Test ID: EHRL
Ehrlichia/Anaplasma, Molecular Detection, PCR, Blood

Evaluating patients suspected of acute anaplasmosis or ehrlichiosis

Real-Time Polymerase Chain Reaction (PCR)/DNA Probe Hybridization

(PCR is utilized pursuant to a license agreement with Roche Molecular Systems, Inc.)

Container/Tube: Lavender top (EDTA)

Specimen Volume: 1 mL

Forms: If not ordering electronically, submit a Microbiology Request Form (Supply T244) with the specimen.

Ehrlichiosis and anaplasmosis are a group of emerging zoonotic infections caused by Ehrlichia and Anaplasma species, respectively, which are obligate intracellular, gram-negative rickettsial organisms that infect human leukocytes.

Human granulocytic anaplasmosis (HA) is caused by the tick-borne rickettsia, Anaplasma phagocytophilum, which is transmitted by contact with Ixodes ticks. The white-footed mouse is the animal reservoir, and the epidemiology of this infection 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. The deer is believed to be the animal reservoir, and 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.

In 2009, Mayo Medical Laboratories detected a new species of Ehrlichia DNA in 4 patients (3 from Wisconsin, 1 from Minnesota) using PCR.(5) Sequenced portions of the groEL and rss (18S rRNA) genes show 98% homology to analogous regions of Ehrlichia muris, a species not previously identified in North America. The Ehrlichia muris-like (EML) organism has since been found in Ixodes scapularis ticks and white footed mice in Wisconsin and Minnesota, although it is not known whether these play a role in the life cycle of this organism.

Infective forms of Ehrlichia and Anaplasma are injected during tick bites and the organisms enter the vascular system where they infect leukocytes. They are sequestered in host-cell membrane-limited parasitophorous vacuoles known as morulae. Asexual reproduction occurs in leukocytes where daughter cells are formed and liberated upon rupture of the leukocytes.

Most cases of ehrlichiosis are probably subclinical or mild, but the 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. The 4 patients infected with the EML organism presented with fever, headache, myalgia, and leukopenia.

Diagnosis of ehrlichiosis 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, intragranulocytic 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. PCR techniques allow direct detection of pathogen-specific DNA from patients' whole blood during the acute phase of disease. This is currently the test of choice for the newly described EML organism.

Serologic testing is usually done only 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 the EML organism, and cross-reactivity with the other Ehrlichia species by serology may be unreliable.

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.

Negative

Positive results indicate presence of specific DNA from Ehrlichia chaffeensis, Ehrlichia ewingii, Ehrlichia muris-like organism, or Anaplasma 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 it does not exclude the presence of the organism 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.

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

Analytical Specificity:

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.

Precision:  

Interassay precision was 97% and intra-assay precision was 96%.

Reference Range:  

Fifty whole blood specimens from normal donors were tested and found to be negative for targeted or detectable Ehrlichia and Anaplasma species. 

Reportable Range:  

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).

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

Nucleic acid is extracted from the pathogens in blood using the automated MagNA Pure LC system. The extract is then transferred to individual self-contained capillary cuvettes for amplification. The LightCycler is an automated instrument that amplifies and monitors the development of target nucleic acid (amplicon) after each cycle of PCR. The DNA target for PCR assay is groEL, the open reading frame gene segment of the heat-shock protein operon (groEL), which is present at a frequency of 1 copy per organism in pathogenic species of Anaplasma and Ehrlichia. A specific base pair DNA target sequence is amplified by PCR. The detection of amplicon is based on fluorescence resonance energy transfer (FRET), which utilizes a hybridization probe with a donor fluorophore, fluorescein, at the 3' end and a second hybridization probe with an acceptor fluorophore, LC-Red 640, at the 5' end. When the target amplicon is present, the LC-Red 640 emits a measurable and quantifiable light signal at a specific wavelength. Presence of the specific organism nucleic acid may be confirmed by performing a melting curve analysis of the amplicon. Using features of the melting curve analysis, the assay primers and specific hybridization probes are able to detect and differentiate among Anaplasma phagocytophilum, Ehrlichiosis chaffeensis, Ehrlichia muris-like organism, and Ehrlichia ewingii/canis. Due to close proximity of the melting curves of Ehrlichia ewingii and Ehrlichia canis, this assay cannot distinguish between these 2 organisms.(Cockerill FR, Uhl FR: Applications and challenges of real-time PCR for the clinical microbiology laboratory. In Rapid Cycle Real-Time PCR. Edited by U Reischl, C Wittwer, F Cockerill. Springer, NY, 2002)

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