Introduction
Ehrlichia species are tick-transmitted intracellular bacteria closely related to
Anaplasma,
Neorickettsia and
Rickettsia.
Ehrlichia chaffeensis was first described as a human pathogen in 1986 and
E. ewingii in 1996 [
1], and recent evidence suggests that other species of
Ehrlichia might also cause illness [
2,
3]. A new species of
Ehrlichia, the Panola Mountain
Ehrlichia species, was recently discovered in the USA [
4]. Clinical signs of ehrlichial infection are often non-specific, and the most common signs are fever, headache, myalgia and malaise [
1]. Laboratory diagnosis of ehrlichiosis depends on the detection of ehrlichiae in samples collected during the acute illness or on demonstration of a significant rise (four-fold or greater) in antibody titer between the acute and convalescent phases of the illness [
1,
5]. Serology is limited because acute serology alone is insufficient to diagnose infection, paired serology does not provide a diagnosis until 3 to 4 weeks after empirical treatment has been initiated, and only
E. chaffeensis is available for serologic testing of humans [
1]. Diagnosis of ehrlichiosis during the acute infection increasingly relies on polymerase chain reaction (PCR) [
1,
6]. According to the official case definition used by the Centers for Disease Control (CDC) and the USA National Notifiable Diseases System, a positive PCR reaction, with confirmation of the amplicon identity, is sufficient for laboratory confirmation of a case of human ehrlichiosis [
5].
Case presentation
On 23 September 2005, a 31-year-old Caucasian man from Atlanta, Georgia, USA presented with a complaint of neck soreness for 3 weeks. The patient reported hiking at Panola Mountain State Park in Georgia on 31 August 2005 and later removing a partially engorged nymphal tick from his upper arm on 3 September. The patient stored the tick in an empty vial at room temperature, and the tick was later identified as Amblyomma americanum. On 8 September, the patient began suffering from a persistent sore neck, characterized by musculoskeletal pain upon turning his head and insomnia due to pain. The pain was refractory to anti-inflammatory medications, including acetaminophen, aspirin and ibuprofen. Physical examination on 23 September was unremarkable. Pyrexia was not observed and no erythema or edema was noted at the site of the tick bite; however, the patient had taken 500 mg aspirin prior to examination. The patient was treated for a presumptive tick-borne illness with 100 mg of oral doxycycline twice daily for 10 days. The patient reported that neck soreness was improved by 48 to 60 hours after doxycycline therapy was initiated.
Laboratory testing
Blood was drawn from the patient on 23 September for PCR testing for tick-borne diseases, on 26 September for a complete blood count (CBC) and acute serology, and on 15 October for convalescent serology. Whole blood from 23 September and sera from 26 September and 15 October were submitted to the CDC for tick-borne disease testing. The CBC was performed by Quest Diagnostics (Nichols Institute, Chantilly, VA), and CBC results (Table
1) were within the normal reference range for this laboratory.
Table 1
Complete blood count results for blood drawn on 26 September 2005
Red blood cell count | 5.32 × 107/μl |
Hemoglobin | 15.9 g/dl |
Mean corpuscular volume | 87.8 fl |
Mean corpuscular hemoglobin | 29.9 pg |
Mean corpuscular hemoglobin concentration | 34.1 g/dl |
Red blood cell distribution width | 12.7% |
Platelet count | 272,000/μl |
White blood cell count | 7500/μl |
Absolute neutrophils (%) | 4868 cells/μl (64.9%) |
Absolute lymphocytes (%) | 2243 cells/μl (29.9%) |
Absolute monocytes (%) | 233 cells/μl (3.1%) |
Absolute eosinophils (%) | 143 cells/μl (1.9%) |
Absolute basophils (%) | 15 cells/μl (0.2%) |
For PCR testing, DNA was extracted from 100 μl of clotted blood and from the dead tick, using an IsoQuick Nucleic Acid Extraction Kit (ORCA Research Inc., Bothell, WA). We detected DNA from
Ehrlichia using a genus-specific, hemi-nested PCR with the outer primers EC12A and HE3 [
4], followed by a hemi-nested reaction using the 'Forward' primer [
7] and HE3. DNA from
Rickettsia species was detected using primer-1 and primer-2 [
8]. We assessed the quality of the tick DNA using primers T1B and T2A [
9]. Positive and negative controls were used for all assays and consisted of genomic DNA from
Rickettsia rickettsii, Ehrlichia ewingii or distilled water. All PCR products were purified with a QIAquick PCR Purification Kit (Qiagen, Valencia, CA) and sequenced in duplicate using PCR primers and a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA). Sequences were determined using an ABI 3100 (Applied Biosystems). Primer sequences were removed and sequences assembled with Seqmerge (Accelrys, San Diego, CA).
Using the hemi-nested PCR, an amplicon from the 16S rRNA gene of an Ehrlichia species was obtained from the acute blood sample. The amplicon was sequenced, and the 361-bp sequence (GenBank accession number DQ217573) was 100% identical to the sequence reported from the Panola Mountain Ehrlichia species (Ehrlichia species P-Mtn, GenBank accession number DQ324367). The amplicon was not identical to sequences from any other species represented in GenBank. No DNA from Rickettsia was detected in the patient's blood. The tick was poorly preserved by the patient, and DNA could not be amplified from it.
For acute and convalescent serology, sera from 26 September and 15 October were tested using indirect immunofluorescence assays (IFA), performed as previously described [
10], to detect antibodies against
Anaplasma phagocytophilum,
Borrelia burgdorferi,
Coxiella burnetii,
Francisella tularensis,
Rickettsia africae,
R. akari, '
R. amblyommii',
R. conorii,
R. parkeri,
R. prowazekii,
R. rickettsii and
R. typhi, and antibodies cross-reactive with
E. chaffeensis. We could not test the patient's serum against the Panola Mountain
Ehrlichia species because this emerging agent has not yet been cultured. Antibody was detected using isotype-specific goat antihuman immunoglobulin G (IgG) and human immunoglobulin M (IgM), labeled with fluorescein isothiocyanate (FITC) (KPL, Gaithersburg, Maryland). Prior to testing for IgM, sera were depleted of IgG by use of a recombinant Protein G kit (Rapi-Sep-M, Pan-Bio, Columbia, MD). Acute and convalescent samples were tested side-by-side, and positive, negative and diluent controls were assayed with the test samples and gave expected results. Serology did not support recent infection with any of the agents tested. With
E. chaffeensis antigen, the patient's serum reacted with a small proportion of the organisms on the slide, as compared with positive control sera, and was considered cross-reactive in both acute and convalescent samples. Titers are expressed as the reciprocal of the last dilution exhibiting specific fluorescence and were as follows: IgM 32 (26 September), and IgG 16 (26 September) and IgG 32 (15 October). Convalescent IgM data were not available.
Conclusion
We report that an emerging pathogen, the Panola Mountain
Ehrlichia species, was detected in blood from a human patient following the bite of a nymphal
Amblyomma that was probably acquired at Panola Mountain State Park in Georgia in the United States of America. The Panola Mountain
Ehrlichia species was originally described from a goat fed upon by
A. americanum collected at this park [
4], but this is the first report associating the agent with human illness.
The Panola Mountain
Ehrlichia species is genetically closely related to
E. ruminantium and more distantly related to
E. chaffeensis [
4]. The patient exhibited myalgia for 3 weeks prior to presentation, had ehrlichemia, which was confirmed by DNA sequencing at presentation, and rapidly recovered after treatment with doxycycline. Although PCR and serological testing for other tick-borne agents was negative, suggesting that ehrlichiosis was the cause of illness, we cannot conclusively rule out the possibility that the patient's symptoms were caused by an unknown factor. Serological confirmation of infection with the Panola Mountain
Ehrlichia species could not be obtained, with only a two-fold rise in IgG titer between the two serum samples. This might be due to the initiation of antibiotic therapy prior to optimal immune response or due to the lack of an appropriate antigen; antibodies against ehrlichial agents are often, but not always, cross-reactive with other species of
Ehrlichia [
3]. In this case, PCR testing of whole blood was of significantly greater diagnostic value than serological testing.
Acknowledgements
Diagnostic laboratory work was supported by the CDC and Department of Health and Human Services. The conclusions in this report do not necessarily represent the views of the funding agencies. We thank Gregory Dasch, Herbert Thompson, Robert Massung, Tonya Mixson and Martin Schreifer for their assistance. The use of trade names in this document does not constitute an official endorsement or approval of the use of such commercial hardware or software.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
WKR identified the tick, isolated DNA from the blood sample, performed sequencing reactions, analyzed data, and was a major contributor in writing the manuscript. ADL tested the DNA from the blood sample, recorded patient information, and was a major contributor in writing the manuscript. WLN performed serological tests and reviewed drafts of this manuscript. AGC was the attending physician for the patient and contributed to drafts, collecting clinical specimens, and patient treatment and observations. All authors read and approved the final manuscript.