Page 1461 - Small Animal Internal Medicine, 6th Edition

CHAPTER 91 Laboratory Diagnosis of Infectious Diseases 1433

can also be used to amplify RNA (RT-PCR). In general, therapeutic responses for some agents (Low et al., 2007; Veir
molecular diagnostic assays are usually more sensitive than and Lappin, 2010). However, minimal data exist for use of
VetBooks.ir other organism demonstration techniques. These assays can quantitative PCR for these purposes and the reader is
directed to specific agent chapters for further information.
be of great benefit for documentation of infectious agents
that are difficult to culture (e.g., Ehrlichia spp.) or cannot be
carefully assess the predictive values of currently available
cultured (e.g., hemoplasmas). Specificity can be quite high Because of these findings, small animal practitioners must
depending on the primers used in the reaction. For example, PCR assays and the expertise and reliability of the laboratory
primers can be designed to detect one genus but not others. that will be performing the assays.
Primers can also be designed to identify only one species.
For example, a PCR assay can be developed to detect all ANIMAL INOCULATION
Ehrlichia spp. and Anaplasma spp. or just one species such as Animal inoculation can be used to identify some infectious
Ehrlichia canis. Assays that contain multiple sets of primers diseases. For example, oocysts of T. gondii cannot be distin-
to detect nucleic acids of many different infectious agents guished morphologically from those of Hammondia ham-
can also be used. mondi or Besnoitia darlingi; only T. gondii is infectious for
Another use of molecular diagnostics is FISH. In this human beings. T. gondii can be differentiated from the other
molecular technique, nucleic acids of infectious agents can coccidians by inoculation of sporulated oocysts into mice
be identified within tissues. One recent infectious disease and monitoring for T. gondii–specific antibody production.
example using FISH showed that Borrelia burgdorferi was However, because live animals are required, animal inocula-
not in the renal tissues of dogs with presumed Lyme tion is rarely used in small animal practice.
nephropathy, supporting the hypothesis that this clinical
syndrome is likely to have an immune-mediated component ELECTRON MICROSCOPY
(Hutton et al., 2008). In addition, FISH was used to help Electron microscopy is a highly sensitive procedure for
prove that Boxer colitis was a syndrome associated with organism identification in body fluids and tissues.
Escherichia coli (Simpson et al., 2006). Glutaraldehyde-containing fixatives are used most com-
Because of the inherent sensitivity of the reaction, molec- monly. One of the most clinically relevant uses of electron
ular diagnostic assays can give false-positive results if sample microscopy is for the detection of viral particles in feces of
contamination occurs during collection or at the laboratory animals with gastrointestinal signs of diseases.
performing the procedure. False-negative results can occur
if the sample is handled inappropriately or if the patient is
receiving antibiotics that are effective against that specific ANTIBODY DETECTION
organism; this is of particular importance for detection of
RNA viruses by reverse transcriptase polymerase chain reac- SERUM
tion (RT-PCR). Results may also be affected by treatment. A variety of different methods exists for detecting serum
Another potential problem is that minimal standardization antibodies against infectious agents; complement fixation,
exists among commercial laboratories offering molecular hemagglutination inhibition, serum neutralization, aggluti-
diagnostic assays. nation assays, agar gel immunodiffusion, indirect fluorescent
Although molecular diagnostic assays can be one of the antibody assay (IFA), ELISA, and Western blot immunoas-
most sensitive for documentation of infections, positive test say are commonly used methods. Complement fixation,
results do not always prove that the infection is causing clini- hemagglutination inhibition, serum neutralization, and
cal illness. For example, because the technique detects DNA agglutination assays generally detect all antibody classes in a
of both live and dead organisms, positive test results may be serum sample. Western blot immunoassay, IFA, and ELISA
achieved even if the infection has been controlled. When the can be adapted to detect specific immunoglobulin (Ig) M,
organism being tested for commonly infects the background IgG, or IgA responses. Western blot immunoassay can be
population of healthy pets, interpretation of results for a used to identify the immunodominant antigens recognized
single animal can be difficult. For example, FHV-1 com- by the humoral immune responses (Fig. 91.8).
monly infects cats and is commonly carried by healthy cats. Comparison of IgM, IgA, and IgG antibody responses
Thus although PCR is the most sensitive way to document against an infectious agent can be used to attempt to prove
infection by FHV-1, the positive predictive value for disease recent or active infection. In general, IgM is the first antibody
of a FHV-1 PCR result is actually quite low. In one study, produced after antigenic exposure. Antibody class shift to
more positive FHV-1 PCR results were detected in the IgG occurs in days to weeks. Serum and mucosal IgA
healthy control group than in the group with conjunctivitis immune responses have also been studied for some infec-
(Burgesser et al., 1999). In addition, the currently available tious agents, including T. gondii, feline coronaviruses, and
PCR assays for FHV-1 also amplify modified-live vaccine Helicobacter felis.
strains, so a positive result does not even indicate presence Timing of antibody testing is important. In general,
of a pathogenic strain. Real-time PCR can be used to deter- serum antibody tests in puppies and kittens cannot be inter-
mine the amount of microbial DNA or RNA in a sample. The preted as specific responses until at least 8 to 12 weeks of age
nucleic acid load may correlate to the presence of disease or because of the presence of antibodies from the dam passed