Guidelines on the Prevention and Control of Tuberculosis in Ireland

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1♦<strong>Guidelines</strong> on the Prevention and Control of Tuberculosis in Ireland 2010HSE/HPSCClinicians need to be aware of the limitations of these tests particularly in relation to the types ofspecimens analysed, the commercial system in use and the quality assurance programmes in place. 159-162Application of NAAT for the diagnosis of TB in sputa, CSF, and the detection of rifampicin and isoniazidresistance in primary and reference specimens should be provided only where there is an adequateinfrastructure, primarily sufficient space for a unidirectional workflow and dedicated equipment. 163Standardised methods with adequate and appropriate positive and negative controls should be used. 112A cost effective approach is to base these tests in laboratories in Ireland with expertise in molecularbiology. For procedural and economic reasons, NAAT might be impractical for laboratories with a smallvolume of testing. Referral of samples for NAAT to high-volume laboratories might be preferable toimprove cost-efficiency, proficiency and turnaround times. 1584.4 Processing of Positive CulturesIdentification methodsPhenotypic methodsMicroscopic and colonial morphology and other growth characteristics are useful in making a preliminaryidentification of an acid-fast bacillus. Colonies of the tubercle bacilli are described as being “rough, toughand buff” on solid media and colonies tend not to emulsify easily for making smears. MTC form serpentinechords in liquid media and are easily observed with ZN staining. These characteristics can lead to the mostrelevant tests being performed on each isolate to obtain identification. 127Probe techniquesProbe detection methods such as the AccuProbe, targeting ribosomal RNA gene, can identify the MTC,M. avium, M. intracellulare, M. avium complex (MAC), M. gordonae and M. kansasii. The technique isrelatively simple and the results are available within hours. The specificity has been shown to be 100% butthe sensitivity can vary within the species or species complexes. 124PCR and restriction endonuclease analysisIn 1993, Telenti 164 modified the Polymerase Chain Reaction (PCR)-Restriction Endonuclease Analysistechnique, subsequently known as the PRA method, to allow for the rapid identification of mycobacteriato the species level. The method has been used extensively for mycobacterial identification. The principaldisadvantage is that it is not commercialised and does not have US Food and Drug Administration (FDA)approval or carry CE3F markings.PCR and reverse hybridisation techniquesNew molecular biology techniques based on PCR and reverse hybridisation procedures have recently beenmarketed for mycobacterial identification. The hybridisation technique is performed on nitrocellulose stripsonto which probe lines are fixed in parallel. This format enables simultaneous detection and identificationof different mycobacterial species. At present, two systems with this design are commercially availablein Europe, the INNO-LiPA TM Mycobacteria v2 test, designed to amplify the mycobacterial 16S-23S rRNAspacer region, and the HAIN GenoType TM Mycobacteria assay, targeting the mycobacterial 23S rRNA. 165Both methods can be completed within two working days and allow precise identification of the majority ofmycobacteria usually isolated in clinical laboratories. They are expensive to perform but this can be offsetby the reduction in turnaround and labour times. These methods are available in the United States as FDAapprovedtests and bear the CE mark in the European Community.DNA sequencingThe availability of DNA sequencing technologies constitutes a great benefit for mycobacterialidentification, owing to the slow growth of these organisms. Recent improvements in automation of targetamplification and sequence analysis have led to the practical implementation of DNA sequencing in someclinical laboratories. 124 The technique requires expensive equipment and is best reserved for referencelaboratories.♦The CE marking (also known as CE mark) is a mandatory conformity mark on many products placed on the single market in theEuropean Economic Area (EEA).-51-
<strong>Guidelines</strong> on the Prevention and Control of Tuberculosis in Ireland 2010HSE/HPSCMycobacterium tuberculosis complex (MTC)The Mycobacterium tuberculosis complex now consists of the following strains; M. tuberculosis sensustricto, M. bovis, M. bovis BCG, M. africanum 166 M. canettii 167 , M. bovis subsp. caprae 168;169 , M. microti 170 ,and M. pinnipedii. 171 All are known to cause infections in humans but they differ in their primary host,geographic range and pathogenicity. M. microti grows very slowly, often requiring up to 16 weeksincubation in liquid and solid culture media.Various methods may be used in order to differentiate members of the MTC. Specific inhibitors can beadded to a culture medium and growth or the lack of growth determined e.g. thiophen-2-carboxylichydrazide (TCH). M. tuberculosis is resistant to TCH while the other members of the complex are sensitive.Similarly, susceptibility to pyrazinamide is useful in differentiating M. bovis from other members of thecomplex in the majority of cases.More recently, a new commercially available DNA strip, GenoType MTBC, has been developed andevaluated. 172;173 Results demonstrated that the assay could unambiguously differentiate all of the MTC,with the exception of M. tuberculosis, M. africanum type II and M. canettii. The latter is considered to bea smooth variant of M. tuberculosis 167 and M. africanum II has not been successfully differentiated fromM. tuberculosis using molecular techniques suggesting that it likely represents phenotypically atypical M.tuberculosis strains. 174The GenoType MTBC was found to:• Enable a very rapid identification of the MTC• Fit into the work flow of a routine laboratory and• Be conducted in laboratories that do not carry out sophisticated biochemical tests fordifferentiation of the MTC.The results of these studies challenge the use of biochemical characteristics for classifying these organismsin diagnostic laboratories. 174Susceptibility testing of M. tuberculosisCDC has recommended that mycobacteriology laboratories work towards the goal of reporting first-linesusceptibility results for MTC within three to four weeks of receipt of the initial diagnostic specimen.Ideally, susceptibility results should be available within seven to 14 days after isolation of an MTC isolate.M. tuberculosis complex resistance is defined as “a decrease in sensitivity of sufficient degree to bereasonably certain that the strain concerned is different from a sample of wild strains of human type thathave never come into contact with the drug”. 175 Methods of drug susceptibility are not designed merelyto detect drug resistant mutants but are also to show that the great majority of bacilli in a culture are assusceptible to a given drug as one or more known susceptible strains. The object of susceptibility testing istherefore to determine whether an isolate is as likely to respond to standard therapy as one or more knownsusceptible strains. 127There are five methods in current use as outlined below:1. The absolute concentration method which is popular in some parts of Europe2. The proportional method also used in Europe and using Middlebrook 7H10 agar is the “goldstandard” method in the USA3. The disk diffusion method4. The resistance ratio method is used in the UK and those countries that are influenced by UKpractice and5. Commercially available systems, including the FDA approved Bactec 460TB, Bactec MGIT 960 andthe Versa TREK systems.The first four methods rely on conventional media and thus have the great disadvantage that there is along delay before results are available. In developed nations commercially available systems are used,so that results are made available more rapidly. 127 If resistance is detected the test may be repeated forconfirmation purposes, however, a report of the initial result should not be delayed while the repeattesting is being performed. The report should indicate that the drug resistance findings are preliminary andconfirmatory testing has been initiated.The first isolate of MTC obtained from every patient should be tested but also each isolate recovered-52-
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Guidelines on the Prevention and Control of Tuberculosis in Ireland

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