Remarks

• 1.These recommendations apply to the use of LPAs for testing sputum smear-positive specimens (direct testing) and cultured isolates of MTBC (indirect testing) from both pulmonary and extrapulmonary sites.
• 2.LPAs are not recommended for the direct testing of sputum smear-negative specimens.
• 3.These recommendations apply to the detection of MTBC and the diagnosis of MDR-TB, but acknowledge that the accuracy of detecting resistance to rifampicin and isoniazid differs and, hence, that the accuracy of a diagnosis of MDR-TB is reduced overall.
• 4.These recommendations do not eliminate the need for conventional culture-based DST, which will be necessary to determine resistance to other anti-TB agents and to monitor the emergence of additional drug resistance.
• 5.Conventional culture-based DST for isoniazid may still be used to evaluate patients when the LPA result does not detect isoniazid resistance. This is particularly important for populations with a high pretest probability of resistance to isoniazid.
• 6.These recommendations apply to the use of LPA in children based on the generalization of data from adults.

Implementation considerations Adopting LPAs to detect rifampicin and isoniazid resistance does not eliminate the need for conventional culture and DST capacity. Culture and phenotypic culture-based DST have critical roles in monitoring patients’ responses to treatment and detecting additional resistance to second-line agents.

• The adoption of LPA should be phased in, starting at national or central reference laboratories, or those with proven capability to conduct molecular testing. Expansion could be considered, within the context of a country’s plans for laboratory strengthening, the availability of suitable personnel in peripheral centres and the quality of specimen transport systems.
• Adequate and appropriate laboratory infrastructure and equipment should be provided, to ensure that the required precautions for biosafety and the prevention of contamination are met – specimen processing for culture and procedures for manipulating cultures must be performed in biological safety cabinets in TB-containment laboratories.
• Laboratory facilities for LPAs require at least three separate rooms, one each for DNA extraction, pre-amplification procedures, and amplification and post-amplification procedures. To avoid contamination, access to molecular facilities must be restricted, a unidirectional workflow must be implemented and stringent cleaning protocols must be established.
• Appropriate laboratory staff should be trained to conduct LPA procedures. Staff should be supervised by a senior staff member with adequate training and experience in molecular assays. A programme for the external quality assessment of laboratories using LPAs should be developed as a priority.
• Mechanisms for rapidly reporting LPA results to clinicians must be established, to provide patients with the benefit of early diagnosis. The same infrastructure used for performing LPAs can be used also to perform second-line LPAs.
• LPAs are designed to detect TB and resistance to rifampicin and isoniazid in the direct testing of processed sputum samples, and in the indirect testing of culture isolates of MTBC. The use of LPAs with other respiratory samples (e.g. from bronchoalveolar lavage or gastric aspiration) or extrapulmonary samples (e.g. tissue samples, CSF or other body fluids) have not been adequately evaluated.
• The availability of second-line agents is critical in the event that resistance to rifampicin or isoniazid, or both, is detected.
• For patients with confirmed MDR/RR-TB, second-line LPAs are recommended to detect additional resistance to second-line anti-TB agents.