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/HPSCfrom each different anatomical site in the same patient. Susceptibility tests should be repeated if there isclinical evidence of failure to respond to therapy or if cultures fail to convert to negative after two monthsof therapy. For patients with resistant isolates, including resistance to the lower, critical concentration ofisoniazid, referral to or consultation with a specialist in TB treatment should be considered. 175The application of two commercially available DNA line probe assays, Genotype MTBDR TM and INNO-LiPA Rif TM to detect resistance to isoniazid and rifampicin (HAIN) or rifampicin alone (INNO LiPA) can beperformed when there is a strong suspicion of MDR-TB. Neither is 100% sensitive and results must beconfirmed by conventional testing. 176 In vitro susceptibility tests are very satisfactory for isoniazid, rifampicinand pyrazinamide and slightly less so for streptomycin. Results from ethambutol (E) and second line drugsmay vary depending on the test method used.Monitoring of anti-mycobacterial drug serum levelsThis may be occasionally required in suspected cases of non-compliance, malabsorption or toxicity. Thisservice can be provided by prior arrangement with the Antimicrobial Reference Laboratory in SouthmeadHospital, Bristol (see appendix 7 for contact details).Molecular typing of M. tuberculosisThere are currently three prominent methods for typing of M. tuberculosis strains. The current “goldstandard” is the IS6110-based restriction fragment length polymorphism fingerprinting. 177 This technique istechnically demanding and requires abundant amounts of growth of isolates. The remaining two methodsare polymerase chain reaction (PCR)–based genotyping tests, mycobacterial interspersed repetitive units(MIRU) typing 178 and spoligotyping. 179 In combination, the latter two tests (MIRU typing and spoligotyping)provide a highly discriminatory method to identify strains and will be used in the CDC TB GenotypingProgramme to enable rapid genotyping of isolates from every patient in the United States. 180 The IMRLintends to use MIRU typing in the first instance supported by spoligotyping.Genotyping of isolates can assist in the clinical and public health management of patients in severalsituations: 112• Genotyping allows evaluation of isolates with different patterns of drug susceptibility. Theoriginal organism may develop drug resistance during or after anti-TB therapy or the patientmay be re-infected with a different strain. The former may be due to non-adherence to therapyor reduced concentrations of anti-TB drugs as a result of malabsorption or drug interaction.The latter may be due to re-infection which would require further contact tracing investigationsas a public health issue.• Evaluation of an outbreak can be more clearly delineated or previously unrecognised contactsdetected• Genotyping can help to establish where resources might best be directed in a TB controlprogramme• On average 3% of patients from whom M. tuberculosis is apparently isolated in clinicallaboratories do not have TB. These positive cultures are due to cross-contamination. 1814.5 False Positive CulturesA review of reports of false positive cultures for M. tuberculosis showed that false positives were identifiedin 93% of studies that evaluated more than 100 patients. 181 The median false positive rate was 3.1%, with arange of 2.2% - 10.5%, and even higher rates (13.6%) have since been published. 182 The mechanism of falsepositive cultures can be many and include contamination of clinical equipment, clerical error and laboratorycross-contamination.For the purposes of further investigation, the source laboratories should not delay the forwarding ofpossible false positive M. tuberculosis complex isolates to the IMRL. The IMRL should perform DNAfingerprinting on all positive M. tuberculosis isolates and not delay the reporting of test results back to thesource laboratory. Confirmed false positives should be reported back to the clinicians as soon as possible.Clinicians should balance laboratory test results with their clinical judgement on whether or not a patienthas TB and inform the laboratory of any doubts.-53-
<strong>Guidelines</strong> on the Prevention and Control of Tuberculosis in Ireland 2010HSE/HPSCLaboratory cross-contaminationOften the most significant laboratory feature is that the false positive culture is the only positive culture froma patient. However, single positive cultures also occur among patients who meet the clinical criteria for adiagnosis of TB. Prospective monitoring of single-positive cultures detected two outbreaks of laboratorycross-contamination that had not been recognised by clinicians or laboratory personnel. 181The quality assurance programme for mycobacteriology laboratories should include a plan for theidentification and review of possible false positive cultures. Criteria that might prompt a review shouldinclude a patient with a single culture positive specimen, cultures with a very low colony count on solidmedia and isolates with unexpected drug resistance.Possible causes of laboratory cross contaminationThe most common causes of laboratory cross-contamination include: 183• Contamination of multiple-use equipment for dispensing reagents• Aerosols• Splashing• Sampling equipment• Reprocessing of contaminated specimens and• Mislabelling.The general principle is to isolate each specimen completely so that there are no opportunities to transferan inoculum from one sample to another via pipettes, the lips, caps of tubes, splashes or common reservoirsof reagents or containers used for discarded materials. Examples of good laboratory practice include thefollowing: 184• Only bring the required numbers of items such as loops, swabs, pipettes, universal containers, etc.into the safety cabinet for each session of work• When using pipettes to deliver reagents, use a separate pipette for each specimen and each timethat the reagent bottle is entered• Individually wrapped sterile plastic pipettes should be used• Reagents, such as sodium hydroxide, should be supplied or prepared in individual sterile vials. Usea separate vial to add a reagent to each sample rather than dispensing it from a common container.• Remove and replace the cap from each specimen tube sequentially during the addition of reagentsto specimens, so that only one tube is open at a time and so that tube caps do not becomeinterchanged• Avoid spills and splashes when decanting supernatants• Clean and disinfect the exposed surfaces of the safety cabinet after each session of work• Where possible ensure that specimens are processed in the order of smear negative to smearpositive specimens. This requires the keeping of an up-to-date list of known smear positivepatients.• Process positive culture vials only when all the work on specimens has been completed for the day• At the end of the working day dispose of any used items in the cabinet and clean and disinfect theinterior surfaces of the safety cabinet• If possible, use a separate safety cabinet for specimens and all positive cultures, whether liquid orsolid 185• Investigate for possible cross-contamination when specimens in proximity to one another becomepositive. Cross-contamination may not occur with all samples in sequence, such that negativecultures occasionally may be found between positive cultures 183• Ensure that written procedures for the processing of cultures include detailed instructions and thatstaff have a very good understanding of the rationale for all aspects of the procedure.4.6 Interferon Gamma Release Assays (IGRA)The TST is a long established test that uses a relatively crude mixture of antigens from M. tuberculosis todetect the immune response to infection with M. tuberculosis (past or present). As a result false positivereactions can occur because of previous BCG vaccination or sensitisation to non-tuberculous mycobacteria.It has many limitations and requires well-trained personnel to both administer and interpret the test(see chapter 2).-54-
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Guidelines on the Prevention and Control of Tuberculosis in Ireland

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