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Veterinary Forum September 2008 (Vol 25, No 9)

Clinical Report — PCR technology today

by Sophia Yin, DVM, MS (Animal Science)

    Veterinarians frequently treat patients that they suspect have ehrlichiosis, babesiosis or another tick-borne infection because they cannot wait to obtain a diagnosis from culture and sensitivity testing. Likewise, they may see a patient with positive tick-borne disease titers and wonder whether the results indicate an active or previous infection.

    When veterinarians are faced with one of these or similar dilemmas, polymerase chain reaction (PCR) testing may provide a clear benefit, says Edward B. Breitschwerdt, DVM, DACVIM (Companion Animal), co-director of the Vector-Borne Disease Laboratory at the North Carolina State University College of Veterinary Medicine in Raleigh. Although traditionally this technology has been available only at university laboratories, some commercial laboratories now offer PCR testing as well.

    "PCR testing is a way of detecting DNA or RNA through amplification of any gene you wish to target," he explains. "It is frequently used for detecting and diagnosing infectious disease organisms by looking for specific genes or portion of genes in the patient's sample. Detection of the DNA usually correlates with active infection."

    Getting timely Results

    The key to PCR technology is the amplification process, emphasizes Breitschwerdt, who also is a professor of internal medicine and infectious disease at NC State. "No one can visualize a single strand of DNA or gene sequence, but if you have amplified many gene copies (up to millions) through PCR, that allows the laboratory to detect the amplified DNA. You then can have it further characterized, if you wish, by obtaining the DNA sequence."

    In general, PCR technology can be used to amplify any gene that has been identified. However, the advantage of PCR testing is its ability to detect organisms that are difficult or time-consuming to grow. For instance, a standard abscess in a dog can be swabbed and Staphylococcus intermedius or S. aureus organisms identified by culture within 24 hours. Antimicrobial sensitivity then can be determined. As currently used, PCR offers no advantage here. However many vector-borne agents, including Ehrlichia canis, can take up to 3 months to isolate in a dedicated research laboratory. "Now with PCR technology," Breitschwerdt says, "in 2 to 4 hours, we can tell with a high degree of sensitivity whether the blood sample contains Ehrlichia organisms."

    To devise such tests, researchers first find a gene that is unique to the agent of interest. Then they create primers — short starter sequences — and add them to the sample to help find these sequences so they can be amplified. In some cases, tests are devised with multiple primers and many genes, making it possible to detect more than one organism. Other PCR assays can be designed to detect organisms in two closely related genera, such as Anaplasma and Ehrlichia. Then if either genus is identified by the screening PCR, species-specific primers are used for more specific identification of the infecting species. This approach has facilitated documentation of infection with more than one Ehrlichia species in the same canine patient.

    Overcoming limitations

    At present, Breitschwerdt indicates that PCR is the most sensitive test available for molecular detection of many infectious organisms, but he cautions that PCR assays are not perfect. For instance, a negative PCR cannot rule out infection. The submitted sample or portion of the sample used for testing may not include the offending species, or the number of organisms may be below the limit of detection.

    Researchers at the NC State laboratory, however, have found a way to help address this for at least one species by developing an enrichment culture medium for growing Bartonella organisms.1 The laboratory takes blood, cerebrospinal fluid or effusion samples and runs PCR testing immediately but also reserves a portion of the sample for growth in medium. After 7 to 14 days in culture, PCR testing is conducted on the enriched sample.

    "Based on our experience to date, we believe that we've gone from detecting less than 10% of Bartonella infections in humans and dogs to detecting 70% to 80% of them," Breitschwerdt says. This type of combination assay is especially useful with Bartonella organisms, he adds, indicating that with Ehrlichia and Babesia infections in sick dogs, there frequently are enough organisms in the blood to be detected by PCR without using an enrichment culture approach, but with Bartonella infections, even sick animals may not harbor enough organisms in the sample to be detected without using that approach.

    Another limitation with PCR testing is that the chosen gene target must be unique. For instance, there are PCR assays for many infectious viruses, including distemper and feline infectious peritonitis (FIP). But in the case of FIP, the historical problem has been identifying a unique gene target for FIP that is not shared by the enteric coronavirus. The extent to which specific laboratories have overcome this problem is not entirely clear.

    In addition, because commercial laboratories recognize the value of using PCR to identify infectious agents, diagnose genetic diseases and target oncogenes, many have now developed or adopted PCR tests but, Breitschwerdt warns, all of them might not be using the controls needed for validating the ability of their assays to identify infectious organisms.

    "For example," Breitschwerdt continues, "companies can go to the GenBank Database [NIH] and look at the complete E. canis genome, then target any of the genes in E. canis and develop a PCR assay for detection of E. canis infection. However, this does not mean that the PCR assay will work to identify only E. canis in patient samples. To increase the sensitivity of the PCR assay, you want to target a multicopy gene for which you have 10 copies [or at least several] per organism. Also, to confirm the specificity of the assay, you have to make sure that the gene you have chosen isn't similar or identical to a gene in some other organism."

    To elaborate on this, Breitschwerdt explains that at the NC State laboratory, researchers found that one of their genetic targets for Bartonella yielded false-positive results because it was identical to a gene found in the plant bacterium, Mesorhizobium, which can be present in sterile water.2 The solution? "We revised the test and chose a different area of the gene to avoid false-positive findings," he says.

    Veterinarians can guard against problems by asking questions. First, ask about the laboratory's analytic sensitivity for PCR testing, which is how many copies of the gene the PCR reaction can detect in a patient sample. Sensitivity can vary with each PCR assay. Breitschwerdt expects at minimum the detection of five genome copies per reaction. Second, ask how many related organisms the laboratory has PCR-tested to prove there is no cross-priming. By the nature of its design, PCR specificity should be 100%. The laboratory is looking for a DNA fingerprint that is specific for the particular bacterium, fungus or virus. If the gene being targeted is selected for uniqueness, it should be 100% specific.

    If the precautions discussed here are taken, PCR testing can be very useful to clinicians.

    For more information:

    For additional information about PCR testing, visit the website http://www.cvm.ncsu.edu/vth/ticklab.html.

    1. Duncan AW, Maggi RG, Breitschwerdt EB. A combined approach for the enhanced detection and isolation of Bartonella species in dog blood samples: pre-enrichment culture followed by PCR and subculture onto agar plates. J Microbiol Meth 2007;69:273-281.

    2. Maggi RG, Breitschwerdt EB. Potential limitations of the 16S-23S rRNA intergenic region for molecular detection of Bartonella species. J Clin Microbiol 2005;43(3):1171-1176.

    References »

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