Ehrlichiae infections pose difficult diagnostic challenges to both clinicians and laboratorians, and the availability of confirmatory assays is limited. Therefore, treatment decisions should be based on epidemiologic and clinical clues, and should never be delayed while waiting for confirmation. Fundamental understanding of the signs, symptoms, and epidemiology of the disease is crucial in guiding requests for tests for ehrlichiosis and interpretation of testing results.
Routine clinical laboratory tests indicative of ehrlichiosis include low white blood cell count, low platelet count, and elevated liver enzymes. Therefore, pancytopenia, aplastic anemia or thrombocytopenia would be consistent with E. canis infection. The organisms can be demonstrated in smears of blood, buffy coat, bone marrow aspirates and spleen aspirates by staining with Diff-Quik or Giemsa. But lightmicroscopic evidence of the pathogens is only successful during the acute phase of the disease and is strongly influenced by the type of material used. Laboratory confirmation of ehrlichiosis requires serologic, molecular, or culture-based methods.
Indirect immunofluorescence assay test (IFAT)
Serologic diagnosis utilizing the indirect immunofluorescent antibody test (IFAT) is currently recommended as the ‘serological gold standard’ for confirming an exposition to E. canis. Antibodies in the serum bind to the organisms on a slide and are detected by a fluorescein-labeled conjugate. Although IFAT remains the principal diagnostic tool for the detection of ehrlichial infection, there is no standardized antigen, conjugate, or agreement on what constitutes a positive result among the various laboratories providing these tests. Individual laboratories should be consulted as to their test threshold levels. The test provides a high sensitivity, but no opportunity to differentiate between current acute or chronic infection or previousi nfection in wich the organisms were immunologically or therapeutically eliminated. Another disadvantage is the cross reactivity with different Ehrlichia species and thus a low specificity.
Blood specimens taken late (convalescent) in the disease course represent the preferred specimens for evaluation. Relatively few data describe the kinetics of IFAT-detectable antibodies for the Ehrlichia species. As E. ewingii has not been successfully cultured to date, an IFA test is not available.
Dogs generally become seronegative within 3 to 9 months after effective treatment, although some dogs maintain persistent and stable titers for years.
Antibodies reactive with one Ehrlichia species can be cross-reactive with other species of Ehrlichia.
Polymerase chain reaction (PCR)
Second to serologic methods, amplification of the ehrlichial DNA by polymerase chain reaction (PCR) is the most frequently used method for detecting infection or to confirm the therapeutic elimination. PCR tests remain unstandardized, and analytical and diagnostic sensitivity and specificity may vary among individual assays. EDTA blood is required for PCR and should optimally be collected prior to or after cessation of antibiotics.
Isolation and cultivation
Direct isolation of the organism remains the gold standard for confirmatory diagnosis, but is the most difficult and time-consuming approach. For example, E. chaffeensis has been recovered from the blood of acutely ill patients by using a variety of cell lines, most frequently canine DH82. Isolation of E. canis out of monocytes has been successful already two days after infection. The disadvantages of the technique are the high costs, the high laboratory standard needed and especially the time until the result is available (14-34 days). Thus the method is not useful as routine test. Nevertheless for the confirmation of therapeutic success after antibiosis the method is optimal due to its high sensitivity.
EDTA-treated blood is the most common specimen for isolation attempts, as well as the most frequent sample used for PCR analysis. Samples should be processed as quickly as possible, although successful recovery of viable ehrlichiae may result from samples refrigerated at 4°C for as long as 1 week.
Dot-enzyme linked immunosorbent assay (Dot-ELISA)
A dot-ELISA detecting circulating antigen showed reduced sensitivity and also cross reactivity, similar to the IFAT, so that the test is hardly used in diagnostics.
New techniques, including enzyme immunoassays using recombinant ehrlichial antigens and fluorometric PCR, are currently under investigation, and these tests may eventually have broader application in public health laboratories.
- Breitschwerdt EB, Hegarty BC, Hancock SI: Sequential evaluation of dogs naturally infected with Ehrlichia canis, Ehrlichia chaffeensis, Ehrlichia equi, Ehrlichia ewingii, or Bartonella vinsonii. J Clin Microbiol. 1998, 36, 2645-51
- Neer TM, Breitschwerdt EB, Greene RT, et al.: Consensus statement on ehrlichial disease of small animals. . J Vet Intern Med. 2002, 16, 309-15