Validation of molecular-diagnostic techniques in the parasitological laboratory

Diagnostic laboratories today often operate according to standard quality management procedures such as ISO/IEC 17025. This requires that only validated methods are used. Validation procedures help to document that a particular protocol used by the accredited laboratory has a guaranteed performance in that particular laboratory. Several study designs exist for validation procedures. Computer programmes are available to help with the statistical analysis of validation results. The agreement beyond chance of results obtained in the protocol that is to be validated can be compared to those achieved in an already established test (agreement). For a method that is used under routine conditions or for epidemiological studies, it is necessary to assess the diagnostic sensitivity and diagnostic specificity of the technique. These parameters can be estimated by comparing the method that needs to be validated with an existing reliable method (‘gold standard’). This is done by testing a standard set of well-documented samples using both techniques in parallel. Approaches using Bayes’ theorem are used to perform gold standard-free validations. Many PCR-based methods are characterised by an excellent analytical sensitivity and are thus good candidates for diagnostic tools of the required diagnostic sensitivity. However, the high level of analytical sensitivity can also make molecular techniques susceptible to cross-contamination and carry-over problems leading to false-positive results. Moreover, the presence of inhibitors can cause false-negative results. After an initial validation, test performance needs to be continuously monitored, e.g. by using combined Shewhart-CUSUM control routines, and test results compared to those obtained by other laboratories (proficiency testing).