Guidelines on the Prevention and Control of Tuberculosis in Ireland

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<strong>Guidelines</strong> on the Prevention and Control of Tuberculosis in Ireland 2010HSE/HPSC• Both viable and non-viable organisms will stain acid fast• Microscopy has a sensitivity of approximately 90% for the most infectious cases of presumed TB.It therefore provides information to support respiratory isolation of patients and other infectioncontrol measures that prevent transmission of disease.• The grading of a smear can give some indication to the probability of a positive culture 124;147 (seetable 4.3)• The minimum number of bacilli necessary to produce a result has been estimated to be between5,000 and 10,000 per millimetre of sputum. The incremental yield of AFB from serial smearexamination has been shown to be 80-82% for the first, 10-14% for the second and 5-8% for thethird specimen. 124;147• 50-80% of patients with pulmonary TB will have positive smears 110• It is a useful tool for initiating treatment and monitoring the progress of anti-TB drug therapy• Fluorescence microscopy is more sensitive and as specific as conventional AFB microscopy 148• A minimum of 75 fields (250x) or 300 fields (1000x) should be examined by fluorescence and ZNrespectively 145• Re-examination of negative smears from specimens with positive culture can dramatically decreasea laboratory’s rate of false negative smears 144• Supervisory review of doubtful results in newly diagnosed patients can markedly decrease theincidence of false positive smears 144;145• Optimal results for sputum are obtained when auramine phenol stain is applied to a liquefied,concentrated sample and examined before the decontamination process 149• In laboratories with good expertise the predictive value of a positive smear can be as high as 90%and the predictive value of a negative smear 96% (based on good quality specimens) 144• The laboratory should participate in an external quality assurance scheme e.g. UK NEQAS AFBscheme.Table 4.3: Relationship of acid-fast smear and culture yieldNo. of AFB seen by staining method and magnificationReport ZN stain FluorochromeX 1,000 X 250 X 450Estimatedconcentration ofbacilliProbability of aculture positiveresultDoubtful;repeat1-2/300F 1-2/30F 1-2/70F 5,000-10,000 50%1+ 1-9/100F 1-9/10F 2-18/50F About 30,000 80%2+ 1-9/10F 1-9/F 4-36/10F About 50,000 90%3+ 1-9/F 10-90/F 4-36/F About 100,000 96.2%4+ > 9/F >90/F >36/F About 500,000 99.95%DecontaminationMany of the specimens sent for the isolation of Mycobacterium tuberculosis are contaminated withcommensal flora originating from the specimen site. This is particularly true of specimens from therespiratory and genitourinary tracts. Sodium hydroxide is the most common decontaminating agent usedin mycobacteriology. A contamination rate of between 2% to 5% is deemed acceptable. 115 An incidence ofless than 2% may suggest excessive decontamination and above 5% may suggest that cultures which mightcontain mycobacteria may be overgrown by commensal organisms. 110Samples from cystic fibrosis (CF) patients present a significant problem in that Pseudomonas aeruginosa,isolated from approximately 80% of CF patients can hinder and even prevent the recovery of-49-
<strong>Guidelines</strong> on the Prevention and Control of Tuberculosis in Ireland 2010HSE/HPSCMycobacterium tuberculosis. 150 A two-step approach is advised where samples are initially decontaminatedwith N-acetyl-L-cysteine NaOH (NALC/NaOH) and only those samples which remain contaminated aresubjected to a second round of decontamination with NALC/NaOH and oxalic acid. Results suggest thatthis protocol could improve the recovery of mycobacteria from heavily contaminated specimens. 151CultureCulture is the bedrock and most reliable tool for the diagnosis of TB 152 and has a detection limit of 10 1to 10 2 viable organisms. 124 It is essential so that isolates are available for susceptibility testing, the resultsof which are necessary for the proper management of the patient. A confirmed case of TB is a case withculture confirmed disease due to M. tuberculosis complex. 23Isolates are also necessary in genotyping studies which are required for epidemiological investigationsand tracking laboratory cross-contamination. 153 Mycobacterium tuberculosis is a slow growing bacteriumand special media and procedures, not required for the culture of other organisms, are necessary. 127 Agarbased and egg based media, Middlebrook broths or solid media, the “traditional” methods of culture,have been superseded by the semi automated Bactec 460 and more recently by the continuous automatedmonitoring in liquid culture techniques (CAMLiC) 154 e.g. the Bactec MGIT960 TM , MB/BacT Alert 3D ‐TM andthe VersaTREK TM systems. These are now the basis of the “gold standard” in the isolation, culture anddefinitive diagnosis of mycobacterial disease.Each of the Bactec 460TB and CAMLiC systems has advantages and disadvantages and the choice ofsystem is often based on non-microbiological factors, e.g. size of equipment, quality of manufacturer’sservice and maintenance or in-built electronic data management systems. The microbiological factorsinclude:• Sensitivity of the detection system• Performance in recovery of Mycobacterium tuberculosis complex (MTC) and non-tuberculousmycobacteria (NTM)• Speed to positivity• Ability to perform susceptibility tests to anti-mycobacterial drugs and• Chemical agents, safety and cross-contamination.It is emphasised that in order to get optimal recovery of mycobacteria from clinical specimens usinga CAMLiC system, a combination of liquid and solid media is essential especially in non-specialisedlaboratories with a low incidence of M. tuberculosis. 155-157Recommendation:It is recommended that solid media be used in combination with a liquid culture system.Nucleic acid amplification tests (NAAT)CDC has produced an updated algorithm which offers useful guidance on the use of nucleic acidamplification tests (NAAT) 158 directly on specimens. CDC now recommends that NAAT should beperformed on at least one respiratory specimen from each patient with signs and symptoms of pulmonaryTB for whom a diagnosis of TB is being considered but has not yet been established, and for whom thetest result would alter case management or TB control activities such as contact tracing. 158Recommendation:All those wishing to undertake NAAT on suspected cases of pulmonary TB should seek advicefrom the local consultant microbiologist. It is recommended that NAAT should be madeavailable at the IMRL.-50-
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Guidelines on the Prevention and Control of Tuberculosis in Ireland

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