Festschrift : Molecular revolution in TB diagnostics

Times have changed! The molecular revolution in TB diagnostics and detection of drug resistance
HJ Koornhof, GJ Coetzee

Hendrik Koornhof, Gerrit Coetzee, National Tuberculosis Reference Laboratory, National Institute for Communicable Diseases; Division of Virology and Communicable Diseases Surveillance, University of the Witwatersrand, Johannesburg. Correspondence to: Prof HJ Koornhof, e-mail: hendrik.koornhof@nhls.ac.za

During the late 1970s, before Prof Schoub went to the National Institute of Allergy and Infectious Diseases in Bethesda, USA to study under the famous rotavirus expert Dr Kapikian, the young Barry Schoub and Prof Hendrik Koornhof shared an interest in the aetiology of gastroenteritis in infants and children and were joint authors of research papers on this topic. One of these was on experimental techniques in the determination of the aetiology of diarrhoeal disease. Although not involved in tuberculosis (TB) research at the time, Prof Koornhof was co-author with Drs Helmuth Kleeberg and H Palmhert of a laboratory manual on tuberculosis methods published in 1970, with a second edition in 1980. The review that follows below deals with recent developments in molecular tests in the field of TB diagnosis and management, an area of interest to the National Tuberculosis Reference Laboratory (NTBRL), where both Prof Koornhof and Dr Gerrit Coetzee work at present. This article is dedicated to Prof Schoub, with fond memories of a time when both he and Prof Koornhof realised the importance of modern and reliable methods required for their research at the time, and which continue to be the cornerstone of activities of the NTBRL and National Institute for Communicable Diseases at present.
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The phenotypic era

For more than a century the world reaped the benefit of sputum smear microscopy for acid-fast bacilli (AFB), a simple, rapid and inexpensive test for the diagnosis and treatment monitoring of tuberculosis (TB). The more sensitive and informative, but much slower, culture of Mycobacterium tuberculosis from clinical specimens, first on solid and subsequently in liquid culture medium systems, and drug susceptibility testing (DST), based on growth inhibition in the presence of critical concentrations of anti-TB agents, were advances introduced over decades during the 20th century. The introduction of colorimetric methods employing oxidationreduction indicator dyes1 and organism-related metabolic characteristics, such as ability to reduce nitrates to nitrites on which the nitrate reductase assay is based,2 were attempts to shorten turnaround times of culture-linked tests. Further improvements were the introduction by Becton Dickinson Microbiology Systems (Sparks, Md, USA) of the BACTEC 460 System, based on a 13C isotope-labelled carbon-containing substrate which releases radioactive CO2 in the atmosphere above the medium when metabolised by multiplying M. tuberculosis organisms3, and the environmentally friendly and fully automated BACTEC Mycobacteria Growth Indicator Tube System (BACTEC 960 MGIT; Becton Dickinson) in which growth is detected by a fluorescent sensor that responds to the concentration of oxygen in the medium.4 More recently,

the microscopic observation drug susceptibility (MODS) assay for the detection of M. tuberculosis and its susceptibility status in sputum samples was found to be inexpensive and rapid (results available in seven days), but rather labour intensive.5 Further examples of interesting developments are mycobacteriophage-based methods for rapid DST of M. tuberculosis strains, including the commercial FASTPlaqueTBRIF assay (Biotech Labs Ltd, Ipswich, UK),6 which is based on replicating D29 phages from test strains, causing holes (plaques) in a lawn of rapidly growing Mycobacterium smegmatis; and the luciferase reporter phage assay, which uses genetically engineered recombinant mycobacteriophages carrying the firefly luciferase gene to demonstrate, on fluorescence microscopy or flow cytometry, luminescent antimicrobial-resistant M. tuberculosis organisms in the presence of the antimycobacterial agent.7

Genotypic detection of M. tuberculosis and drug resistance

Developments in phenotypic testing have recently been overtaken by a remarkable upsurge in molecular-based research involving M. tuberculosis, which led to the development of novel methods for rapid detection of the TB organism and its susceptibility to rifampicin and other antiTB drugs. Detection of M. tuberculosis by culture is likely to remain the gold standard for TB diagnosis for some time to

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come, but nucleic acid amplification assays have finally come of age. Exciting methodologies have been developed for rapid detection of M. tuberculosis-specific nucleotide sequences, with mutations in genes involved in the action of anti-TB drugs presently being the main targets for the demonstration of drug resistance.
Methodologies underlying new assays

Real-time PCR

Initial extraction of DNA from clinical specimens and amplification of target nucleotide sequences by the polymerase chain reaction (PCR) or other amplification methods, together with amplicon detection, form the basis for all molecular assays for TB diagnosis and resistance detection. In the case of realtime PCR (RT-PCR) techniques, the amplification process and amplicon detection take place simultaneously. Critical aspects of nucleic acid amplification techniques (NAATs) are selection of primers from target genes, the method of amplification, and amplicon detection.
Selection of primers

Real-time PCR is based on simultaneous amplification of different DNA targets employing fluorimetric detection by labeled probes.21 The Roche Diagnostics LightCycler 2.0 system performs multiplex PCR and combines melting curve analysis with six fluorescence detection channels. It performs quantification analysis by measuring fluorescence signals during the exponential increase (log-linear) phase of the PCR process.21,22 Amplicon detection methods used in real-time PCR include the 3-minor groove binder-DNA probe23 which, in addition to binding to its specific target sequence, binds to the minor groove of the double-stranded DNA helix, enhancing the stability of the probe-amplicon binding and allowing relatively short probes to have melting points of ~60C.15 This method is useful to detect point mutations or single-nucleotide polymorphisms in M. tuberculosis, of which the genome has a high GC content, predisposing to probes with a high melting temperature.23 The real-time PCR-based Cobas TaqMan MTB technology utilises exonuclease probes, with cleavage of a dual-labelled oligonucleotide target detection probe which is fluorescencelabelled at the 5-end with a fluorescence quencher at the 3end.21,24 While the probe is bound to its homologous sequence, the RT-PCR instrument does not measure fluorescence, because the quencher absorbs the energy of the dye. With the elongation PCR step, the 5-3 exonuclease activity of the Taq polymerase releases the fluorescence dye, resulting in emission of fluorescence which is measured by the instrument. Fluorescence is only emitted if the probe is correctly anchored to its homologous site to allow the Taq enzyme to release the dye.21 Molecular beacons-based probes21,25 have a stem-loop structure, consisting of a nucleotide sequence which is homologous with the target DNA, and a fluorescent dye and a quencher in the 5 and 3 ends of the probes. The stem-loop structure is disrupted when the beacon binds to its homologous sequence in the target DNA, emitting fluorescence. When a mutation is covered by the probe, its binding to the probe is impaired and the beacon fails to emit fluorescence. In the GeneXpert system (Cepheid), five coloured molecular beacons-based fluorogenic nucleic acid hybridisation probes are used to cover the M. tuberculosis-specific 81 bp rpoB core region, which contains >95% of mutations linked to rifampicin resistance. The molecular beacons hybridise only to targets associated with rifampicin resistance that do not contain mutations so that, in the case of rifampicin-susceptible strains, all five beacons would emit coloured fluorescence from the beacons.25
Detection of amplification products

For M. tuberculosis detection, genes encoding IS6110,8,9 16S rRNA,9,10 23S rRNA11 and internal transcribed spacer sequences (ITS) flanking 16S and 23S rRNA12 genes, as well as the B-subunit of RNA polymerase (rpoB gene)13 and the hsp65 gene,14 have traditionally been targeted with appropriate primers, while the 81-bp hypervariable core region of the rpoB gene provides primer sequences for rapid detection of rifampicin, and the katG and inhA genes for isoniazid resistance.15
Amplification techniques

Apart from conventional PCR, several isothermal amplification methods have been developed. The polymerase enzyme replicates DNA with the aid of various accessory proteins. Their identification resulted in the development of new in vitro isothermal DNA amplification methods, including the BD ProbeTec MTB Test (Becton Dickinson, Sparks, MD), using strand displacement technology8 which also forms the basis of the loop-mediated isothermal amplification (LAMP) method.16 Other amplification methods that have been used are the ligase chain reaction in the LCx M. tuberculosis assay (Abbott Laboratories, Chicago, IL, USA)17 and the transcription-mediated amplification (TMA) in the Amplified M. tuberculosis Direct Test (AMTD; Gen-Probe Inc. San Diego, CA),17 as well as the recently described cross-priming amplification technique (Ustar, Hangzchou, China).18 Another example is nucleic acid sequence-based amplification targeting the 23S RNA gene in the GenoType CM/AS (Hain Lifescience, Nehren, Germany) test,19 and the 16S-23S spacer (ITS) region in the INNO-LiPA Mycobacteria (Innogenetics, Ghent, Belgium)20 for the identification of mycobacterial species. This approach is linked to solid phase reverse hybridisation, in which PCR products are hybridised to a nitrocellulose strip with immobilised probes for different Mycobacterium species.

DNA sequencing of amplified target DNA can be considered the reference method for amplicon detection, as it provides

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the most complete and accurate information linked to the target recognition, for example mycobacterial species identification and/or drug resistance status. Amplicon detection can be performed during the final phase or endpoint of the conventional PCR reaction, for example through ethidium-gel detection,21 or during the early logarithmic phase of amplification in RT-PCR.22 Several molecular approaches targeting specific nucleotide sequences have been described, including pyrosequencing,26 DNA heteroduplex analysis,27 nucleotide mismatch analysis28 and single-stranded conformational polymorphism.29 Pyrosequencing is a DNA synthesising technique, where a sequencing primer is hybridised to a DNA template and pyrophosphate linked to a luciferase-mediated light emitting system is released on nucleotide base pair binding following addition of the four nucleotide bases in a defined order (C, G, A, and T).26 The widely used and interdisciplinary PCR-based amplification refractory mutation system (ARMS)30 for point mutation and small deletion detection has been employed for rapid detection of rpoB mutations in the 81-bp rifampicin resistance-determining region (RRDR) in M. tuberculosis isolates in China,30 and point mutations in the 23S rRNA gene linked to high-level resistance to clarithromycin in Mycobacterium avium complex isolates in a recent Japanese study.31 Detection of denatured PCR products using highperformance liquid chromatography (HPLC) is a cheap and effective method for high-throughput screening of rifampicin resistance.32 Hybridisation in oligonucleotide microarrays, as a tool for rapid detection of drug resistance in M. tuberculosis, has been studied extensively.21,33 In a Centers for Disease Control and Prevention (CDC)-based evaluation conducted on a relatively small sample set of 29 in vitro-selected mutants of M. tuberculosis and 75 clinical isolates with known rifampicin resistance status from their culture collection, specificity and positive predictive values of 100% were reported, with a sensitivity index of 80%.33 Short oligonucleotide chip systems, for the detection of mutations in the RRDR of the rpoB gene for rifampicin resistance, in a study involving 60 rifampicin-resistant clinical isolates from the Wuhan region in China, showed a sensitivity of 91.7%, with 87.3% accuracy and 83.6% concordance for rifampicin resistance detection.34 In a South Korean study comprising 243 M. tuberculosis isolates, for detection of mutations in the rpoB gene for rifampicin resistance and the katG and inhA genes for isoniazid resistance, the sensitivity for detection of rifampicin resistance was 93%, and for isoniazid resistance 71.4%.35 The line probe assays (LPAs) based on reverse hybridisation for Mycobacterium species identification by the Innogenetics and Hain Lifescience systems have already been mentioned.19,20 The same firms developed the INNO-LiPA Rif TB Assay (LiPA) for rifampicin resistance detection,36 and the GenoType

MRBDRplus Assay (Hain LPA) for the demonstration of rifampicin and isoniazid resistance in M. tuberculosis isolates,37 targeting rpoB mutations for rifampicin and katG and inhA mutations for isoniazid resistance, respectively. The range of the Hain Lifescience technology has been expanded by the addition of a new GenoType M. tuberculosis drug resistance second-line (MTBDRsl) assay for the detection of resistance to ofloxacin, amikacin/capreomycin and/or ethambutol.38 The genes targeted in the MTBDRsl assay are gyrA, rrs, and embB. The sensitivity of the assay was recorded as 29 out of 32 strains (90.6%) for ofloxacin, 84.8% for amikacin, 86.7% for capreomycin and 69.2% for ethambutol, while the specificities for the drugs approached 100%.38 The LiPA system for rifampicin resistance showed excellent specificity approaching 100%, but sensitivity ranged from 70-100% in various studies, with sensitivity in sputum smear-negative specimens of 47% in one study.39 In studies conducted in South Africa40,41 and elsewhere,37 evaluating the Hain LPA on sputum smear-positive specimens, sensitivities for rifampicin resistance ranged from 95-99%, and for isoniazid from 9399%, while specificities for the detection of resistance to either of the two drugs exceeded 99%. Amplification product (amplicon) detection methods employed in all these rapid molecular tests are intriguing, and some of the widely validated commercially available methods include reverse hybridisation used in the LPA methods INNO-LiPA Rif. TB (Innogenetics) and GenoType MTBDRplus kit (Hain Lifescience) mentioned earlier.36,37 High-resolution melting point analysis of amplicons in the LightCycler systems (Roche Diagnostics)22 and the five-coloured molecular beacons-based fluorogenic nucleic acid hybridisation probes of the GeneXpert system (Cepheid) proved to be highly efficient.42 Among the many exciting new technologies, the loop-mediated isothermal amplification (LAMP) method showed early promise for large-scale use in high-burden TB-endemic countries, like South Africa.16 It is designed to detect M. tuberculosis directly in specimens and because of its simplicity, low cost and near site testing potential, it will be singled out for further elaboration.
Loop-mediated isothermal amplification method

Loop-mediated isothermal amplification (LAMP) methodology43 recognises six primer pairs in eight distinct regions of the IS6110 and 16S rRNA genes as targets for M. tuberculosis detection, in an isothermic amplification process. Abundant amplicons are produced as a result of autocycling in the process of strand displacement DNA synthesis, mediated by the Bst DNA polymerase enzyme obtained from Bacillus stearothermophilus. The enzyme functions at isothermal conditions ranging from 60-65C, and amplifies stem-loop DNA structures, with several inverted repeats of the target forming cauliflower-like structures with multiple loops. It produces large amounts of amplified products which enable simple visual detection in a single reaction tube through turbidity produced by precipitation of

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pyrophosphate during the amplification process, or the use of DNA-binding dyes producing a colour change, or fluorescence in the reaction mixture. The process obviates the use of timeconsuming thermocycling of conventional PCR. With all these characteristics, LAMP lends itself to point-of-care testing at clinic and bedside level. It has also been shown in a recent study to perform very well in the rapid and reliable diagnosis of TB meningitis.43 LAMP has been shown in several studies to be highly sensitive in smear-positive sputum samples (>95%), with sensitivities varying from ~50% to >90% in smear-negative specimens and specificities in excess of 90%.16 LAMP has also been developed to detect non-tuberculous mycobacteria (NTM), but, in a recent study using mycobacterial culture lysates the sensitivity of LAMP was only 44.7% in the conventional 60 minutes amplification cycle, improving to 85.7% at 90 minutes and 91.7% in a 120-minute cycle.44 This study, as well as others used for the rapid diagnosis of diseases such as malaria45 and trypanosomiasis,46 has shown varying sensitivities, suggesting that the test may need further refinement and evaluation before it could be used on a large scale in national TB control programmes.

laboratories of the National Health Laboratory Service (NHLS), and embarked on a training programme of technologists/ technicians earmarked for the task of performing the tests for the NTBCP. Initially it was anticipated that the Hain LPA would play a major role in the laboratory diagnosis and treatment monitoring of TB in South Africa but, with the advent of the GeneXpert, a new, less prominent role for the Hain LPA is envisaged.
GeneXpert (Xpert MTB/RIF) assay

The Cepheid Xpert MTB/RIF assay is a hands-free sputum processing system with rapid on-demand, near-patient technology to detect M. tuberculosis and rifampicin resistance simultaneously.42 The sample processing system and an automated hemi-nested real-time PCR assay are integrated into a single disposable cartridge, and the assay can be performed directly from a clinical sputum sample or from a decontaminated sputum pellet. In the performance of the test, a sample reagent (lysis buffer) is added to sputum in a 2:1 ratio, or decontamination pellet in a ratio of 3:1, and the mixture agitated twice during a 15-minute period at room temperature. Two millilitres of the inactivated material is then transferred to the test cartridge, which is loaded into the MTB/RIF platform where the real-time amplification process is completed in one hour 45 minutes and the test result is made available in printed form. MTB/ RIF platforms are available in GX modules of 4, 16 and 48 (Infinity platform) capable of processing 16, 64 and 192 specimens, respectively.35 Investigation of containment of the risk of infection through aerosol formation by the Xpert MTB/ RIF, and the killing capacity of its sample reagent, suggested that bench top use of the system limits infection risk.47 The Xpert MTB/RIF assay was shown to have a limit of detection with 95% confidence of 131 colony-forming units per ml of sputum and 4.5 genomes per reaction,42 and in a multi-centre study involving 1,730 patients, demonstrated an overall sensitivity of 97.6%, with a 72.5% sensitivity index for smear-negative patients and a specificity of 98.1%.48 In a recent study published in The Lancet on the implementation of the GeneXpert in 1,033 consecutive culture-confirmed cases of TB in urban health centres in the Western Cape and five other tuberculosis-endemic countries, GeneXpert had a specificity of 99% and an overall sensitivity of 90.3% (76.9% in smear-negative cases) for the detection of M. tuberculosis.40 For rifampicin resistance detection, the sensitivity of the test was 94.4% and the specificity 98.3%.49 Based on these excellent results, a World Health Organization (WHO) expert group recommended in September 2010 that Xpert MTB/RIF should be used as an initial diagnostic test in individuals suspected of having multidrug-resistant (MDR)TB or HIV-associated TB, and that the test may be used as a follow-on test to microscopy where MDR and/or HIV is of lesser concern, especially in smear-negative specimens. In

Introduction of molecular techniques into national TB control programmes

Two methods which detect M. tuberculosis and rifampicin resistance as a marker for multiple drug resistance in sputum samples have been approved by the Foundation for Innovative New Diagnostics (FIND) for use in TB highly endemic countries: the GenoType MTBDRplus Hain LPA, which also detects isoniazid resistance in a large percentage of cases and has been approved for use on smear microscopy-positive sputum specimens,37 and the exciting FIND-approved GeneXpert,42 which has been extensively validated in a multicentre study involving three countries where TB is highly prevalent, including South Africa, and is presently being phased into the TB control programme of this country. Their performance in various studies and phasing into the South African TB control programme will be briefly outlined below. GenoType MTBDRplus assay The GenoType MTBDRplus line probe assay (Hain LPA) has been extensively evaluated in South Africa. In studies conducted in the Western Cape (536 culture isolates) and Gauteng (731 isolates), sensitivities of 98.7% and 94.2% for detection of rifampicin and isoniazid resistance, respectively, were recorded in the Western Cape,40 and 95.0% and 93.4% in Gauteng.41 The respective specificities for resistance to rifampicin and isoniazid were 99.4% and 99.7% for the Western Cape,40 and 99.7% and 100% for Gauteng.41 Based on these findings and a subsequent large demonstration study in South Africa, endorsed by FIND, the National TB Control Programme (NTBCP) in South Africa, in collaboration with the NTBRL, decided to introduce the Hain LPA in approximately 30

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December 2010, WHO endorsed Xpert MTB/RIF for the rapid diagnosis of TB and MDR-TB and decided to develop a global roadmap for the rapid uptake of the technology in a systematic and phased approach. Mechanisms were proposed to monitor and assess the roll-out and to document the impact on case detection, MDR-TB response scale-up and cost effectiveness. It was also decided to negotiate with FIND prices for tests and four-module instruments for the public sector in low- and middle-income and high-burden countries. South Africa initiated a phased roll-out programme of this technology in 2010 and by World TB day on 24 April 2011, 23 targeted sites in 10 "hotspot" regions with a particularly high prevalence of TB were enrolled in the country. Further phasing in of the roll-out programme will follow. An algorithm for the diagnosis of TB in South Africa has been proposed, and there is little doubt that Xpert MTB/RIF will play a pivotal role in the NTBCP of the country in the near future.

Mycobacterium tuberculosis. J Clin Microbiol 2002; 40: 150-154 5 Moore AJ, Evans CAW, Gillman RH, et al. Microscopic-observation drug-susceptibility assay for the diagnosis of TB. N Engl J Med 2006; 355: 1539-1550 6 Albert H, Hydenrych A, Mole R, et al. Evaluation of FASTPlaqueTB-RIF, a rapid,manual test for the determination of rifampicin resistance from Mycobacterium tuberculosis cultures. Int J Tuberc Lung Dis 2001; 5: 1-6 7 Piuri M, Jacobs WR, Hatfull GF. Fluorobacteriophages for rapid, specific, and sensitive antibiotic susceptibility testing of Mycobacterium tuberculosis. PLoS ONE |www.plosone. org; 2009; 4(3):e4870 8 Kim S-Y, Park Y-J, Kang S-J, et al. Comparison of the BDProbeTec ET system with the Roche COBAS AMPLICOR system for detection of Mycobacterium tuberculosis complex in the respiratory and pleural fluid specimens. Diagn Micrbiol Infect Dis 2004; 49: 13-18 9 Libana E, Aranaz A, Francis B, Cousins D. Assessment of genetic markers for species differentiation within the Mycobacterium tuberculosis complex. J Clin Microbiol 1996; 34: 933-938 10 Tevere VJ, Hewitt PL, Dare A, et al. Detection of Mycobacterium tuberculosis amplification with pan-Mycobacterium primers and hybridization to an M. tuberculosis-specific probe. J Clin Microbiol 1996; 34: 91 11 Richter E, Rsch-Gerdes S, Hillemann D. Evaluation of the GenoType Mycobacterium assay for identification of mycobacterial species from cultures. J Clin Microbiol 2006; 44: 17691775 12 De Beenhouwer H, Liang Z, de Rijk P, et al. Detection and identification of mycobacteria by DNA amplification and oligonucleotide-specific capture plate hybridization 13 Blakemore R, Story E, Helb D, et al. Evaluation of the analytical performance of the Xpert MTB/RIF assay. J Clin Microbiol 2010; 48: 2495-2501 14 Telenti A. Marchesi F, Balz M, et al. rapid identification of mycobacteria to the species level by polymerase chain reaction and restriction enzyme analysis. J Clin Microbiol 1993; 31: 175-178 15 Palomino JC. Molecular detection, identification and drug resistance detection in Mycobacterium tuberculosis. FEMS Immunol Med Microbiol 2009; 56:103-111 16 Boehme CC, Nabeta P, Henostroza G, et al. Operational feasibility of using loop-mediated isothermal amplification for diagnosis of pulmonary tuberculosis in microscopy centers of developing countries. J Clin Microbiol 2007; 45:1936-1940 17 Piersimoni C, Scarparo C. Relevance of commercial amplification methods for direct detection of Mycobacterium tuberculosis complex in clinical samples. J Clin Microbiol 2003; 41: 5355-5365 18 Fang R, Li X, Hu L, et al. Cross-priming amplification for rapid detection of Mycobacterium tuberculosis in sputum specimens. J Clin Microbiol 2009; 47: 845-847 19 RussoC, Tortoli E, Menichella D. Evaluation of the new GenoType Mycobacterium Assay for identification of mycobacterial species. J Clin Microbiol 2006; 44: 334-339 20 Tortoli E, Mariottini A, Mazzarelli G. Evaluation of INNO-LIPA MYCOBACTERIA v2: Improved reverse hybridization multiple DNA probe assay for mycobacterial identification. J Clin Microbiol 2003; 41: 4418-4420 21 De Viedma DG. Rapid detection of resistance in Mycobacterium tuberculosis: a review discussing molecular approaches. Clin Microbiol Infect 2003; 9: 349-359 22 Burggraf S, Reischl U, Malik N et al. Comparison of an internally controlled, large-volume LightCycler assay for detection of Mycobacterium tuberculosis in clinical samples with the COBAS AMPLICOR assay. J Clin Microbiol 2005; 43: 1564-1569 23 Van Doorn HR, Claas EC Templeton KE et al. Detection of point mutation associated with high-level isoniazid resistance in Mycobacterium tuberculosis by using real-time PCR technology with 3-minor groove binder-DNA probes. J Clin Microbiol 2003; 41: 4630-4635 24 Holland PM, Abramson RD, Watson R, Gelfand DH. Detection of specific polymerase chain reaction product by utilizing the 5.3 exonuclease activity of Thermus aquaticus DNA polymerase. Proc Nat Acad Sc USA 1991; 88: 7276-7280 25 El-Hajj HH, Marras SAE, Tyagi S, Kramer FR, Alland D. Detection of rifampin resistance in Mycobacterium tuberculosis in a single tube with molecular beacons. J Clin Microbiol 2001; 39: 4131-4137 26 Van Doorn HR, Claas EC, Templeton KE, et al. Detection of a point mutation associated with high-level isoniazid resistance in Mycobacterium tuberculosis by using real-time PCR technology with 3- minor groove binder-DNA probes. J Clin Microbiol 2003; 41: 4630-4635 27 Jureen P, Engstrand L, Eriksson S, et al. Rapid detection of rifampin resistance in Mycobacterium tuberculosis by pyrosequencing technology. J Clin Microbiol 2006; 44: 1925-1929 28 Nash KA, Gaylan A, Inderlied CB. Detection of rifampin resistance in Mycobacterium tuberculosis by use of a rapid, simple, and specific RNA/RNA mismatch assay. J Infect Dis 1997; 176: 533-536 29 Telenti ANH, Cole ST. Detection of mutations in mycobacteria by PCR-SSCP (Single-Strand Conformation Polymorphism). In: Stoker Ng, Parish T, eds. Mycbacteria protocols. Totowa, New Jersey: Humana Press, 1998: 423-230 30 Fan X-Y, Hu Z-Y, Xu F-H, et al. Rapid detection of rpoB gene mutations in rifampin-resistant Mycobacterium tuberculosis isolates in Shanghai by using the amplification refractory mutation system. J Clin Microbiol 2003; 41: 993-997 31 Inagaki T,Yagi T, Ichikawa K, et al. Evaluation of a rapid detection method of clarithromycin resistance genes in Mycobacterium avium complex isolates. J Antimicrob Chemother 2011; 66: 722-729 32 Evans JT, Parveen A, Smith GE, et al. Application of denaturing HPLC to rapidly identify rifampin-resistant Mycobacterium tuberculosis in low- and high-prevalence areas. 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Future prospects
The advent of the GeneXpert assay is likely to be regarded as an historic milestone in TB diagnosis and management, and its performance and usefulness will be difficult to surpass or even emulate. It is probably unnecessary to further improve the specificity of molecular technology based on known unique sequences of M. tuberculosis complex-specific targets. With regard to sensitivity, the estimated level of detection of 4.5 genome copies per PCR reaction of the Xpert MTB/ RIF is very low but compared to culture, the ~131 CFU/ml in sputum leaves room for some improvement. It is possible that the sensitivity of the GeneXpert, as well as other and new methods, could be improved by better preservation of DNA integrity and by reducing inhibition of DNA amplification by inhibitory substances in sputum. The determination of the genetic basis of drug resistance not yet discovered for antiTB agents will lead to greater sensitivity of tests for the rapid detection of drug resistance. Further research on the performance of newly described methods and exploitation of microarray-based nucleotide chip and molecular beacon technology may well result in improvement of existing methods, or lead to the development of new tests. With the tremendous amount of ongoing research in the field of molecular diagnostics, methods to improve patient management even further are likely to continue to materialise in the future.

References
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41 Matsoso LG, Veriava Y, Poswa X, et al. Validation of a rapid tuberculosis PCR assay for detection of MDR-TB patienta in Gauteng, South Africa. South Afr J Epidemiol Infect 2010; 25(2): 12-15 42 Helb D, Jones MR, Story E, et al. Rapid detection of Mycobacterium tuberculosis and rifampin-resistance by use of on-demand near-patient technology. J Clin Microbiol 2010; 48: 229-237 43 Nagdev KJ, Kashyab RS, Parida MM, et al. Loop-mediated isothermal amplification for rapid and reliable diagnosis of tuberculous meningitis. J Clin Microbiol 2011; 49: 1861-1865 44 Iwamoto T, Sonobe T, Hayashi K. Loop-mediated isothermal amplification for direct detection of Mycobacterium tuberculosis complex, M. avium, and M. intracellulare in sputum samples. J Clin Microbiol 2003; 41: 2616-2622 45 Poon LLM, Wong BWY, Ma EHT, et al. Sensitive and inexpensive molecular test for falciparum malaria: detecting Plasmodium falciparum DNA directly from heat-treated blood by loopmediated isothermal amplification. Clin Chem 2006; 52: 303-306 46 Njiru ZK, Mikosza ASJ, Matovu E, et al. African trypanosomiasis: Sensitive and rapid detection of the sub-genus trypanozoon by loop-mediated isothermal amplification (LAMP) of parasite DNA. Int J Parasitol 2008; 38: 589-599 47 Banada PP, Sivasubramani SK, Blakemore R, et al. Containment of bioaerosol infection risk by the Xpert MTB/RIF assay and its applicability to point-of-care settings. J Clin Microbiol 2010; 48: 3551-3557 48 Boehme CC, Nabeta P, Hillemann D, et al. Rapid molecular detection of tuberculosis and rifampin resistance. N Engl J Med 2010; 363: 1005-1015 49 Boehme CC, Nicol M, Nabeta P, et al. Reasibility, diagnostic accuracy, and effectiveness of decentralized use of the Xpert MTB/RIF test for diagnosis of tuberculosis and multidrug resistance: a multicentre implementation study. Lancet 2011; 377: 1495-1505

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