Sample Adequacy Control (SAC) Lowers False Negatives and Increases the Quality of Screening: Introduction of “Non-Competitive” SAC for qPCR Assays

Abstract

Sample Adequacy Control (SAC) has critical analytical, clinical and epidemiological value that increases confidence in a negative test result. The SAC is an integral qPCR assay control, which ensures that all pre-analytical and analytical steps are adequate for accurate testing and reporting. As such, a negative SAC with a negative result on pathogen screen specifies that the result should be reported as inconclusive instead of negative. Despite this, many regulatory approved tests do not incorporate SAC into their assay design. Herein, we emphasize the universal value of SAC and offer for the first time, a simple technical strategy to introduce non-competitive SAC which does not interfere with the limit of detection for the screened pathogen. Integration of SAC can provide key benefits towards identifying, isolating, quarantining and contact tracing infected individuals and in turn can improve worldwide efforts in infection control.

Importance of Adequate qPCR Controls in Infection Control

“…The accuracy of diagnostic test results are dependent on adequate samples [ 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 ]. Current clinical protocols allow for a variety of samples types to be used for the detection of respiratory pathogens, including various anatomical sites and sampling techniques, each having its own respective interpretation of sampling adequacy [ 1 , 6 , 10 , 11 , 12 , 13 , 14 ].…”

“…Current clinical protocols allow for a variety of samples types to be used for the detection of respiratory pathogens, including various anatomical sites and sampling techniques, each having its own respective interpretation of sampling adequacy [ 1 , 6 , 10 , 11 , 12 , 13 , 14 ]. Nasopharyngeal swabbing is one of the most common methods for obtaining clinical specimens [ 4 , 9 , 13 , 15 , 16 , 17 , 18 ]. However, the human genome equivalents present in the respiratory sampling can vary over one million-fold, while the ratio of virus genome equivalents to human genome equivalents can differ by up to one billion-fold (from (1/3 × 10) 4 to 3 × 10 4 ratios) [ 3 , 4 , 19 , 20 ].…”

“…Nasopharyngeal swabbing is one of the most common methods for obtaining clinical specimens [ 4 , 9 , 13 , 15 , 16 , 17 , 18 ]. However, the human genome equivalents present in the respiratory sampling can vary over one million-fold, while the ratio of virus genome equivalents to human genome equivalents can differ by up to one billion-fold (from (1/3 × 10) 4 to 3 × 10 4 ratios) [ 3 , 4 , 19 , 20 ]. This inherent variability in both human and virus genome equivalents can be measured with high resolution techniques, like quantitative PCR, by following the quantitative signal of sample-specific biomarkers that must be present in every sample [ 2 , 3 , 4 , 6 , 7 , 8 , 21 , 22 ].…”

“…To estimate the impact of sampling variability on respiratory swab results, we measured positivity rates (numbers of positive tests/total number of tests) of three common respiratory viruses (influenza A, influenza B, and RSV) as a function of the number of human genome equivalents present in the sample. The concentration of single-copy-human-gene (RNase P) is typically chosen to present the quantitative measure of SAC [ 3 , 4 , 6 , 7 , 20 , 22 , 23 ]. In the case of symptomatic respiratory infections, it is usually assumed that the quantity of a virus-specific biomarker is a few log values higher than the sample adequacy biomarker.…”

“…For example, during the viral incubation period, the ratio between viral and human genome equivalents can be very low (~1/10,000) [ 3 ], which can differ greatly from what is seen in a patient during a typical symptomatic infection. Thus, in the early stages of viral infections, determination of sample adequacy can be extremely valuable [ 4 , 17 , 18 , 24 , 25 , 26 , 27 ] by ensuring the quality and quantity of sampling was sufficient, and greatly reducing the possibility of a false negative result. In addition, consistent use of SAC allows earlier detection of positive cases and improves infectious control measures.…”