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Clinical microbiology laboratory is responsible for the isolation or detection of microorganisms to establish the diagnosis of infection. Rapid and accurate identification of these organisms and subsequent antibacterial drug susceptibility tests also guide antibiotic therapy. Although these goals can be fulfilled most of the time, some bacteria may be difficult to be identified due to fastidious growth, morphological variations, unusual biochemical reactions, lack of previous recognition, or a combination of these. Subculture failure, though it rarely happens, virtually makes routine identification impossible. Fortunately, technological advances have largely overcome these limitations for bacterial identification. One of the advances realized in the past decade or so has been the analysis of the nucleotide sequences of the 16S ribosomal RNA gene (16S rDNA), which has emerged as the single best method to identify bacteria (Kolbert and Persing, 1999; Drancourt et al., 2000). This chapter reviews its theoretical basis, methodology, clinical application, and limitations. A thorough and in-depth review with many practical points has just been published elsewhere (Clarridge, 2004).

“Blood banking has become a manufacturing industry, an industry that must conform to high standards and quality control requirements comparable to those of pharmaceutical companies or other regulated industries,” said David A. Kessler, M.D., former FDA commissioner (Revelle, 1995). Screening donated blood for infectious diseases that can be transmitted through blood transfusion is very important in ensuring safety. The United States has the safest blood supply in the world (Revelle, 1995), and the Food and Drug Administration (FDA) is striving to keep it safe by decreasing the risk of infectious disease transmission. The regulatory agency is continuously updating its requirements and standards for collecting and processing blood. An important step in ensuring safety is the screening of donated blood for infectious diseases. In the United States, tests for infectious diseases are routinely conducted on each unit of donated blood, and these tests are designed to comply with regulatory requirements (Table 21.1). The field of clinical microbiology and virology is now moving into the focus of molecular technology. Currently, nucleic acid testing techniques have been developed to screen blood and plasma products for evidence of very recent viral infections that could be missed by conventional serologic tests. It is time for all blood safety staffs to use molecular detection techniques. This approach can significantly aid in blood safety to reduce the risk of transmission of serious disease by transfusion. This chapter will review the current antigen/antibody–based technology, molecular biological technology, and published regulatory policy data for blood safety.

Sexually transmitted diseases (STDs) constitute the most common infectious diseases around the world and bear significant consequences for both the individual and public health of the community. More than 20 STDs have now been identi- fied, and they affect more than 13 million men and women in the United States each year (CDC, 2002). Data from the Centers for Disease Control and Prevention (CDC) show that more than 7 million cases of infection and more than 350,000 cases of infection were reported in 2000 (CDC, 2001). In the past decade, the rapid development of molecular techniques has gradually shifted the paradigm of laboratory diagnosis from traditional biological to molecular amplification and detection of major causative agents of sexually transmitted infections.

The first technique for diagnosing tuberculosis (TB) was reported in 1882 when Dr. Robert Koch, along with Dr. Paul Erlich, developed the acid-fast stain as a means to identify (MTB). TB remains a disease associated with crowded living conditions, depressed immunity, and poverty.MTB infects one third of the world’s population, and approximately 8 million new tuberculosis cases are reported each year, with a resultant 2 million deaths (Dye ., 1999).

is a major pathogen responsible for both nosocomial and community-acquired infections. Although the first isolates displaying resistance to methicillin (MRSA) were reported in the early 1960s (Barber, 1961), endemic strains of MRSA carrying multiple resistance determinants did not become a worldwide nosocomial problem until the early 1980s (Hryniewicz, 1999). The presence of MRSA in an institution is paralleled by an increased rate of bacteremia or other severe MRSA infections (Harbarth et al., 2000). MRSA-related bacteremia carries a threefold attributable cost and a threefold excess length of hospital stay when compared with methicillin-susceptible (MSSA) bacteremia (Abramson and Sexton, 1999).

Researchers have focused on developing specific assays for conclusively identifying and measuring the levels of bacteria, fungi, protozoa, viruses (microbes), and their associated products (biomarkers) that cause disease in humans and animals (Murray et al., 2003). Traditional methods using microscopy and chemical or immunological stains, test cultures with selective media or target cells, or serological assays have been used effectively to identify infectious agents in biological specimens or environmental samples. However, due to increasing veterinary, medical, and public health concerns, faster and more accurate diagnostic tools have been sought. Multiplex array-based assays allow for a range of biomarkers to be rapidly and simultaneously measured within specimens (Robertson and Nicholson, 2005). Recently, multiplex bead-based flow cytometric immunoassays have been developed and applied that show great promise for improving the study, diagnosis, and therapeutic management of infectious diseases (Alvarez-Barrientos et al., 2000; Jani et al., 2002).

Microbial strain typing is increasingly important in routine clinical microbiology laboratories as a method to track hospital-acquired infections. Although many methods can be used, this chapter focuses on one particular method used for typing of bacteria and fungi: repetitive sequence–based PCR (rep-PCR). A number of valuable reviews are available for more in-depth discussion of other technologies (Olive and Bean, 1999; Soll, 2000; Zaidi et al., 2003). The technology behind rep-PCR will be introduced with a review of traditional, manual rep-PCR. The latest advances will be highlighted with a discussion of the DiversiLab System, Bacterial Borcodes, Inc., Athens, GA, which is an automated rep-PCR technology. Additionally, we describe a comparison of the technologies, current applications in clinical microbiology laboratories, and future potential of rep-PCR as a routine clinical test.

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.

In the 21st century, one of the greatest challenges to public health and clinical microbiologists is the rapid detection and identification of emerging and reemerging pathogens. Complex factors such as genetic variation in the host and pathogen, environmental changes, population pressures, and global travel can all influence the emergence of infectious diseases. The SARS epidemic of 2003 highlighted the potential of an emerging pathogen to spread globally in a very short time frame (Peruski and Peruski, 2003). The diagnostics of such infectious diseases has been greatly affected in the past 20 years. No longer is cultivation and microscopy the only means of detecting infectious agents. With the introduction of molecular diagnostics, the ability to detect minute amounts of microbial nucleic acids in clinical specimens has revolutionized clinical microbiology. In particular, the utility of PCR allows the detection and quantitation of specific agents in a matter of hours. PCR sequencing of specific segments of nucleic acid allows for the determination of specific drug resistance that now aids in guiding viral therapies.