Diagnosis of multidrug-resistant tuberculosis and extensively...

The emergence of multidrug-resistant tuberculosis (MDR-TB)and, more recently, extensively drug-resistant TB (XDR-TB)

is a major threat to global TB control (1-4). MDR-TB is resistantto isoniazid (INH) and rifampicin (RIF). While MDR-TB hasbeen documented in the past (3), the term XDR-TB appeared inthe literature for the first time in March 2006, in a report jointlypublished by the World Health Organization (WHO) and theUS Centers for Disease Control and Prevention. This reportdescribed a severe form of disease caused by strains of

Mycobacterium tuberculosis which were resistant not only to INHand RIF but also to at least three of the six classes of second-lineanti-TB drugs (fluoroquinolones, aminoglycosides, polypeptides,thioamides, cycloserine and para-aminosalicylic acid) (1).

Because the initial XDR-TB definition was dependent ondrug susceptibility testing (DST) of second-line drugs, which isknown to be unreliable, and because some forms of drug-resistantTB are more treatable than others, it was subsequently revised bythe WHO XDR-TB Task Force in October 2006. XDR-TB is

Can J Infect Dis Med Microbiol Vol 19 No 2 March/April 2008 169

1WHO Collaborating Centre for TB and Lung Diseases, Fondazione S Maugeri, Care and Research Institute, Tradate; 2Institute of Infectious andTropical Diseases, University of Brescia; 3Supranational Reference Laboratory, S Raffaele Institute, Milano, Italy; 4McGill University andMontreal Chest Institute, Montreal, Quebec

Correspondence: Dr Madhukar Pai, Department of Epidemiology, Biostatistics and Occupational Health, McGill University, 1020 Pine Avenue West, Montreal, Quebec H3A 1A2. Telephone 514-398-5422, fax 514-398-4503, e-mail [email protected]

Received for publication November 27, 2007. Accepted January 18, 2008

©2008 Pulsus Group Inc. All rights reserved

SPECIAL ARTICLE

Diagnosis of multidrug-resistant tuberculosis andextensively drug-resistant tuberculosis:

Current standards and challenges

Giovanni Battista Migliori MD PhD1, Alberto Matteelli MD PhD2, Daniela Cirillo MD PhD3, Madhukar Pai MD PhD4

GB Migliori, A Matteelli, D Cirillo, M Pai. Diagnosis ofmultidrug-resistant tuberculosis and extensively drug-resistanttuberculosis: Current standards and challenges. Can J Infect DisMed Microbiol 2008;19(2):169-172.

INTRODUCTION: The emergence of multidrug-resistant tubercu-losis (MDR-TB) and, more recently, extensively drug-resistant TB(XDR-TB) is widely considered a serious threat to global TB control.Over 400,000 new cases of MDR-TB occur each year and, althoughtheir rates are currently unknown, XDR-TB cases have been detectedin every country where there is capacity to detect them (includingCanada). METHODS: The present article provides a narrative overview of thevarious diagnostic options available for XDR-TB, including conven-tional tools and newer rapid tests for drug resistance. Available datasuggest that automated liquid cultures are highly accurate and theiruse is rapidly expanding. Newly developed phenotypic tests includeTK Medium (Salubris Inc, USA), microscopic-observation drug-susceptibility assay, FASTPlaque-Response bacteriophage assay(Biotec Laboratories Ltd, UK), colorimetric redox indicator methodsand the microcolony method. These tests are usually cheaper but notalways simple to perform, with some requiring high standards ofbiosafety and quality control. Among the newly developedphenotypic methods, reverse hybridization-based assays, referred to asline probe assays, represent a useful tool because of their superior accu-racy and cost-effectiveness.CONCLUSIONS: To effectively address the threats of MDR-TB andXDR-TB, global initiatives are required to scale-up culture and drugsusceptibility testing capacities, especially in high-burden countrieswhere such capacity is scarce. In parallel, efforts are needed to expandthe use of novel and emerging technologies (ie, molecular diagnostics)for the rapid determination of drug resistance.

Key Words: Diagnosis; Drug resistance; MDR-TB; Tuberculosis;

XDR-TB

Le diagnostic de tuberculose multirésistante etde tuberculose ultrarésistante : Les normes etles défis actuels

INTRODUCTION : L’émergence de la tuberculose multirésistante(TBM) et, plus récemment, de la tuberculose ultrarésistante (TBU), estlargement perçue comme une grave menace au contrôle mondial de la TB.Plus de 400 000 nouveaux cas de TBM se déclarent chaque année, et bienqu’on n’en connaisse pas le taux, on a recensé des cas de TBU dans tousles pays en mesure de les déceler (y compris le Canada).MÉTHODOLOGIE : Le présent article contient un aperçu narratif desdiverses possibilités diagnostiques de la TBU, y compris les outils classiqueset les tests rapides récents de pharmacorésistance. Selon les donnéesdisponibles, les cultures liquides automatisées sont très précises, et leurutilisation prend une rapide expansion. Les nouveaux tests phénotypiquesincluent le TK Medium (Salubris Inc, États-Unis), la méthode depharmacosensibilité par observation microscopique, le dosagebactériophage FASTPlaque-Response (Biotec Laboratories Ltd, Royaume-Uni), les méthodes colorimétriques par indicateur Redox et la méthodedes microcolonies. Ces tests sont généralement moins coûteux, mais pastoujours faciles à exécuter, car certains exigent des normes élevées debiosécurité et de contrôle de la qualité. Parmi les nouvelles méthodesphénotypiques, les dosages par hybridation inversée, ou méthodes parsonde en direct, constituent un outil utile en raison de leur précisionsupérieure et de leur rapport coût-efficacité.CONCLUSIONS : Pour affronter la menace de la TBM et de la TBUavec efficacité, il faudra entreprendre des initiatives mondiales pourdévelopper les capacités des tests de culture et de susceptibilité auxmédicaments, notamment dans les pays où le fardeau est élevé et où lescapacités sont déficientes. Parallèlement, il faudra consentir des effortspour généraliser l’usage des technologies novatrices et émergentes (p. ex.,diagnostics moléculaires), afin de déterminer rapidement lapharmacorésistance de la maladie.

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now defined as resistance to, at least, INH and RIF and, inaddition, to any fluoroquinolones and to at least one of thethree following injectable drugs – capreomycin, kanamycinand amikacin (1,5). These classes of drugs are the most potentand the least toxic options for second-line therapy. In addition,DST is more reliable for fluoroquinolones and injectable drugsthan for other second-line drugs.

LABORATORY DIAGNOSIS OF MDR-TB AND XDR-TB

Drug-resistant TB often goes undetected and untreated inmany countries. With the exception of a few developed coun-tries, most national TB programs worldwide do not routinelyprovide diagnostic services based on culture and DST. The lab-oratory is an essential component in TB control programs, andbroader access to DST is a priority for most countries. Earlychoice of appropriate treatment is an essential determinant offavourable outcome, and rapid determination of drug resist-ance can allow a customized approach to treatment early in thecourse of the disease and can potentially reduce morbidity,mortality and infectiousness (6).

The diagnosis of MDR-TB and XDR-TB is hampered bythe absence of effective and affordable rapid diagnostic tech-niques for drug sensitivity. Several approaches, phenotypic andmolecular, have been explored to develop rapid, reliable andaccurate methods for the rapid detection of drug resistance inM tuberculosis. These methods should also be evaluated andapplied in high-incidence areas.

CONVENTIONAL CULTURE-BASED METHODSUsing standardized DST procedures with conventional methods,eight to 12 weeks are required to identify drug-resistantmicroorganisms on solid media (ie, Lowenstein-Jensen medium).In general, such methods assess inhibition of M tuberculosisgrowth in the presence of antibiotics to distinguish betweensusceptible and resistant strains.

The proportion method allows precise determination of theproportion of resistant mutants to a certain drug; the resistanceratio method compares the resistance of an unknown strain withthat of a standard laboratory strain. While relatively inexpensiveand undemanding of sophisticated equipment, results usuallytake weeks and this is challenging; inappropriate choice of treat-ment regimen may result in death within weeks of initiation,such as in the case of XDR-TB (especially in HIV-infectedpatients). In addition, delayed identification of drug resistanceresults in inadequate treatment, which may generate additionaldrug resistance and continued transmission in the community.

LIQUID CULTURE-BASED METHODSAutomated liquid culture systems are more sensitive than solidmedia cultures, and they significantly reduce turnaround time.However, even with liquid cultures, two to four weeks are stillneeded to obtain results, and their substantially higher cost is anissue for resource-limited countries. The BACTEC 460 TBradiometric system (Becton Dickinson, USA) was considered tobe a major advancement when it was introduced, but has beenreplaced by the Mycobacteria Growth Indicator Tube system(Becton Dickinson, USA). Several published studies have shownthe excellent performance of the Mycobacteria Growth IndicatorTube system for the rapid detection of resistance to first- andsecond-line anti-TB drugs (7). Detection of drug resistance can

be accomplished in days rather than weeks, although still con-strained by high cost (equipment and consumables).

In 2007, the WHO issued policy guidance on the use ofliquid TB culture, DST and rapid species identification in low-resource settings (8). The WHO policy recommends phasedimplementation of these systems as a part of a country-specificcomprehensive plan for laboratory capacity strengthening, andaddresses key issues including biosafety, customer support, stafftraining, maintenance of infrastructure and equipment, speci-men transport and reporting of results.

NOVEL, RAPID PHENOTYPIC METHODSAmong novel, rapid phenotypic methods, the microcolonymethod is relatively low cost. It has been adapted for the rapiddetection of drug resistance directly from sputum samples, andhas been shown in early studies to be accurate for the detectionof MDR-TB compared with the reference proportion method,with results available in one week (9). Newly developedphenotypic tests such as TK Medium (Salubris Inc, USA),microscopic-observation drug-susceptibility assay (MODS) andFASTPlaque-Response bateriophage assay (Biotec LaboratoriesLtd, UK) are usually cheaper but not always simple to perform,with some requiring high standards of biosafety and qualitycontrol (10).

TK Medium is a novel colorimetric system that indicatesgrowth of mycobacteria by changing the colour of the growthmedium. Metabolic activity of growing mycobacteria changesthe colour of the culture medium, and this allows for an earlypositive identification before bacterial colonies appear. TKMedium also permits susceptibility testing for drug resistance,and can allow for differentiation between M tuberculosis andnontuberculous mycobacteria. Unfortunately, there is insuffi-cient published evidence on the field performance of this testin developing countries (10).

The MODS assay is based on the observation of thecharacteristic cord formation of M tuberculosis that is visualizedmicroscopically in liquid medium with the use of an invertedmicroscope (11). MODS uses simple light microscopy todetect early growth of M tuberculosis as ‘strings and tangles’ ofbacterial cells in the broth medium with or without antimicro-bial drugs (for DST) (12). The agreement between MODS andthe reference standard for drug susceptibility testing is 97% forINH, 100% for RIF, and 99% for INH and RIF combined(MDR). Lower values of agreement were obtained for ethamb-utol (95%) and streptomycin (92%). One minor disadvantageof MODS is the requirement for an inverted microscope forobservation of the mycobacterial growth.

FASTPlaque-Response is a phage amplification-based test,and has been developed for direct use on sputum specimens.Drug resistance is diagnosed when M tuberculosis is detected insamples that contain the drug (ie, RIF). A recent meta-analysisof the accuracy of phage-based methods for detecting RIF resist-ance in M tuberculosis concluded that these assays performed onM tuberculosis culture isolates have high sensitivity, but variableand slightly lower specificity (13). Not enough evidence is avail-able on the accuracy of these assays when performed directly onsputum samples. Safety and quality control issues related to theuse of this technique should also be addressed carefully.

Several colorimetric methods have also been proposed inthe past few years for the rapid detection of drug resistance inM tuberculosis. A recent systematic review and meta-analysis(14) of colorimetric redox indicator methods found evidence

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of high sensitivity and high specificity for the rapid detectionof MDR-TB. Colorimetric methods represent a good alterna-tive for the rapid detection of drug resistance in laboratorieswith limited resources. However, these tests cannot be directlyused on clinical specimens.

Overall, large multicentric studies defining the accuracy ofphenotypic DST methods are still unavailable. Practical issues,such as quality controls and training requirements, have notbeen adequately addressed under field conditions. The applica-tion of these approaches to support individualized treatmentthrough determination of second-line drug susceptibilityprofiles remains largely unexplored, implying that their appli-cation in support of individualized treatment of MDR-TB (andespecially for XDR-TB) remains uncertain.

NOVEL, RAPID MOLECULAR METHODSThe identification of specific mutations responsible for drugresistance has facilitated the development of novel, rapidmolecular tools for DST. The detection of RIF resistance istraditionally used as a predictor of MDR-TB – its positive pre-dictive value is a function of the sensitivity and specificity ofRIF resistance testing and the prevalence of MDR and non-MDR RIF resistance, which is highest among previouslytreated cases in settings with high MDR prevalence and lownon-MDR RIF resistance. Molecular tools are based on nucleicacid amplification in conjunction with electrophoresis,sequencing or hybridization. Although most of the techniqueswere initially developed to detect drug resistance in TB com-plex isolates, they are being evaluated for direct detection ofTB complex isolates and identification of alleles related todrug resistance in clinical specimens (such as sputum). Theirpotential advantage is that there is no need for growth of theorganism and DST results can be determined in days ratherthan weeks; research suggests that they can be highly reliable.

Direct sequencing is another approach to detecting muta-tions, but it is an expensive and time-consuming process.Techniques, such as real-time polymerase chain reaction, thatmake use of wild-type primer sequences to amplify genes andenable the use of specific probes (ie, molecular beacons) to iden-tify mutations are expensive and complicated, even if highlysensitive and specific. Reverse hybridization-based assays,referred to as line probe assays, represent a useful tool for theirsuperior cost-effectiveness. These tests are based on thehybridization of specific probes for wild-type and mutatedsequences of genes involved in drug resistance, and they showhigh specificity and medium/high sensitivity.

Commercially available line probe assays include the INNO-LiPA Rif. TB kit (Innogenetics, Belgium) and the GenoTypeMTBDR assay (Hain Lifescience, Germany). A recent meta-analysis summarized the results obtained for the INNO-LiPARif. TB test, and showed that this line probe assay has highsensitivity and specificity when culture isolates are used (15).The majority of studies had sensitivities of 95% or greater, andnearly all were 100% specific. The results, however, are less

accurate when the test is directly applied to clinical specimens(ie, sputum). There is a paucity of data on the application of thistest directly to clinical specimens.

The GenoType MTBDR test is able to detect mutations inthe rpoB gene for RIF resistance, and the most frequentmutation at codon 315 of the katG gene for INH resistance,either in isolates or clinical specimens. The specificity and sen-sitivity of the assay for RIF resistance were nearly 100%; forINH-resistance, despite a high specificity (approximately100%), the sensitivity of the test ranged from 70% to 90%,depending on the prevalence of the particular mutation at thekatG locus (16,17). GenoType MTBDRplus (Hain Lifescience,Germany), an advanced version of the assay, includes probesfor the identification of other mutations in the hotspot regionof the rpoB gene for RIF resistance, and probes to detect muta-tions in the promoter region of the inhA gene involved in INHresistance. These improvements facilitate the detection ofanother 10% to 20% of INH-resistant cases, with an enhance-ment in rapid MDR-TB diagnosis.

Overall, line probe assays are accurate and useful for rapiddetection of drug resistance directly in clinical specimens.However, the number of genes that can be analyzed remainslimited and the test fails to distinguish insertion mutations.Furthermore, they retain a lower sensitivity among acid fastbacilli-negative samples. In general, line probe assays are expen-sive and require sophisticated laboratory infrastructure. Theirrole and utility in low-income, high-burden countries will needto be evaluated in field studies.

CONCLUSIONSEffective control of MDR-TB and XDR-TB will require massivescaling-up of culture and DST capacity, and the expanded useof novel and rapid assays for drug resistance. Overall, molecularapproaches are still insensitive for many of the mutations thatallow some TB strains to remain resistant to second-line drugsdue to our limited understanding of the underlying biologicalmechanisms. Furthermore, all genotypic tests require DNAextraction, gene amplification and detection of mutation andare, therefore, relatively expensive and demand resources andskills that are usually unavailable in most regions where rates ofMDR-TB and XDR-TB are high. The challenge, therefore, is tonot only develop new tools, but to also make sure that benefitsof promising new tools actually reach the populations that needit most, but can least afford them.

Agencies such as the Stop TB Partnership, Foundation forInnovative New Diagnostics, the Special Programme forResearch and Training in Tropical Diseases, and the WHO, arewell placed to address these challenges (18). Thanks to variousinitiatives, the new diagnostics pipeline has rapidly expanded(10,19,20). Funding and international support for the newGlobal Plan to Stop TB, 2006–2015 (21) and the RetoolingTask Force (22) of the Stop TB Partnership will greatlyenhance the development and implementation of new toolsfor TB control.

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20. Perkins MD, Cunningham J. Facing the crisis: Improving thediagnosis of tuberculosis in the HIV era. J Infect Dis 2007;196:S15-27.

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22. World Health Organization. New technologies for tuberculosiscontrol: A framework for their adoption, introduction andimplementation <http://whqlibdoc.who.int/publications/2007/9789241595520_eng.pdf> (Version current at March 20, 2008).


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