The clinical presentations for most infectious agents are often not specific enough to allow for a definitive diagnosis. Coughing and fever, for example, are symptoms that may be caused by many different bacterial or viral infections. Thus, for better treatment and disease control, a molecular differential diagnostic (MDD) assay that can identify, differentiate, and pinpoint the offending pathogen associated with a clinical syndrome (Fig. 27.1) is needed. MDDs are essential tools for effective infectious disease surveillance, biodefense, and personalized medicine.

MDDs are needed for emerging infectious disease surveillance and control. When outbreaks such as SARS occur, public health officials and laboratory scientists often struggle for weeks, if not longer, to identify the offending pathogen. With molecular differential diagnostic assays available, scientists involved in an outbreak investigation can quickly rule out many pathogens associated with similar clinical symptoms and focus on new, emerging infections. An MDD test can also aid in the management of a public health crisis. It can help health care personnel in triaging patients and determining which patients should be isolated, as well as identifying environmental sources of contamination within an intensive care unit (ICU) or patient room.

MDDs are needed for homeland security and biodefense. With the current global political atmosphere, biodefense threats are a reality. A first-response technology could quickly identify a bioterrorism agent and control the spread of the pathogen. Without the availability of MDDs for rapid pathogen identification, the bioterror-ism agent may go undetermined for days. Every hour wasted in determining the causative agent provides a greater opportunity for pathogen spread and for global panic to occur.

MDDs are needed for delivering true personalized medicine. Personalized medicine focuses on treating the patient, rather than the disease. It is genotype-based medicine, rather than phenotype- or symptom-based. An MDD test also makes it possible to practice theranosis (therapy guided by a diagnosis) by developing or reclassifying drugs that specifically target the molecular cause of the disease. If pharmacogenomics is the development of drugs based on individual

Pathogen Syndrome MDD Cause

Pathogen Syndrome MDD Cause

FIGURE 27.1. Multiple Pathogens can lead to the same clinical syndrome. One MDD test should be able to differentiate and identify the real cause.

FIGURE 27.1. Multiple Pathogens can lead to the same clinical syndrome. One MDD test should be able to differentiate and identify the real cause.

genotypes, then theranosis is the administration of drugs based on individual (or infectious agent) genotypes. It is clear that MDDs are needed, but in order to make the assays practical, we want them to have the following advanced features:

  • Multiplex capabilities. The definition of multiplexing is "receiving multiple signals from the same source." For MDDs, multiplexing refers to the ability to conduct multiple genotyping tests at the same time and within the same sample. We want multiplexing because it requires only small amounts of precious patient sample; it allows the clinician to run fewer tests while acquiring more relevant information; it reduces the amount of reagents, consumables, and time involved; and most importantly, it can save lives. For infectious diseases MDDs, we want a multiplex test that can identify all pathogens related to a clinical syndrome or that can detect all the genes and mutations responsible for the drug-resistance phenotype.
  • Specificity. Even though multiple microorganisms are studied simultaneously, we want only the pathogens associated with the infection be identified with a high level of confidence.
  • Sensitivity. We want a MDD to be able to identify a pathogen or drug resistance directly from a patient sample or enrichment culture. Using the patient sample directly reduces the time required for bacterial or viral culture preparation and enzymatic testing. Yet, bypassing this propagation step forces the assay to be sensitive enough to detect only a small amount of pathogen material present in the patient sample.
  • Reliability. For clinical application of MDDs, a consistent performance from assay to assay and from lot to lot is required.
  • Speed. For an MDD to be practical for infectious disease diagnosis and treatment, it must be locally available and produce results within a few hours.
  • Simplicity. An MDD should not require a Ph.D. laboratory scientist to conduct the assay. An MDD should be user-friendly and even automatable. No special training should be required to perform the assay. The MDD system should be easily integrated into standard molecular laboratory practice.
  • Affordability. MDDs should be efficient and cost-effective.

The technology advancements in this post-genomic era have made sequence information readily available for almost all known pathogenic microorganisms. Based on this information and armed with standard molecular tools, scientists have developed molecular assays, usually PCR-based, for almost every infectious pathogen. A simple Internet keyword search for a pathogen name and the word "PCR" will produce several pages referencing specific tests for that pathogen. From this exercise, it seems possible that the basic needs for molecular differential diagnosis can be met. However, to produce the MDD assay we really want, some unique technical challenges should be addressed.

The most difficult challenge of all is multiplexing. PCR technology has been established for nearly 20 years. However, multiplex PCR is still very difficult to accomplish. The following is a list of common challenges associated with multiplexing:

  • Incompatible loci. Each target in a multiplex PCR demands its own optimal condition; therefore, increasing the number of multiplex targets becomes difficult and, in many instances, impossible.
  • Lack of specificity. Multiple sets of high-concentration primers in a system often generate primer dimers or give nonspecific, background amplification. Lack of specificity also adds operational burdens by requiring post-PCR clean-up and multiple posthybridization washes.
  • Lack of sensitivity. Crowded primers reduce amplification efficiency and waste resources by occupying enzymes and consuming substrates.
  • Uneven amplification. Differences in amplification efficiency may lead to large discrepancies in amplicon yields. In a multiplex system, some loci may amplify very well, whereas others may amplify poorly or even fail to amplify. Uneven amplification also makes it impossible to accurately perform end-point quantitative analysis.
  • Lot-to-lot variation. Due to the fact that large amounts of primers are consumed in each reaction and that manufacturers can generate only a limited amount of assays per lot, quality control and quality assurance can be difficult.

In the following discussions of this chapter, we will present a new multiplex PCR technology developed by Genaco (Huntsville, AL, USA) scientists, called Tem-plex. The Templex technology answers many of the challenges that have already been described and delivers what infectious disease control professionals truly want. This chapter will also discuss technology integration strategies and application examples. Finally, the implementation and impact of MDDs (also called MD2 by Genaco) will be discussed.

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