Molecular Biological Techniques for Detection of Multidrug Resistant Tuberculosis (MDR) and Extremely Drug Resistant Tuberculosis (XDR) in Clinical Isolates of Mycobacterium tuberculosis

Tuberculosis (TB) is a contagious disease caused by Mycobacterium tuberculosis (M. tuberculosis). When people infected with tuberculosis cough, sneeze, talk or spit, the bacilli are propelled into the air. Each person with active TB disease will infect on average between 10 and 15 people every year. But people infected with TB bacilli will not necessarily become sick with the disease. The immune system "walls off" the TB bacilli which, protected by a thick waxy coat, can lie dormant for years. When the immune system is weakened, the chances of the infection progressing to disease are higher.

caused by TB bacilli resistant to isoniazid and rifampicin, the two most powerful anti-TB drugs.Rates of MDR-TB are high in some countries, especially in the former Soviet Union, and threaten TB control efforts [WHO, 2010].
While drug-resistant TB is generally treatable, it requires extensive chemotherapy (up to two years of treatment) with second-line anti-TB drugs which are more costly than first-line drugs, and which produce adverse drug reactions that are more severe, though manageable.Quality assured second-line anti-TB drugs are available at reduced prices for projects approved by the Green Light Committee [WHO, 2010].
The emergence of extensively drug-resistant (XDR) TB, particularly in settings where many TB patients are also infected with HIV, poses a serious threat to TB control, and confirms the urgent need to strengthen basic TB control and to apply the new WHO guidelines for the programmatic management of drug-resistant TB.
The important ramifications in the laboratory diagnosis of tuberculosis and drug resistant tuberculosis are: the delay in the isolation of the bacilli in culture, low sensitivity/detection limit of the direct smears and lack of technically trained personnel.Identification of tuberculosis using molecular techniques namely, polymerase chain reaction (PCR), PCR based restriction fragment length polymorphism (PCR-RFLP) and PCR based DNA sequencing are very rapid, more sensitive and reliable when compared to the conventional culture.There are several in house nested PCR (nPCR) standardized for the detection of M. tuberculosis from clinical specimens targeting MPB64 [Therese KL et al, 2005], IS6110 [Wang et al, 2004] and 16S rRNA [Ninet et al, 1996] genes.MPB64 is an immunogenic protein produced by M. tuberculosis and a few strains of M. bovis and BCG strain.IS6110 is an insertion element present in single or multiple copies in M. tuberculosis complex isolates.There are studies which reported on the lack of IS6110 insertion element in strains isolated from Asian population.Also there are strain variations among the M. tuberculosis isolates.Thus multiplex PCR or nPCR targeting more than one gene target will be a better tool for the detection of M. tuberculosis from direct clinical specimens.
Drug resistance in M. tuberculosis is caused by mutations in relatively restricted regions of the genome.Mutations associated with drug resistance occur in rpoB for rifampicin (RIF) katG and the promoter region of the mabA (fabG1)-inhA operon for Isoniazid (INH), embB for ethambutol (EMB), pncA for pyrazinamide (PZA), rpsL and rrs for streptomycin (STR) and gyrA, gyrB for fluoroquinolones (FQs) such as ofloxacin (OFX) and levofloxacin (LVX) [Musser et al, 1995;Zhang et al, 2000].

Worldwide reports on MDR-TB
In 2008, there were an estimated 440,000 (range, 390 000-510 000) MDR-TB cases emerging worldwide.About 250,000 of these cases (range: 230 000-270 000) should have been reported to WHO, if countries had tested all the TB patients that they notified for drug resistance.However, only just over 30,000 MDR-TB cases (12%) were actually notified globally in 2009 [WHO, November 2010].
Almost 50% of MDR-TB cases worldwide are estimated to occur in China and India.In 2008, MDR-TB caused an estimated 150,000 deaths.Since 1994, 114 countries have reported surveillance data on MDR-TB: 42 perform continuous surveillance of anti-TB drug resistance based on routine testing of all TB patients; 72 rely on periodic surveys of representative samples of TB patients.The Russian Federation, which was able to provide high-quality continuous surveillance data from 12 of its oblasts and republics, reported proportions of 23.8-28.3%MDRTB among new TB cases in three of its oblasts in the northwest part of the country.Other Russian oblasts were found to have proportions of MDR-TB as low as 5.4% among new TB cases.Tajikistan, in its first ever survey, found proportions of 16.5% MDR-TB among new TB cases and 61.6% MDR-TB among previously treated TB patients in Dushanbe city and Rudaki district, the highest proportion ever reported among previously treated TB patients.To date, 12 countries have reported nationwide or subnational proportions of MDR-TB of 6% or more among new TB cases [WHO, November 2010].
Five of these countries also report MDR-TB proportions of 50% or more among previously treated cases.All of these settings are located in the eastern part of Europe or in Central Asia.

Solid-phase Hybridization techniques
There are currently two commercially available solid-phase hybridization techniques: the Line Probe Assay (INNO-LiPA Rif TB Assay; Innogenetics, Ghent, Belgium) for the detection of rifampicin resistance and the GenoType MTBDR assay (Hain Lifesciences, Nehren, Germany) for the simultaneous detection of isoniazid and rifampicin resistance.The LiPA assay was introduced several years ago and is based on the hybridization of amplified DNA from cultured strains or clinical samples to 10 probes covering the core region of the rpoB gene of M. tuberculosis, immobilized on a nitrocellulose strip.The GenoType MTBDR on the other hand, detects resistance to isoniazid and rifampicin in culture samples based on the detection of the most common mutations in the katG and rpoB genes respectively.Both assays have now been evaluated in different settings, giving encouraging results.In a recent study Hillemann et al evaluated the GenoType MTBDR assay and found that 99% of MDR strains with mutations in the rpoB gene and 88.4% of strains with mutations in the codon 315 of the katG gene were correctly identified.Correlation with DNA sequencing was 100% and compared with conventional tests good sensitivity and specificity were also obtained.Both solid hybridization methods have shown to be relatively simple to perform although basic expertise in molecular biology and PCR techniques is required.As with other genotypic methods the sensitivity of the test depends on the amount of DNA present in the sample and also the presence of inhibitors could cause false-negative results.
INNOLiPA Rif.TB kit: INNOLiPA Rif.TB kit simultaneously detects the M. tuberculosis complex and the presence of mutations in the rpoB gene associated with resistance to rifampicin which is considered a marker for MDR-TB strains.The strip contains 5 probes for detection of sensitive genotypes (S1-S5) [Morgan et al, 2005;Makinen et al, 2006] and 4 probes for detection of resistance genotypes (R2,R4a, R4b and R5).Rifampicin resistance is indicated by the absence of one or more sensitive probes, possible accompanied by the appearance of one or more mutant probes.
The GenoType MTBDR test is able to detect mutations in the rpoB gene for RIF resistance, and the most frequent mutation at codon 315 of the katG gene for INH resistance, either in isolates or clinical specimens.The specificity and sensitivity of the assay for RIF resistance were nearly 100%; for INH-resistance, despite a high specificity (approximately 100%), the sensitivity of the test ranged from 70% to 90%, depending on the prevalence of the particular mutation at the katG locus [Hilleman et al, 2007].
GenoType MTBDRplus (Hain Lifescience, Germany), an advanced version of the assay, includes probes for the identification of other mutations in the hotspot region of the rpoB gene for RIF resistance, and probes to detect mutations in the promoter region of the inhA gene involved in INH resistance.These improvements facilitate the detection of another 10% to 20% of INH-resistant cases, with an enhancement in rapid MDR-TB diagnosis.
The line probe assays are accurate and useful for rapid detection of drug resistance directly in clinical specimens.However, the number of genes that can be analyzed remains limited and the test fails to distinguish insertion mutations.Furthermore, they retain a lower sensitivity among acid fast bacilli-negative samples.In general, line probe assays are expensive and require sophisticated laboratory infrastructure.Their role and utility in lowincome, high-burden countries needs to be evaluated in field studies.

Real-time Polymerase Chain Reaction techniques
Real-time PCR techniques have also been introduced for rapid detection of drug resistance.Different probes have been used like the TaqMan probe, fluorescence resonance energy transfer (FRET) probes, molecular beacons and bioprobes.The main advantages of real-time PCR techniques are the speed of the test and a lower risk of contamination.The main disadvantages would be the requirement for expensive equipment and reagents, and the need for skilled technical personnel.Real-time PCR was initially applied to M. tuberculosis strains but more recently it has been successfully applied directly in clinical samples.Results could be obtained in an average of 1.5-2.0h after DNA extraction.Realtime PCR could eventually be implemented in reference laboratories with the required capacity to properly set up the technique and in settings where it can contribute to the management of TB patients.Detection of antitubercular drug resistance is vital to effective patient management.Realtime PCR offers the potential to detect gene mutations responsible for drug resistance within hours from patient specimens compared with the average of 2 weeks required for traditional susceptibility test methods.The rpoB and katG genes are the most common M. tuberculosis targets utilized in real-time PCR methods and well-known mutations in these genes correlate with resistance to rifampin and isoniazid, respectively [Edwards et al, 2001;El-Hajj et al, 2001;Garcia de Viedma et al, 2002;O'Mahony et al, 2002;Piatek et al, 2002;Torres et al, 2000;Torres et al, 2003;van Doorn et al, 2003].The significance of other gene targets such as kasA, ahpC-oxyR, and inhA for the prediction of isoniazid resistance is somewhat controversial [Piatek et al, 2000].Torres et al used two sets of FRET hybridization probes to detect rpoB mutations in 24 rifampin-resistant strains of M. tuberculosis and another set of FRET hybridization probes to detect katG mutations in 15 isoniazid-resistant M. tuberculosis strains [Torres et al, 2000] Viedma et al, 2002].Since not all gene mutations conferring drug resistance are well characterized and are thus not amenable to PCR assay development, traditional culture-based susceptibility testing methods are still required.However, the ability to predict rifampin and isoniazid resistance up to 2 weeks sooner than current methods for some isolates should have significant benefit for patient care.

Microarray
An array is an orderly arrangement of samples where matching of known and unknown DNA samples is done based on base pairing rules [Heyman et al, 1999].An array experiment makes use of common assay systems such as microplates or standard blotting membranes.The sample spot sizes are typically less than 200 microns in diameter usually contain thousands of spots.Thousands of spotted samples known as probes (with known identity) are immobilized on a solid support (a microscope glass slides or silicon chips or nylon membrane [Heyman et al, 1999].The spots can be DNA, cDNA, or oligonucleotides.These are used to determine complementary binding of the unknown sequences thus allowing parallel analysis for gene expression and gene discovery.An experiment with a single DNA chip can provide information on thousands of genes simultaneously.An orderly arrangement of the probes on the support is important as the location of each spot on the array is used for the identification of a gene [Heyman et al, 1999].
Microarray technology is used in the detection of drug resistant M. tuberculosis rather than the detection of M. tuberculosis from clinical specimens.The TB Biochip oligonucleotide microarray is the most widely used Microarray for the detection of isoniazid and rifampicin resistance simultaneously [Garcia de Viedma D et al, 2003].

TB-Biochip oligonucleotide microarray for the detection of rifampicin resistant M. tuberculosis
The TB-Biochip oligonucleotide microarray system is designed to detect and identify 29 codon substitutions and 1 codon deletion distributed over 10 codon positions (507,511,512,513,515,516,522,526,532,533) within the rifampicin resistant determining region (RRDR).Each element of the microarray contains an immobilized oligonucleotide whose sequence matches that of either a wild-type or mutated segment of the RRDR.The use of acrylamide gel pads increases the robustness of the hybridization reaction.Hybridization of the microarray with fluorescently labeled target DNA produces a spatial pattern of fluorescence intensities corresponding to the efficiencies of hybridization of the labeled target DNA to the various oligonucleotide probes.
In the TB-Biochip system, the fluorescence intensities are recorded using a charge-coupled device camera, and the relative intensities of fluorescence for the elements representing wildtype sequences and mutant sequences for each codon are compared using imaging software and automated computer-assisted interpretation of hybridization results [Garcia de Viedma D et al, 2003;Cavusoglu et al, 2004].The isolate is designated RIF susceptible if the fluorescence of each of the wild-type elements is greater than the fluorescence of any of the corresponding mutant elements.The isolate is designated RIF resistant if the fluorescence of any one of the mutant elements is greater than the fluorescence of its corresponding wildtype element.

Advantange and disadvantage of TB-biochip system
The complete TB-Biochip system may be suitable for use in clinical laboratories with molecular biology expertise because it requires relatively little hands-on time for experimental manipulations or data analysis, tests can be run individually or in batches, and specialized training is not required.The observed discrepancies between the results of conventional DST and the TB-Biochip system (all were falsely called susceptible with the TB-Biochip system) likely result from the large number of mutations found in RIF-resistant isolates and the limited range of mutations included on the biochip.
Although technically a solid-phase-type hybridization assay, microarrays, also known as biochips, have been proposed as new molecular methods for detecting drug resistance in M. tuberculosis.They are based on the hybridization of DNA obtained from clinical samples to oligonucleotides immobilized in a solid support, such as miniaturized glass slides.They have been mainly used to detect resistance to rifampicin.In a recent evaluation using oligonucleotide microarrays for analysis of drug resistance, Gryadunov et al. detected over 95% rifampicin resistant and almost 80% isoniazid resistant M. tuberculosis isolates within 12 h in a sample of drug-resistant isolates and clinical samples.For the time being and due to the high cost involved, the use of microarrays for detecting drug resistance in TB is still beyond the reach of most clinical mycobacteriology laboratories.

DNA sequencing
Sequencing DNA of PCR amplified products has been the most widely used method; it is accurate and reliable and it has become the gold standard for mutation detection.It has been performed by manual and automated procedures although the latter is now the most commonly used.It has been widely used for characterizing mutations in the rpoB gene in rifampicin-resistant strains and to detect mutations responsible for resistance to other antituberculosis drugs.It would be rather difficult, however, to implement it routinely for detection of drug resistance mutations for several drugs since it would involve several reactions for each isolate, making the cost high.

DNA extraction
DNA from clinical M. tuberculosis isolates can be extracted by using Qiagen kit (Germany) and also by keeping the MGIT suspension at 80˚C for 10 minutes.After 10 minutes, centrifuged at 3000 rpm and the supernatant can be used as template DNA for PCR.The procedure for extraction of DNA using Qiagen kit is mentioned below.


Carefully open the QIAamp mini spin column and add 500l buffer AW2 without wetting the rim.Close the cap and centrifuge at 14,000 rpm for 3min, followed by an empty spin at 14,000 rpm for 1min.


Place the QIAamp mini spin column in a clean 1.5 ml microfuge tube and discard the tube containing filtrate.Carefully open the QIAamp mini spin column and add 200l AE Buffer.Incubate at room temperature for 1min, and then centrifuge at 8,000 rpm for 1min.Discard the column and store the DNA at -20°C.

PCR protocol
Reagents and other accessories required:

PCR protocol for amplification of embB gene using primers targeting 640-1577 region
PCR targeting embB gene was standardized with a specialized Taq DNA polymerase enzyme called "Z Taq" enzyme (Takara Bio, Ohtsu, Shiga, Japan).The Z-Taq polymerase offers unmatched PCR productivity, with a processing speed five times faster than those of other commercially available Taq polymerases.The total PCR cycle takes only 29 minutes.
All the reagents for PCR (dNTP, 10X, Z Taq) will be provided along with the buffer.Each 50 µl reaction contained 2.5mM dNTP, 10X, 1pM of forward and reverse primers and 2.5 Units of Z taq enzyme.The details of primer targeting drug resistance genes, their thermal profile used and the expected amplicon size are given in the Table2.

Detection Of Amplified Products:
The amplified product is subjected to electrophoresis on 2% agarose gel incorporated with 0.5 µg/ml ethidium bromide for visualization by UV transilluminator (Vilber Lourmat -France).

DNA sequencing of amplified products
The term DNA sequencing refers to sequencing methods for determining the order of the nucleotide bases-adenine, guanine, cytosine, and thymine-in a molecule of DNA.DNA sequencing enables us to perform a thorough analysis of DNA because it provides us with the most basic information of all: the sequence of nucleotides.The classical chaintermination method requires a single-stranded DNA template, a DNA primer, a DNA polymerase, radioactively or fluorescently labeled nucleotides, and modified nucleotides that terminate DNA strand elongation.Dye-terminator sequencing utilizes labelling of the chain terminator ddNTPs, which permits sequencing in a single reaction, rather than four reactions as in the labelled-primer method.In dye-terminator sequencing, each of the four dideoxynucleotide chain terminators is labelled with fluorescent dyes, each of which with different wavelengths of fluorescence and emission.Owing to its greater expediency and speed, dye-terminator sequencing is now the mainstay in automated sequencing.Its limitations include dye effects due to differences in the incorporation of the dye-labelled chain terminators into the DNA fragment, resulting in unequal peak heights and shapes in the electronic DNA sequence trace chromatogram after capillary electrophoresis.This problem has been addressed with the use of modified DNA polymerase enzyme systems and dyes that minimize incorporation variability, as well as methods for eliminating "dye Table 2. Primer sets used in the study to sequence the different loci of target genes with their thermal profile and expected amplicon sizes [Sekiguchi et al, 2007;Siddiqi et al, 2002;Sreevatsan et al 1997] blobs".The dye-terminator sequencing method, along with automated high-throughput DNA sequence analyzers, is now being used for the vast majority of sequencing projects.
DNA sequencing involves the following steps, Holding Temp -4˚C

Purification of Cycle Sequenced Product
The cycle sequenced products are purified to remove the unincorporated dye terminators before the samples are analyzed.


The vials are then dried at 37˚C (incubator) until ethanol completely evaporates.Presence of ethanol will prevent complete dissolving of DNA in formamaide.

Loading into DNA sequencer
Once the ethanol is completely dried, add 10 µl of formamide.This is denatured at 95˚C (Thermal cycler) for 3 minutes and immediately snap cooled in ice.The sequence of the PCR amplified DNA is deduced with the help of the ABI Prism 3100 AVANT (Applied Biosystems, USA) genetic analyzer that works based on the principle of Sanger dideoxy sequencing.The amplified products with the dye at the terminated 3'end is subjected to capillary electrophoresis by an automated sample injection.The emitted flurorescence from the dye labels on crossing the laser area are collected in the rate of one per second by cooled, charge-coupled device (CCD) camera at particular wavelength bands (virtual filters) and stored as digital signals on the computer for processing.

Basic Local Alignment Search Tool (BLAST) analysis
The sequences are analysed by sequence analysis softwares such as Bio Edit sequence alignment software or Chromas software.BLAST analysis, using pubmed, http://www.ncbi.nlm.nih.gov/BLAST is done to confirm the sequenced data with the standard strains and to determine the percentage homology.

Multalin analysis
Multalin analysis (http://multalin.toulouse.inra.fr/multalin/) to be done to identify the presence of polymorphism or mutation by comparing with the reference strain from genbank (Accession No. L27989 for rpoB, U41314 for katG, MTU16243 for inhA and oxyR-ahpC, X70995 for rrs and rpsL, AY743320 for pncA and MTU68480 for embB).An example of multalin analysis targeting rpoB and katG are shown in the figure 1&2.    3. Primer sets used in the study to sequence the different loci of target genes with their thermal profile and expected amplicon sizes [Sekiguchi et al, 2007;Siddiqi et al, 2002;Sreevatsan et al 1997] For PCR protocol and DNA sequencing protocol, please refer to procedures 2.3.1 to 2.3.7.

Conclusion
The expectation that molecular techniques would surpass conventional methods for diagnosis of TB or phenotypic susceptibility testing has not yet been realized.The genetic basis of resistance must be understood before achieving such a goal.However, the clinician now has a variety of new tools to improve the diagnosis of TB and drug resistance.Most of them still require detailed and systematic evaluations using standard techniques as references before their widespread application in clinical settings.Most of these techniques require trained personnel and specialized equipment, hindering their application in field conditions, but they can be used in reference laboratories as part of the TB control programs.
Stock dNTPs dilution: 100 mM concentration of dNTPs-dATP, dCTP, dTTP and dGTP  Working standard dNTP (200µM): 2 µl of each of the stock dNTP made up to 400 µl using MilliQ water.

Fig. 1 .
Fig. 1.An example of multalin result targeting rpoB gene using forward primer showing the presence of most commonly reported mutation TCG→TTG (Ser531Leu).Inset enlarged view of the mutation.

2. 3 . 8
PCR based DNA sequencing for XDR-TB strains I f a n M D R -T B s t r a i n h a s t h e a b o v e -m e n t i o ned mutations, it should be screened for resistance to Amikacin, capreomycin, kanamycin and the fluoroquinolones by PCR based DNA sequencing targeting rrs, tlyA, thyA, gyrA and gyrB.The details of primer targeting drug resistance genes, their thermal profile used and the expected amplicon size are given in the Table3.

Fig. 2 .
Fig.2.An example of multalin result targeting katG gene using forward primer showing the presence of most commonly reported mutation AGC→ACC (Ser315Thr).Inset enlarged view of the mutation.
. Additionally, Garcia de Viedma et al. used two sets of rpoB probes and one set of katG probes to detect rpoB and katG mutations, but in a single tube, for www.intechopen.comMolecular Biological Techniques for Detection of Multidrug Resistant Tuberculosis (MDR) and Extremely Drug Resistant Tuberculosis (XDR) in Clinical Isolates of Mycobacterium tuberculosis 459 29 resistant M. tuberculosis isolates [Garcia de Briefly centrifuge the micro centrifuge tube to remove drops from inside of the lid. Add 200l of ethanol (96-100%) to the sample, mix by pulse vortexing for 15 seconds.After mixing, briefly centrifuge the 1.5ml microfuge tube to remove drops from inside of the lid. Carefully apply the mixture from step 6 to the QIAamp mini spin column (in a 2ml collecting tube) without wetting the rim.Close the cap, and centrifuge at 8000 rpm for 1 min.Place the QIAamp mini spin column in a clean 2 ml collecting tube and discard the tube containing the filtrate. Carefully open the QIAamp mini spin column and add 500l buffer AW1 without wetting the rim.Close the cap and centrifuge at 8000 rpm for 1min.Place the QIAamp mini spin column in a clean 2ml collecting tube and discard the tube containing filtrate.
Pipette out the supernatant and wash the pellet twice with 250 µl of 70% ethanol at 12,000 rpm for 10 minutes.Care should be taken not to touch the sides of the eppendorf vial during pipetting.