLC CNS specific Not sensitive ELISA Easy sampling Non-specific Immunological

Tests Mantoux test Easy sampling Non-specific

Molecular Biological tests PCR Sensitive Doubtful results

X-ray Sensitive Non-specific CT Sensitive Non-specific

Radiographic and Imaging

MRI Sensitive Cost ineffective

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