TABLE 5.

List of issues to be considered and sources of possible error in context with interpretation and confirmation of results of NAAT

Step	Issue(s) to consider and indicate	Pitfalls and comments
A	Interpretation: what was considered a positive/negative analysis and what was considered a positive/negative result	How many replicates of those tested had to be positive for the sample to be considered positive?
		How was the assay-specific cutoff value defined, e.g., based on clinical data or presentation of the patients; microscopic, culture, or serological results; or just on the assay's lower detection limit?
B	Confirmation: (i) by DNA reextraction and NAAT repetition; (ii) by amplification of different gene; (iii) by independent method	Neither hybridization (irrespective of whether a probe, amplicon, or primers are used) nor dot blotting or sequencing represents an adequate methodology to confirm a specimen as truly positive or negative for a target gene, since these methods would also confirm a positive result accomplished due to amplicon carryover contamination; the specified methods rather ensure specificity of the amplicon produced during PCR and, partly, might enhance assay sensitivity