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 ) were within the normal reference range for this laboratory.
Complete blood count results for blood drawn on 26 September 2005
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.