Real Time PCR in Infectious Disease Diagnosis

Molecular diagnostic tools and detection methods such as nucleic acid amplification are being used increasingly in the clinical microbiology laboratory to enhance the diagnosis of microbial pathogens (Lanciotti, 2001; Mackay, 2004). Nucleic acid-based technology is also used to assess drug resistance and epidemiological surveillance (Piatek, 1998; Makinen, 2001; Huletsky, 2004; Sloan, 2004). The principle of the real-time PCR is primarily used to detect and amplify a unique gene or a signature sequence of the microorganism. Quantitative measurements of viral load can also be made simple. Sensitive detection and accurate identification can speed up reporting of microbial pathogens without reliance on their phenotypic characteristics or viability after antibiotic treatment.

The application of real-time PCR in infectious diseases enables the diagnosis of microbial pathogens both with accuracy and expediency. The clinical significance of using molecular diagnosis of infectious agents can be characterized by the following aspects. (1) Pathogens that show fastidious slow growth or inability to grow in vitro: Mycobacterium, Legionella, Bartonella, Leptospira, Borrelia, Bordetella, Mycoplasma, and Tropheryma whippelii may require days or weeks of incubation under specific conditions; (2) obligatory intracellular organisms (Chlamydia, Rickettsia, Coxiella, Ehrlichia, DNA and RNA viruses); (3) prior antibiotic use; (4) biochemically inert for phenotypic characterization; (5) additional waiting time for drug-resistance determination, (6) diagnostic speed from bench to bedside.

Qualitative real-time amplification has outpaced conventional culture methods in detection of a long list of specific pathogens that are difficult to cultivate: Bartonella henselae, Bordetella pertussis, Borrelia burgdorferi, Coxiella burnetii, Ehrlichia spp., Legionella spp., Mycoplasma pneumoniae, Chlamydia trachomatis, Rickettsia, Toxoplasma gondii, Microsporidium, Cryptosporidium, Tropheryma whippelii, Mycobacterium tuberculosis and its drug-resistant determinants (Franzen, 1999; Pretorius, 2000; Hammerschlag, 2001; Bell, 2002; Fournier, 2002; Gerard, 2002; Kovacova, 2002; Exner, 2003; Templeton, 2003; Wang, 2003; Fenollar, 2004; Koenig, 2004; Simon, 2004; Wada, 2004; Khanna,

2005). Furthermore, the rapid turn-around time supported by real-time PCR may directly benefit the patient care and reduce mortality in areas of invasive infections caused by common pathogens. The examples are infective meningitis of bacterial or viral etiology, such as Streptococcus pneumoniae, Streptococcus agalactiae, Neisseria meningitidis, Haemophilus influenzae, Listeria monocytogenes, enteroviruses, herpes simplex viruses (HSV), and so forth (Corless, 2001; van Haeften, 2003; Archimbaud, 2004; Bryant, 2004; Guarner, 2004; Mengelle, 2004; Mohamed, 2004; Picard, 2004; Uzuka, 2004; Aberle, 2005).

Quantitative measurement of viral load using real-time PCR is another significant methodology improvement, and its diagnostic implication is infinite. First of all, HIV viral copy numbers in blood and body fluids are important disease and treatment markers directly tied into actions of clinical management. RNA reverse transcription and PCR (RT-PCR) can be established in a single-tube reaction, and copy numbers can be extrapolated from a standard curve in a single run (Kostrikis, 2002; Erikkson, 2003; Lee, 2004; Watzinger, 2004). Similarly, other viral etiology such as cytomegalovirus (CMV; Jebbink, 2003), HSV-1, HSV-2, varicella-zoster virus (VZV), Epstein-Barr virus (EBV; Legoff, 2004), parvovirus B19 (Hokynar, 2004; Plentz, 2004; Liefeldt, 2005), human polyomaviruses of BK and JC, and human herpesviruses 6,7, and 8 can be measured both qualitatively and quantitatively according to clinical needs (Whiley, 2001; Beck, 2004; Watzinger, 2004). RT-PCR can be performed to detect and quantify hepatitis A virus (HAV Costa-Mattioli, 2002), hepatitis B (HBV; Payungporn, 2004; Sum, 2004; Yeh, 2004; Pas, 2005; Zhao, 2005), and hepatitis C (HCV; Candotti, 2004; Castelain, 2004; Cook, 2004; Koidl, 2004; Walkins-Riedel, 2004) in whole-blood samples. Real-time RT-PCR panels are increasingly becoming commonplace for respiratory viral diagnosis of influenza A and B viruses, parainfluenza viruses, human adenoviruses, human metapneumovirus, and respiratory syncytial virus, respectively (Kahn, 2003; Boivin, 2004; Cattoli, 2004; Daum, 2004; Frisbie, 2004; Moore, 2004; O'shea, 2004; Stone, 2004; Templeton, 2004; Ward, 2004).

Despite apparent high sensitivity and specificity, molecular amplification techniques are not error-free. Contamination as a result of amplicon carry-over is a top concern of its practice in clinical diagnostics. Physical and chemical control of amplified products has to be designed and implemented before the tests are validated. Strict separation of pre- and post-PCR (negative-pressure room for post-PCR analysis) environments through laboratory design and personnel training has to be the first step. Amplification chemistry employing uracil and uracil-N-glycosylase is an effective end-product degradation control (Pennings, 2001; Pierce, 2004). Second, microbial DNA extraction is a rate-limiting step deciding the ultimate test sensitivity. The wide spectrum of cell wall makeup pertaining to specific microorganisms makes it impossible to limit the method of extraction to any single standard approach. Specific emphasis has to be made to optimally recover DNA materials from certain species of bacteria or parasites. Sonication and/or freeze-thaw methods can be used in conjunction with enzyme digestion for DNA extraction from mycobacteria and cyst-forming protozoa parasites (Harris, 1999; Kostrzynska, 1999; Lanigan, 2004). Third, sampling error is intrinsic to PCR-based approach due to its small specimen input nature. A false-negative reaction can be a result of low copy-number of the target material or simply missing the target material from the infected foci. Fourth, microbial genome database is rapidly growing but incomplete. Diagnosis based on single target amplification, followed by sizing, melting peak analysis, or even sequencing may not be sufficient to pin down a specific microbial agent. Sequence polymorphism as well as unknown etiology has repeatedly surprised the interested microbial miner in the field of infectious diseases. Finally, molecular sequence-based diagnosis does not provide viable organisms for additional phenotypic or genotypic investigation. Traditional culture methods remain of indispensable value for biological genetic research of emerging virulence or drug-resistance traits and epidemiological surveillance.

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