2.  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).

3.  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.

4.  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.

5.  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.

6.  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.

7.  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.

8.  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.

9.  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.

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

11.  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.

12.  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).

13.  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.

14.  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.

15.  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.

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

17.  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.

18.  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.

19.  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.

20.  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.

21.  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.

22.  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.

23.  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.

24.  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).

25.  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.

26.  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.

27.  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.

28.  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.

29.  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.

30.  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.

31.  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.

32.  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.