Quality Control

Quality control for any clinical laboratory procedure is critical for ensuring the accuracy of patient results, and ensuring the quality of molecular methods in the clinical microbiology laboratory is equally important. The sensitivity of molecular methods is so high that even one molecule of target can be used as a template. Thus, ensuring that the integrity of specimens is maintained, i.e., that specimens are not contaminated by other specimens or with the products of previous amplification procedures, is critical to avoid false positives. On the other hand, it is equally important to ensure that the lack of a product in an amplification procedure is due to the absence of the target organism and not the presence of inhibitors preventing the amplification of target sequences (false negative).

The incorporation of positive controls in a nucleic acid amplification assay shows that the assay system is functioning properly. A sensitivity control that is positive at the lower limit of detection demonstrates sensitivity of qualitative assays. Two positive controls, one at the lower limit and the other at the upper limit of detection, should be run in quantitative assays to test the dynamic range of the assay. Reagent blank or contamination controls are critical for monitoring reagents for carry-over contamination. These controls contain all of the reagents except target sequences and should always be negative. For typing and other studies that might include nontarget organisms, a negative template control containing nontarget organism^) should also be included. With regard to amplification controls (see below), the negative template control should have a positive amplification control signal, whereas the reagent blank should be negative for target and amplification. The presence of an amplicon in the negative control negates the assay, and the source of the contamination must be found.

In order to rule out false negatives due to amplification failure, an amplification control aimed at a target that is always present can be incorporated into an amplification assay. If the amplification control is amplified, then the fact that the target did not amplify can be more confidently interpreted as a true negative result. Amplification controls are usually housekeeping genes or those that are always present in a human sample. Housekeeping genes that are used as internal controls include prokaryotic genes such as groEL, rpoB, recA, and gyrB19 and eukary-otic genes such as p-actin, glyceraldehyde-3-phosphate, interferon-7, extrinsic homologous control, human mitochondrial DNA and peptidylprolyl isomerase A.15,20

Internal controls are amplification controls that monitor particular steps of an amplification method. Internal controls can be either homologous extrinsic, heterolo-gous extrinsic, or heterologous intrinsic. A homologous extrinsic control is a wild-type-derived control with a nontarget-derived sequence insert. This control is added to every sample after nucleic acid extraction and before amplification. The amplification of this control occurs using the same primers as for the target. It is good for ensuring that amplification occurs in the sample, but it does not control for target nucleic acid degradation during extraction. Heterologous extrinsic controls are nontarget-derived controls that are added to every sample before nucleic acid extraction. This control will ensure that extraction and amplification procedures were acceptable, but a second set of primers must also be added to the reaction for this control to be amplified. Use of this control requires that the procedure be optimized such that the amplification of the control does not interfere with the amplification of the target.

Heterologous intrinsic controls are eukaryotic genes. Human gene controls serve to ensure that human nucleic acid is present in the sample in addition to controlling for extraction and amplification. The use of this control requires that either two amplification reactions are performed on the sample, one for the control and the other for the target gene, or that the amplification procedure be multiplexed, which may result in interference of the amplification of the target.

In a procedure that detects a microorganism, a positive result states that the organism is present in that sample, whereas a negative result indicates that the organism is not present (at least not at amounts up to the detection limits of the assay). Although most false positives can be eliminated by preventing carryover contamination, another source of false positives that cannot be controlled in the laboratory is the presence of dead or dying microorganisms in the sample of a patient taking antimicrobial agents. In this situation, the nucleic acid-based tests will remain positive longer than culture assays and thus may appear as a false positive. Repeating the nucleic acid-based assay 3-6 weeks after antimicrobial therapy is more likely to yield a true negative result.15

False-negative results may be more problematic and arise when the organism is present, but the test result is negative. There are a few reasons for obtaining false-negative results on a sample. First, the organism may be present, but the nucleic acid was degraded during collection, transport, and/or extraction. This can be prevented by proper specimen handling, effective transport media, and inhibiting the activity of DNases and RNases that may be present in the sample and in the laboratory. Second, amplification procedures can be inhibited by substances present in the specimen. Hemoglobin, lacto-ferrin, heparin and other anticoagulants, sodium poly-anethol sulfonate (anticoagulant used in blood culture media), and polyamines have been shown to inhibit nucleic acid amplification procedures.15 Attention to nucleic acid isolation procedures and ensuring optimal purification of nucleic acid from other components of the specimen and extraction reagents will help minimize the presence and influence of inhibitors on the amplification reaction. Experimenting with different commercial nucleic acid extraction systems may result in discovering a system that is optimal for a particular purpose.15

Extensive validation must be performed on new molecular-based tests that are brought into the laboratory (see Chapter 16). Controls must be tested, and the sensitivity, specificity, and reproducibility of the assay must be determined. Proficiency testing of personnel should be performed regularly to ensure that the people performing the tests are doing so correctly. The Clinical and Laboratory Standards Institute,19 Association for Molecular Pathology,20 and the Food and Drug Administration (FDA) have guidelines for molecular methods in the laboratory.

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