MOLECULAR DIAGNOSIS OF MULTI DRUG RESISTANT TUBERCULOSIS

Drug resistant tuberculosis has been reported since the early days of introduction of anti-tuberculosis therapy but recently multi drug resistant tuberculosis (MDR-TB) and extensively drug resistanttuberculosis (XDR-TB) has been an area of growing concern and is posing threat to the global efforts of tuberculosis control. Current WHO report states that the prevalence of primary and acquired rifampicin resistance in India is 2.8 % and 17.2 % respectively. Rifampicin resistance is of special concern because it is the most effective bactericidal drug against Mycobacterium tuberculosis. More than 95 % of all mutations are located in a 81 bp (1294—1375 bp) core region (Rifampicin Resistance Determining Region ,RRDR ) of the rpo B gene between codons 507—533 with the most common changes in codons Ser 531 Leu , His 526 Tyr, and Asp 516 Val. These changes occur in more than 70 % of rifampicin resistant. Thus rifampicin resistance acts as a surrogate marker of MDR-TB. The molecular methods to detect rifampicin resistance include methods such as Line Probe Assay (LiPA), DNA sequencing, Single-Strand Conformational Polymorphism (SSCP), Heteroduplex Analysis (HA), Molecular beacons (MB) and Polymerase chain reaction--Enzyme Linked Immunosorbent Assay (PCR-ELISA) .Among these, the Line Probe Assay is most advanced. The major advantage of PCR based molecular methods is the speed by which the result can be obtained.


Introduction
Drug resistant tuberculosis has been reported since the early days of introduction of antituberculosis therapy but recently multi drug resistant tuberculosis (MDR-TB) has been an area of growing concern and is posing threat to the global efforts of tuberculosis control. Prevalence of MDR-TB in a community mirrors the functional state and efficacy of tuberculosis control programme and realistic attitude of the community towards implementation of such programmes 1 . MDR-TB is almost twice as common in tuberculosis patient co-infected with Human Immunodeficiency Virus (HIV) compared to tuberculosis patient without HIV 2 .The mean survival time for patients who are co-infected with HIV and MDR-TB is about 2 months from the time of diagnosis, with 1 year mortality rate of 60 % as compared to 30 % in non-HIV infected persons 3 . Of late, there has been a special concern for conversion of poorly managed MDR-TB to XDR-TB (Extensively drug resistant-tuberculosis) which is the untreatable form of tuberculosis 4 .The combination of XDR-TB and HIV is virtually the death sentence for the patient.
Thus, the alarming increases in MDR-TB,the emergence of XDR-TB and rapid mortality of MDR-TB and XDR-TB patients with HIV coinfection ,have highlighted the urgency for rapid molecular diagnostic methods.

Definitions:
Multi-drug resistant tuberculosis (MDR-TB) is defined as disease due to M.tuberculosis that is resistant to Isoniazid (H) and Rifampicin (R). As per the latest definition, XDR-TB is a subset of MDR-TB with additional resistance to any fluoroquinolone (Ciprofloxacin, Ofloxacin etc) and one of the second line injectables namely Kanamycin, Capreomycin and Amikacin 5  Thus an early and rapid diagnosis is required for the management of tuberculosis, especially the MDR and XDR variety.

Molecular mechanism of multi-drug resistance:
The development of clinical drug resistance in tuberculosis is shown in figure I. Mutation in the genome of Mycobacterium tuberculosis that can confer resistance to antituberculosis drugs occur spontaneously with an estimated frequency of 3.5 × 10¯6 for isoniazid and 3.1 × 10 -8 for rifampicin 8 . The clinical drug resistance is classified as acquired resistance when drug resistant mutants are selected as a result of ineffective treatment or as primary resistance when a patient is infected with a resistant strain. MDR-TB (resistance to isoniazid and rifampicin) will thus occur mainly in circumstances where sequential drug resistance follows sustained treatment failure.

Rifampicin resistance and rpo B gene:
Rifampicin was introduced in 1971 as antituberculosis drug. It is the most potent anti-tuberculosis agent and thus included in all ATT regimes. Rifampicin resistance is of special concern because it is the most effective bactericidal drug against Mycobacterium tuberculosis . It is a better sterilizing agent than isoniazid.It permeates all tissue membrane including the blood-brain and blood-placental barrier and is equally effective against intracellular as well as extracellular bacilli.Rifampicin is the only antitubercular drug that is effective against "persisters" or dormant bacilli which are found in solid caseous lesions,all other drugs being inactive 9,10 . In this context, it has a distinct advantage over isoniazid.This makes the detection of its resistance very important. The complete genome sequence of Mycobacterium tuberculosis consists of 4043 genes 11 . Out of these ,mutation in the rpo B gene is responsible for resistance to rifampicin. The rpo B gene consists of 3267 bp of which the Rifampicin Resistance Determining Region (RRDR) consists of 81 bp as shown in figure II.

Molecular mechanism of action of rifampicin:
Rifampicin interferes with transcription by the DNA dependent RNA polymerase. RNA polymerase consists of four different subunits (α β β I σ).The β subunit is the catalytic site. These four subunits (α β β I σ) are encoded by rpo A,rpo B,rpo C and rpo D genes respectively. Rifampicin binds with the β subunit of RNA polymerase,hindering transcription and thereby killing the bacteria (bactericidal) .So the mutation in the rpo B gene conferred conformational changes leading to defective binding of rifampicin to RNA polymerase making it ineffective as bactericidal agent.  It involves following steps i) DNA extraction from M.Tuberculosis isolates or directly from clinical specimen ii) PCR amplification of the RRDR of rpo B gene using biotynylated primers iii) Labelled PCR products are hybridized with specific oligonucleotide probes immobilized on a strip as shown in figure IV. iv) Captured labeled hybrids are detected by colorimetric development. If mutation is present in one of the target regions, the amplicon will not hybridise with the relevant probe. Mutations are therefore detected by i) Lack of binding to wild type probes (S 1 ,S 2 ,S 3 ,S 4 ,S 5 ) ii) Binding to specific mutant probes (R 2 ,R 4a ,R 4b ,R 5 ) for the most commonly occurring mutations. The post-hybridisation reaction leads to the development of colour bands on the strip at the site of probe binding and can be observed by naked eye. Line probe assay performance have been adequately validated in direct testing of sputum smear-positive specimens and on isolates of M.Tuberculosis complex grown from smear-negative and smear positive specimens. Direct use of LiPA on smear negative-clinical specimens is not recommended 19 . LiPA is a reliable, rapid and informative tool for the early detection and characterization of rpo B gene mutation associated with rifampicin resistance in mycobacterium tuberculosis 16 . But this method is too costly for use in routine laboratories, especially in developing country. LiPA is recommended by WHO for rapid detection of rifampicin resistance since 27 th June 2008 19 . B. Sequencing: PCR amplification followed by DNA sequencing is the most commonly applied technique to characterize mutations in the rpo B gene 18 . The detection of rifampicin resistance by DNA sequencing is rapid, advantageous and provides a confirmatory result thereby aiding in initiation of immediate alternative drug therapy in patients who develop rifampicin resistance. Rifampicin resistance as reported by various researchers is due to mutation in the Rifampicin Resistance Determining Region (RRDR) of rpo B gene.They reported the most frequent mutations in RRDR of rpo B gene are in codon 531 followed by codon 526 and codon 516 16,18,21,22,23 . However, some of the previous researchers 16 also reported codon 526 as the most common site of mutation leading to rifampicin resistance in MDR-TB cases. The advantage of using DNA sequencing as a diagnostic modality for diagnosis of MDR-TB is that it can detect mutation outside RRDR which is responsible for 8-9 % cases of MDR-TB. However, PCR-SSCP analysis is technically demanding and not sufficiently sensitive. Furthermore SSCP conditions must be carefully evaluated since not all mutations will be detected under the same conditions. D. Heteroduplex analysis (HA): HA depends on the conformation of duplex DNA when analysed in native gels. Heteroduplexes are formed when amplicons from known wild type and unknown mutant sequences are heated and reannealed. The DNA strand will form a mismatched heteroduplex if there is a sequence difference between the strands of the wild type and tested DNA. These heteroduplexes have an altered electrophoretic mobility when compared to homoduplexes, since mismatches tend to retard the migration of DNA during electrophoresis. Recently, temperature mediated HA has been applied to the detection of mutations associated with mutation in rpo B gene 24,25 . HA has certain disadvantages in that it is insensitive to G-C rich regions and is very time consuming. E. Molecular beacons: Molecular beacons are single-stranded oligonucleotide hybridization probes which can be used as amplicon detector probes in diagnostic assays 26,27 .

C. Single-Strand
A beacon consists of a stem-loop structure in which the stem contains a fluorophore on one arm and a quencher on the other end of the arm as shown in figure V.
The loop contains the probe which is complementary to the target DNA. If the molecular beacon is free in a solution it will not fluoresce, because the stem places the fluorophore so close to the non-fluorescent quencher that they transiently share electrons, eliminating the ability of the fluorophore to fluoresce. However, in the presence of complementary target DNA the probes undergo a conformational change that enables them to fluoresce brightly. Different colored fluorophores (different primers) can be used simultaneously to detect multiple targets (each target will give a different color) in the same reaction. Molecular beacons are very specific and can discriminate between single nucleotide substitutions. Thus they are ideally suited for genotyping and have been used in the detection of drug resistance in M. tuberculosis 26,27 . But the limitations of molecular beacon technique are that limited genes and sites are targeted. By this method all mutations cannot be detected. F. Polymerase chain reaction--Enzyme Linked Immunosorbent Assay (PCR-ELISA): After PCR amplification of rpo B gene, ELISA is done with the amplicons. The reverse primer is labelled with digoxigenin at the 5 ′ end so that after PCR the amplicons will contain digoxigenin at their 5 ′ end.The ELISA plates are streptavidine coated. In this ELISA wells, capture probes (5 ′ biotinylated), amplicons, enzyme linked antibody and chromogenic substrates are added. If there is change in nucleotide sequence in the rpo B gene then the capture probe will not hybridise with the amplicons and will not produce colour. Thus a positive result shows the absence of any mutation in that region, while a negative result indicate a lack of hybridization and presence of a mutation in the rpo B sequence corresponding to the probe 28,29 . PCR-ELISA cannot identify the specific mutation causing rifampicin resistance but does indicate the region in which the mutation is located 28 . Knowledge of the specific mutation conferring resistance is, however, not necessary for efficient patient management. The benefits of PCR-ELISA system lie in its speed and accuracy in identifying rifampicin resistant strains and it can be considered as a screening method for MDR-TB.

Applications
The major advantages of PCR based molecular methods are the speed by which the result can be obtained. By using the molecular methods the results can be obtained within few days in contrast to the culture and drug sensitivity testing (DST) takes about 6 weeks for diagnosing rifampicin resistance. It is emphasized that the molecular techniques are important for rapid diagnosis of rifampicin resistance and MDR-TB.

Summary
Recently MDR-TB has been a growing area of concern and XDR-TB is virtually the Rifampicin resistance serves as a surrogate marker for MDR-TB since more than 90 % of rifampicin resistant isolates are also isoniazid resistant.The advantage of using PCR based molecular techniques lies in it speed and accuracy in identifying rifampicin resistance strain.The use of molecular techniques will reduce the time required to detect rifampicin resistance . Although molecular methods are more rapid, and can be done directly from a clinical sample, there are important limitations when compared to conventional methods. These include a lack of sensitivity since not all molecular mechanisms leading to drug resistance are known, therefore not all resistant isolates will be detected. Some mutations are silent and do not confer resistance but they can be detected by these molecular techniques and can lead to wrong interpretation.