4.1. SPECIFICITY VS SENSITIVITY Developing testing methodologies that are sensitive and specific can be difficult. Linear primers or probes complementary toward specific areas of the nucleic acid target can result in false-negative results if the target region is polymorphic. These polymorphisms obstruct base-pairing with the primer or probe. Inaccurate quantification resulting from these variations has been demonstrated from studies with human immunodeficiency virus (HIV) (46). These variants can occur naturally or be introduced by enzymes with poor fidelity during the amplification process. Incorporation of erroneous bases during the in vitro replication process can hinder end-point probe-based detection if the mis-incorporated base is introduced early (within the first three cycles) of the reaction. In this case, the resulting amplicon will not reflect the actual target sequence. If the error is present in the sequence complementary to the probe, the probe might not hybridize because of this mismatch. Methodologies that use low annealing temperatures (i.e., 42°C) might nonspecifically bind to related sequences. To address these issues, methods often incorporate a secondary probe detection step to increase specificity.
Targeting conserved regions of viral, fungal, or bacterial genomes allows the identification of novel unculturable infectious entities and results in higher sensitivity. In bacteria, the DNA sequence that encodes the 16s rRNA has regions that are highly conserved and is referred to as ribosomal DNA (rDNA). Using this approach, different bacterial strains or biotypes have been identified for Streptococcus sp., Mycobacterium sp., coryneform bacterial isolates (47,48) as well as other bacterial species (49). The rDNA sequence most amenable to identification by amplification and subsequent sequencing for fungi is the large subunit. Strain identification of the fungi-nail-tissue-associated Trichophyton rubrum and the sinus-cavity-associated Schizophyllum commune have been assisted with the aid of conserved primers, followed by sequencing (50,51). Using this approach, Candida dubliniensis, which is often incorrectly identified as Candidia albicans, has been classified as an emerging yeast pathogen (52). Sequencing need not always be required for the identification of fungal isolates. The information gained through sequencing can be adapted to a more simplistic approach. A line probe assay (LiPA), in which unique probes are affixed to a solid support and hybridized with a labeled product, has been used to identify C. albicans, C. para-psilosis, C. glabrata, C. tropicalis, C. krusei, C. dubliniensis, Cryptococccus neoformans, Aspergillus fumigatus, A. versicolor, A. nidulans, and A. flavus (53).
Similarly, primers designed in this manner can detect over 80 different types of HPV (54,55). Although most interest in HPV-associated cancers have classically been for the types associated with cervical dysplasia, identifying cutaneous types associated with epidermodyplasia verruciformis is useful for confirming the clinical diagnosis. In addition, this approach can be used to detect various herpes viruses associated with lym-phomatoid papulosis (36). Although these approaches cited here use PCR, they underscore the importance of primer design and are amenable to other amplification methods utilizing the alternative methods described previously.
4.2. PROBLEMS WITH ENZYMATIC AMPLIFICATION ASSAYS A caveat with all amplification assays is the use of enzymes to facilitate the increase in the target. Inhibitors are often identified in clinical specimens. Internal standards are necessary to rule out the presence of inhibitors that could lead to false-negative results (56). Even in cases of nonprotein-based enzymatic activity (such as in hammerhead ribozymes), various components can result in enzymatic inhibition (57,58).
4.3. CONTAMINATION The fear of amplicon contamination figures prominently in molecular laboratories utilizing amplification methods. Because small amounts of amplicons can result in widespread contamination, clinical laboratories must implement numerous contamination control protocols such as dedicated pipettors, aerosol-resistant tips, different work areas for master mix preparation, specimen extraction and amplicon detection, and unidirectional workflows. Some commercial kits contain reagents that control contamination by making the amplicon sensitive to enzymatic degradation (amperase; Roche Molecular Systems, Indiannapolis, IN). Closed-tube systems decrease the risk for contamination by physically separating the amplified product from the environment; however, precautions must still be made to avoid inadvertently contaminating the reagent preparation area and specimen extraction area with positive specimens or control material.
4.4. FLEXIBILITY Commercially available kits are not always available for esoteric targets. For these less common agents, the clinical laboratory must develop and validate in-house molecular assays for these tests. User-developed assays for esoteric testing most often use PCR technology. One possible explanation is the higher prevalence of PCR equipment and experience in clinical laboratories. Yet another explanation could be relative ease of optimizing this assay as the higher specificity associated with high-temperature annealing (59,60,61). As other approaches become more frequently used, it is anticipated that users will learn how to modify these platforms to address esoteric testing.
Finally, molecular methods are increasingly being used to assist in the diagnosis of various genetic and infectious diseases. The use of these methods improves patient care by decreasing the turnaround times (relative to culture or serol-ogy) and for confirming diagnoses based on clinical observations. Technology is rapidly advancing to decrease the time required for these assays as well as their costs. Many of the methods discussed in this chapter could facilitate the implementation of automated testing in molecular diagnostic laboratories. In the future, the range of molecular pathology will extend beyond nucleic-acid-based detection systems and will evolve to utilize information derived from the proteome as well to provide information on optimizing treatment and predicting outcomes for patients with both genetic and infectious diseases.
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