Ehrlichia/Anaplasma, Molecular Detection, PCR, Blood
Evaluating patients suspected of acute anaplasmosis or ehrlichiosis
Clinical Information Discusses physiology, pathophysiology, and general clinical aspects, as they relate to a laboratory test
Ehrlichiosis and anaplasmosis are a group of emerging zoonotic tick-borne infections caused by Ehrlichia and Anaplasma species, respectively. These obligate intracellular, gram-negative rickettsial organisms infect leukocytes and cause a potentially serious febrile illness in humans.
Human granulocytic anaplasmosis (HA) is caused by Anaplasma phagocytophilum, which is transmitted through the bite of an infected Ixodes sp. tick. The epidemiology of this infection in the United States is very much like that of Lyme disease (caused by Borrelia burgdorferi) and babesiosis (caused primarily by Babesia microti), which all have the same tick vector. HA is most prevalent in the upper Midwest and in other areas of the United States that are endemic for Lyme disease.
Human monocytic ehrlichiosis (HE) is caused by Ehrlichia chaffeensis, which is transmitted by the Lone Star tick, Amblyomma americanum. Most cases of HE have been reported from the southeastern and south-central regions of the United States. Ehrlichia ewingii, the known cause of canine granulocytic ehrlichiosis, can occasionally cause an HE-like illness in humans. Clinical features and laboratory abnormalities are similar to those of Ehrlichia chaffeensis infection, and antibodies to Ehrlichia ewingii cross-react with current serologic assays for detection of antibodies to Ehrlichia chaffeensis.
Most recently, Mayo Medical Laboratories detected a new species of Ehrlichia in patients with exposure to ticks in Wisconsin and Minnesota. This organism is most closely related to Ehrlichia muris and has therefore been referred to as the Ehrlichia muris-like agent or EMLA. The name E. muris eauclairensis has recently been proposed after the city in which the first case was described. Ehrlichia muris eauclairensis causes a similar disease to ehrlichiosis due to E. chaffeensis and E. ewingii, and may cause more severe disease in immunocompromised hosts.
Most cases of anaplasmosis and ehrlichiosis are subclinical or mild, but infection can be severe and life-threatening in some individuals. Fever, fatigue, malaise, headache, and other "flu-like" symptoms, including myalgias, arthralgias, and nausea, occur most commonly. Central nervous system involvement can result in seizures and coma.
Diagnosis may be difficult since the patient's clinical course is often mild and nonspecific. This symptom complex is easily confused with other illnesses such as influenza, or other tick-borne zoonoses such as Lyme disease, babesiosis, and Rocky Mountain spotted fever. Clues to the diagnosis of ehrlichiosis in an acutely febrile patient after tick exposure include laboratory findings of leukopenia or thrombocytopenia and elevated serum aminotransferase levels. However, while these abnormal laboratory findings are frequently seen, they are not specific. Rarely, intra-granulocytic or monocytic morulae may be observed on peripheral blood smear, but this is not a reliable means of diagnosing cases of human ehrlichiosis or anaplasmosis.
Definitive diagnosis is usually accomplished through PCR and serologic methods. Serologic testing is done primarily for confirmatory purposes, by demonstrating a 4-fold rise or fall in specific antibody titers to Ehrlichia species or Anaplasma antigens. There is not currently a commercially available specific serologic test for E. m. eauclairensis, but cross-reactivity with the other Ehrlichia species by serology may be detected.
PCR techniques allow direct detection of pathogen-specific DNA from patients' whole blood and is the preferred method for detection during the acute phase of illness. The Mayo PCR assay is capable of detecting and differentiating A. phagocytophilum, E. chaffeensis, E. ewingii, and E. muris eauclairensis.
It is important to note that concurrent infection with Anaplasma phagocytophilum, Borrelia burgdorferi, and Babesia microti is not uncommon as these organisms share the same Ixodes tick vector, and additional testing for these pathogens may be indicated
Reference Values Describes reference intervals and additional information for interpretation of test results. May include intervals based on age and sex when appropriate. Intervals are Mayo-derived, unless otherwise designated. If an interpretive report is provided, the reference value field will state this.
Positive results indicate presence of specific DNA from E. chaffeensis, E. ewingii, E. m. eauclairensis organism, or A. phagocytophilum and support the diagnosis of ehrlichiosis or anaplasmosis.
Negative results indicate absence of detectable DNA from any of these 4 pathogens in specimens, but do not exclude the presence of these organisms or active or recent disease.
Since DNA of Ehrlichia ewingii is indistinguishable from that of Ehrlichia canis by this rapid PCR assay, a positive result for Ehrlichia ewingii/canis indicates the presence of DNA from either of these 2 organisms.
Cautions Discusses conditions that may cause diagnostic confusion, including improper specimen collection and handling, inappropriate test selection, and interfering substances
This assay should not be used for screening asymptomatic individuals, and should only be used to test patients with signs and symptoms of ehrlichiosis or anaplasmosis.
A negative result does not indicate absence of disease.
Inadequate specimen draw or improper conditions for storage or transport may invalidate test results.
This test may detect DNA of Ehrlichia canis (reported to cause asymptomatic infection in Venezuela only).
This PCR test does not detect DNA of Rickettsia (formerly Ehrlichia) sennetsu, which has been reported to cause a rare mononucleosis-like illness in humans (in Japan and Malaysia).
The following validation data supports the use of this assay for clinical testing.
Accuracy/Diagnostic Sensitivity and Specificity:
Results from this real-time PCR assay on the LightCycler (LC PCR) were compared to those generated using conventional PCR assay for Anaplasma phagocytophilum on 127 unique, archived whole blood specimens (26 positive and 99 negative specimens by conventional PCR). Using the conventional PCR as the gold standard, the diagnostic sensitivity and specificity for detection of Anaplasma phagocytophilum were 100%. In addition, 12 known Ehrlichia chaffeensis isolates and 2 Ehrlichia ewingii isolates (reference strains) were tested by the LC PCR and were positive.
Supplemental Data (Spiking Studies):
To supplement the above data, 30 negative whole blood samples were spiked with Anaplasma phagocytophilum positive control plasmid at the limit of detection (LoD) (10 copies/microL). The 30 spiked specimens were run in a blinded manner along with 30 negative (nonspiked) specimens. 100% of the spiked specimens were positive, and 100% of the nonspiked specimens were negative.
Analytical Sensitivity/Limit of Detection (LoD):
The lower LoD of this assay for each of the species in EDTA blood is as follows:
-Anaplasma phagocytophilum=approximately 10 targets per microliter
-Ehrlichia chaffeensis=approximately 5 targets per microliter
-Ehrlichia muris-like=approximately 100 targets per microliter
-Ehrlichia ewingii/canis=approximately 10 targets per microliter
No PCR signal was obtained from extracts of the following organisms: herpes simplex virus, Epstein-Barr virus, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pyogenes, Escherichia coli, Pseudomonas aeruginosa, Bartonella henselas, Bartonella quintana, Ricketssia typhi, Ricketssia rickettsii, Toxoplasma gondii, Babesia microti MN, Babesia microti ATCC 53899, Borellia burgdorferi ATCC 51990, Ehrlichia risticii ATCC VR-986, and Anaplasma marginale. Positive results were obtained from nucleic extracts of 2 Ehrlichia canis strains (patient strain and ATCC CRL-10390 strain), with a melting temperature (Tm) of 49.5 degrees C (indistinguishable from Ehrlichia ewingii). A positive melting peak was also noted with Ehrlichia muris (ATCC VR-1411), but the Tm (55.24 degrees C) was easily distinguished from the Tm of the target organisms.
Interassay precision was 97% and intra-assay precision was 96%.
Fifty whole blood specimens from normal donors were tested and found to be negative for targeted or detectable Ehrlichia and Anaplasma species.
This is a qualitative assay, and results are reported as either negative or positive for targeted Ehrlichia/Anaplasma species (positive for Anaplasma phagocytophilum, Ehrlichia chaffeensis, Ehrlichia muris-like or Ehrlichia ewingii).
Clinical Reference Provides recommendations for further in-depth reading of a clinical nature
1. Bakken JS, Dunler JS: Human granulocytic ehrlichiosis. Clin Infect Dis 2000 Aug;31(2):554-560
2. Dunler JS, Bakken JS: Human ehrlichioses: newly recognized infections transmitted by ticks. Ann Rev Med 1998;49:201-213
3. Krause PJ, McKay K, Thompson CA, et al: Disease-specific diagnosis of coinfecting tickborne zoonoses: babesiosis, human granulocytic ehrlichiosis, and Lyme disease. Clin Infect Dis 1999 May 1;34(9):1184-1191
4. McQuiston JH, Paddock CD, Holman RC, Childs JE: The human ehrlichioses in the United States. Emerging Infect Dis 1999 Sept-Oct;5(5):635-642
5. Pritt BS, Sloan LM, Johnson DK, et al: Emergence of a new pathogenic Ehrlichia species, Wisconsin and Minnesota, 2009. N Engl J Med 2011 Aug 4;365(5):422-429
6. Johnson DK, Schiffman E, Davis JP, et al. Human infection with Ehrlichia muris-like Pathogen, United States, 2007-2013. Emerging Infect Dis 2015; 21(10):1794-99