GO TO CMA Home
GO TO Inside CMA
GO TO Advocacy and Communications
GO TO Member Services
GO TO Publications
GO TO Professional Development
GO TO Clinical Resources

GO TO What's New
GO TO Contact CMA
GO TO Web Site Search
GO TO Web Site Map


CMAJ
CMAJ - November 16, 1999JAMC - le 16 novembre 1999

CMAJ Specialty Spotlight · Infectious disease

Molecular diagnosis of infections in the new millennium

Natasha Press, MD; Marc Romney, MD; Satchan Takaya, MD; Brian Conway, MD

CMAJ 1999;161:1294


Technology: Polymerase chain reaction (PCR) (see sidebar)

Use: PCR is a highly sensitive technique that is used to amplify and detect small amounts of specific DNA sequences in biological material. It is made possible by the availability of specific DNA polymerases (e.g., Taq or Tth) that are produced by bacteria in hot springs. These thermostable enzymes allow for repeated cycles of denaturation and DNA synthesis in a test tube, theoretically generating 230 or 1 × 109 copies of a given genetic sequence in a matter of hours. The PCR assay can be used in diagnosis to detect unculturable or fastidious microorganisms that cannot be identified by conventional methods. It provides the rapid identification of common strains that cause epidemics1 and of microorganisms that can take weeks to culture. It was used to identify Bartonella henselae as the cause of bacillary angiomatosis,2 and is commonly used to measure the amount of human immunodeficiency virus in the blood as a marker of disease progression,3 and thus aids in determining the most effective antiviral therapy.

History: Dr. Kary Mullis,4 the genius behind this technology, conceived of the PCR process while working at Cetus Corporation (Emeryville, Calif.) in 1983 and was later awarded the Nobel prize in chemistry for it. The patent for the technology was later sold for $300 million, and by 1994 PCR had been referenced in over 7000 scientific publications.

Promise: The sheer speed of PCR-based assays (often 2­5 hours) means treatment can be implemented earlier. PCR is often the only test that can provide a diagnosis of HIV in neonates in the first week of life;5 the resulting early antiviral treatment may help prevent devastating neurologic complications. Although PCR-based assays are more expensive than conventional diagnostic tests, there is a significant potential for savings through improved health care outcomes.

Problems: The meaning of positive PCR results has not been validated for all infections. For instance, does the detection of cytomegalovirus indicate latent infection or current disease? PCR is also technically more difficult to perform than conventional tests, and false-positive or negative results due to technical problems, sample contamination, lack of specificity or amplification may hinder rather than help the clinician make decisions.6 The lack of standardization and reproducibility of many PCR-based assays have also limited many applications to "in-house" research use.

Prospects: PCR is a powerful tool with many legitimate applications; however, its premature acceptance as the universal standard may lead to diagnostic inaccuracies with significant clinical implications. In particular, its ability to detect nonviable genetic material presents problems of interpretation.

Although PCR-based assays have been available for almost 10 years they have not yet revolutionized the field of infectious diseases. There is a perception that the assays are complex and beyond the expertise of many laboratories. It is likely that the availability of automated workstations to perform sample processing, analysis and product detection will lead many to reconsider the use of PCR assays. The cost has also influenced the decision to retain more traditional and less costly procedures. It is important that the clinical benefit of a test outweigh its cost and complexity and that this is demonstrated in rigorous experimental conditions. In the study of HIV and AIDS, where such evidence exists, PCR-based assays have been embraced, and novel assays developed at Laval University7 may soon lead to widespread acceptance of this approach in bacteriology.

Competing interests: None declared.

Comments Send a letter to the editor
Envoyez une lettre à la rédaction

Reviews of PCR-based assays
  1. Eisenstein BI. The polymerase chain reaction: a new method of using molecular genetics for medical diagnosis. N Engl J Med 1990;322:178-83.
  2. Relman DA. The identification of uncultured microbial pathogens. J Infect Dis 1993;168:1-8.
  3. Fredericks DN, Relman DA. Application of polymerase chain reaction to the diagnosis of infectious diseases. Clin Infect Dis 1999;29:475-88.
Selected Web sites on PCR-based assays

The authors are from the Division of Infectious Disease and the Department of Pharmacology and Therapeutics, University of British Columbia, Vancouver, BC.


References
  1. Bifani PJ, Plikaytis BB, Kapur V, Stockbauer K, Pan X, Lutfey ML, et al. Origin and interstate spread of a New York City multidrug-resistant Mycobacterium tuberculosis clone family. JAMA 1996;275:452-7. [MEDLINE]
  2. Wagar EA. Defining the unknown: molecular methods for finding new microbes. J Clin Lab Anal 1996;10:331-4. [MEDLINE]
  3. Mellors JW, Munoz A, Giorgi JV, Margolick JB, Tassoni CJ, Gupta, P, et al. Plasma viral load and CD4+ lymphocytes as prognostic markers of HIV-1 infection. Ann Intern Med 1997;126:946-54. [MEDLINE]
  4. Mullis K, Faloona F, Scharf S, Saiki R, Horn G, Erlich H. Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harb Symp Quant Biol 1986;51(pt 1):263-73. [MEDLINE]
  5. Owens DK, Holodniy M, McDonald TW, Scott J, Sonnad S. A meta-analytic evaluation of the polymerase chain reaction for the diagnosis of HIV infection in infants. JAMA 1996;275:1342-8. [MEDLINE]
  6. Vaneechoutte M, Van Eldere J. The possibilities and limitations of nucleic acid amplification technology in diagnostic microbiology. J Med Microbiol 1997;46:188-94. [MEDLINE]
  7. Picard FJ, Bergeron MG. Rapid diagnosis of bacterial infections using technologies based on nucleic acid detection. Can J Infect Dis 1999;10(Suppl C): 16C-24C.

© 1999 Canadian Medical Association or its licensors