 Routine viral diagnostics: indirect and direct detection of viruses. ◦ Indirect detection: serological tests; ◦ Direct detection:  Viral antigens;  Viruses or viral components by isolation on cell cultures or through animal experiments;  Viral nucleic acids also referred to as nucleic acid testing (NAT).

 Molecular assays are currently used routinely in almost all routine virological laboratories;  Technological improvements provide the possibility to develop and introduce assays for most viruses of clinical interest.  Reduced risk of contamination, shortened turn-around time to generate results, and improved sensitivity.  Standardization and quality assurance/quality control issues have often remained underdeveloped requiring urgent improvement.

 Reliable viral diagnostics depends on pre-analytical issues: ◦ Choice of the correct specimen; ◦ Optimal sampling time with regard to the course of disease; ◦ And both time and conditions of the specimen transport to the laboratory.

 QC systems have been implemented in many laboratories;  Certification – based on the supervision of description and conformity of processes;  Accreditation – focuses on the competence of the laboratory providing reliable test results and their correct interpretation.

 Quality assurance = careful documentation in the routine diagnostic laboratory.  For each newly implemented test or test system – a standard operation procedure and verification or validation data must be available for each test.

 Validation goes a step beyond verification: Validation determines that we are doing the correct test and Verification confirms that we are doing the test correctly.

 Verification or validation work has to be done if a new test is introduced in the routine diagnostic laboratory.  Any change of an existing test procedure requires further validation work.

 Commercialy available test – the manufacturer is responsible by the processes. Nevertheless, the user must verify that performance characteristics, such as accuracy and precision, are achieved in the lab.

VerificationValidation Accuracy Sensitivity Precision (intra- and inter-assay)Specificity Linearity (if quantitative)Precision (intra- and inter-assay) Linearity (if quantitative)

 ACCURACY = the degree of conformity of a measured or calculated quantity to its actual (true) value and can be estimated by analyses of reference materials or comparisons of results with those obtained by a reference method. When neither is available, other evidence relevant to the ability of the method to measure the analyte is needed.

 PRECISION = the level of concordance of the individual test results within a single run (intra-assay) and from one run to another (inter-assay). It is usually characterized in terms of the standard deviation of the measurements and relative standard deviation (variation coefficient).  LINEARITY = the determination of the linear range of quantification (quantitative tests or test systems).

 Clinical laboratory that develops (home-brewed) tests is responsible for the correct performance of the test.  They must be validated including accuracy, sensitivity, specificity, precision, and, if quantitative, linearity.

 SENSITIVITY = measure how well a test correctly identifies the proportion of true positives.  SPECIFICITY = measure how well a test correctly identifies the proportion of true negatives.

 Simplified validation procedure – it may be applied only if a test for validation is based on scientific publication in a highly recognized journal, calibrators are not commonly accessible or the significance of the parameter to be tested is very limited.

 Calibrators = reference material, patient samples or pooled sera.  Patient samples or pooled sera – they must have been tested earlier with the existing gold standard.

 Calibrators: ◦ Positive controls: For detection of virus specific antibodies and detection of viral antigens defined as more than dilution factor 3 over the lower limit of detection of the test and within the upper limit of linearity. For NAT defined as more than 1 log 10 over the lower limit of detection of the test and within the upper limit of linearity.

 Calibrators: ◦ Low positive controls: For detection of virus specific antibodies and detection of viral antigens defined as up to dilution factor 3 over the lower limit of detection of the test. For NAT defined as up to 1 log 10 over the lower limit of detection of the test. o Negative controls.

 If more than one positive control is necessary to complete testing for a certain performance characteristics, they should always contain different concentrations (within the linearity range as defined above) of the parameter to be tested.

 It is necessary to validate each matrix (sample material) intended to be tested in the routine diagnostic lab.  At least nine specimens (3 positives, 3 low positives, and 3 negatives) must be tested for each additional matrix.

 For home-brewed tests it is proposed to estimated the detection limit.  DETECTION LIMIT = lowest concentration or quantity of an analyte that can be detected with a stated reasonable uncertainty for a given analytical procedure. If there is no reference material available, both the determination of the detection limit and absolute quantification are not possible.

 If an existing test is modified or even replaced, diagnostic accuracy must be included in the evaluation process.  The outcome from a test under evaluation is compared with the outcome from the reference test.  20 specimens (7 positives, 6 low positives, and 7 negatives) must be tested in parallel.

 In case of qualitative tests, one positive and one low positive specimen are used for determination of intra-assay precision. Each sample is tested three times within a run.  For inter-assay precision, one positive and one low positive specimen are used. Each sample is tested once on three different days.

 In case of quantitative tests for detection of virus specific antibodies or viral antigens, four positive and three low positive specimens are used for determination of intra-assay precision (each sample tested three times within a run).  For inter-assay precision, two positive and one low positive specimen are used (each sample tested once on three different days).

 In case of quantitative NAT tests, three positive and three low positive specimens are used for determination of intra-assay precision.  For inter-assay precision, one positive and one low positive specimen are used.  Linearity must be verified by analyzing a serial dilution (10-fold dilution series with at least three dilution steps) of one positive specimen in duplicate.

 Primer and probe sequences (that has already been published in a highly recognized journal) must be checked carefully by use of a genome sequence databank.  The molecular technique employed, the detection format, introduction of an internal control (IC), and quantitation must be addressed.

 Automation reduces hands-on work and thus helps to avoid human error.  To guarantee maximum specificity, introduction of a probe detection format is required.  The IC must be incorporated in every NAT assay to exclude false- negative results.  The IC should be added to the specimen before the start of the nucleic acid extraction procedure to guarantee validation of the entire analytical testing processes.

 Sensitivity = it is determined by testing 10 positive and 10 low positive specimens.  Specificity = it is determined by testing 20 negative but potentially cross-reactive specimens including positives for antibodies against viruses of the same family, sera testing positive for rheumatoid factor, and sera containing auto-antibodies for serological tests.

 For tests based on NAT, sample positive for viruses of the same family and samples spiked with potentially cross-reactive reference material are analyzed.  For each pottentially cross-reactive analyte, one high-positive (at least 10 5 TCID 50 /mL or 10 5 genome equivalents/mL) must be tested.

 Use of six positives instead of three for determination of intra-assay precision and two instead of one for determination of inter-assay precision.  Linearity must be validated additionally by analyzing serial dilutions (10-fold dilution series with at least four dilution steps) of two positive specimens in duplicate on 2 days.

 The cell line should be tested with two concentrations of both a reference virus strain and a wild type isolate.  Tests must be done in triplicate on 3 days.  Precision is performed by using 20 wild type samples which must be tested in parallel on the existing and the newly introduced cell line.  The viability of the cells and the influence of the sample matrix must be recorded carefully.

 Implementation of a new test or test system in the routine diagnostic virological laboratory demands for verification or validation procedures in compliance with a quality management system.