INTRODUCTION

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Ehrlichia species are obligate, intracellular pathogens belonging to the Rickettsiaceae family that are agents of human and veterinary disease in regions throughout the United States and Europe (5, 12). A newly emerging Ehrlichia species, the agent of human granulocytic ehrlichiosis (HGE) has become more frequently diagnosed in regions where Ixodes species tick vectors are abundant (5, 12). Human infection with the HGE agent causes a nonspecific, influenza-like illness that can be difficult to diagnose. Specific etiologic diagnostic tests for HGE include blood smear examination for ehrlichial morulae within neutrophils, PCR with primers based upon the HGE agent 16S rDNA sequence (4, 7), indirect immunofluorescent antibody test (IFA), which detects HGE agent antibodies in patient sera (6), and direct cultivation of the HGE agent from patient blood (8). PCR is a highly useful diagnostic test for HGE since it provides early detection of infection at a time when therapeutic decisions are being made and since there are limitations associated with the other diagnostic tests. Although PCR diagnosis of HGE with the 16S rDNA primers GE9f and GE10r was shown to be highly sensitive (86%) and specific (100%) in a prospective evaluation (7), recent studies have shown a disappointing lack of sensitivity in actual clinical practice (9). Thus, an enhanced PCR method with increased sensitivity for the diagnosis of HGE would be advantageous.

Phylogenetic studies indicate that the HGE agent belongs within a narrow clade including Ehrlichia phagocytophila and Ehrlichia equi (12). The close genetic relationship between these species along with other molecular and biological evidence suggests that they may actually be a single Ehrlichia species that causes granulocytic ehrlichiosis in both humans and animals. Recently, it has been discovered that these Ehrlichia species share a unique gene, epank1, coding for a 153-kDa protein antigen, Epank1, which contains 11 ankyrin repeats of 33 amino acids (11; P. Caturegli, K. M. Asanovich, J. J. Walls, J. S. Bakken, J. E. Madigan, V. L. Popov, and J. S. Dumler, submitted for publication). Recent evidence suggests that the HGE agent contains at least two copies of epank1 in its genome (Caturegli et al., submitted). Considering this evidence, along with the uniqueness of epank1 to the E. phagocytophila genogroup and the fact that the 16S rRNA and groESL genes are highly conserved among prokaryotes, we investigated whether the PCR diagnosis of HGE could be improved by the use of epank1 primers.

(This work was presented in part at the European Working Group-American Society for Rickettsiology Joint Meeting, Marseilles, France, June 1999, abstr. 174.)

	MATERIALS AND METHODS

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Patient specimens. A total of 31 blood samples from patients with suspected HGE agent infection were tested with the epank1 primers. All patient whole-blood samples were previously tested for HGE agent DNA in a PCR assay with the GE9f and GE10r primers (4), and sera were tested for HGE agent antibodies by IFA (2, 6, 10, 13). To exclude false-positive IFA results due to cross-reactivity, patient sera were also tested for Ehrlichia chaffeensis antibodies. Patients were initially evaluated for HGE based upon typical history, clinical, and laboratory findings that included at least some of the following: fever, headache, malaise, myalgia, leukopenia, thrombocytopenia, anemia, elevated serum hepatic transaminases, a history of tick bites or exposure to tick-infected areas, and an illness occurring during a period of active tick feeding. Twenty-four of the patients were from southeastern New York, 7 were from either Minnesota or Wisconsin, and 1 was from the Eastern Shore of Maryland. Twelve patients were PCR positive with the HGE agent 16S rRNA gene primers and had a seroconversion detected by IFA (group A). Ten of the patients were previously PCR negative but had a seroconversion detected by IFA (group B). The remaining 10 patients were previously PCR negative and seronegative (group C); 5 of these patients were diagnosed with Lyme disease, 1 had an influenza-like illness after a tick bite and persistent (>3 months) thrombocytopenia suggestive of idiopathic thrombocytopenic purpura, and another had a self-limited influenza-like illness with no hematologic abnormalities; final etiologic diagnoses were never established for the remaining 3 subjects.

epank1 PCR. DNA was extracted from 300 ml of acute-phase, EDTA-anticoagulated whole blood with the Puregene kit (Gentra Systems, Research Triangle Park, N.C.) and quantitated with a GeneQuant UV spectrophotometer (Pharmacia Biotech, Piscataway, N.J.). Two milligrams of DNA was added to a 100-µl PCR mixture containing (final concentrations) 0.3 mM concentrations of the epank1 primers, 1.5 mM MgCl₂, 0.6 mM deoxynucleoside triphosphates, and 2 µl of eLONGase enzyme mixture (GIBCO/BRL, Gaithersburg, Md.). The forward primer (LA6, 5'-GAGAGATGCTTATGGTAAGAC-3') and the reverse primer (LA1, 5'-CGTTCAGCCATCATTGTGAC-3') were designed to flank a region of epank1 encoding 4 of the 11 ankyrin repeats, a region comprising 444 nucleotides. Each PCR mixture included a known HGE agent positive control (E. equi-infected horse neutrophil DNA) and two negative controls (water blank and known negative human DNA). Thermocycling was performed with preamplification denaturation for 2 min at 94°C, followed by 8 cycles of 30 s at 94°C and 30 s at 72°C; the annealing temperature during these 8 cycles was lowered 2°C every 2 cycles from 62 to 56°C. Thereafter, amplification was performed for an additional 28 cycles at a constant denaturation temperature of 94°C for 30 s, annealing temperature of 54°C for 30 s, and extension temperature of 72°C for 30 s, followed by a final extension at 72°C for 5 min. The resulting amplification yielded an approximately 444-bp product that was visualized by ethidium bromide staining and 0.9% agarose gel electrophoresis on an Eagle Eye UV gel imaging system (Stratagene, La Jolla, Calif.).

Determination of analytical sensitivity. A phagemid that contains the epank1 gene truncated by 90 nucleotides on the 5' end was obtained by screening an E. equi lambda phage Express (Stratagene) genomic library with polyclonal antiserum to E. equi, followed by in vivo excision of the phagemid containing the recombinant insert (Caturegli et al., submitted). The identity of the ehrlichial gene was established by sequence analysis (Caturegli et al., submitted). epank1-containing phagemids were serially diluted from 10⁷ to 10⁰ copies/50 ml of PCR mixture to assess the analytical sensitivity of the epank1 primers in a PCR assay. Additionally, known quantities of HGE agent-infected tissue culture cells were diluted in whole EDTA-anticoagulated blood from a healthy donor, and the extracted DNA was used in a PCR assay.

	RESULTS

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Analytical sensitivity. As shown in Fig. 1, epank1 primers detected as few as 10 plasmid copies and also detected ehrlichial DNA in samples containing less than one infected cell per milliliter.

View larger version (86K): [in this window] [in a new window]   FIG. 1.   Analytical sensitivity of epank1 PCR determined with purified epank1 genes (A) and human blood supplemented with various numbers of HGE agent-infected HL60 cells (B). (A) The PCR master mixture was directly supplemented with between 100 and 107 plasmids containing the epank1 gene or no plasmid (0). (B) Comparison of epank1 PCR (top) and 16S rRNA gene PCR (bottom) performed with nucleic acid templates prepared from human blood supplemented with between 102 and 104 HGE agent-infected HL60 cells. The size of the amplified nucleotide fragment is designated on the right of each panel; +C, genomic HGE agent control DNA prepared from heavily infected HL60 cells; H2O, water only control.

PCR. As shown in Table 1, 10 of the 11 (91%) group A HGE patients who were previously PCR positive with the HGE agent 16S rRNA gene primers were also PCR positive with the epank1 primers. From group B, all 10 (100%) seropositive patients tested were epank1 PCR positive, and from group C, none of the 10 former PCR-negative and seronegative patients were PCR positive when tested with the epank1 primers. The intervals of illness and the mean geometric antibody titers in convalescence were similar (P = 0.34 and P = 0.80, respectively [Student's t test]) for both groups of HGE patients regardless of 16S rRNA or epank1 PCR results.

	DISCUSSION

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All diagnostic tests for HGE have limitations; cultivation of the organism may be difficult and is not timely (9), detection of morulae within a blood smear is not very sensitive (1, 2, 9), and the different antigens used for IFA may give discrepant or ambiguous results (9, 10, 13). PCR is the most useful diagnostic test for active disease, but the current methods need improvement owing to disappointing clinical sensitivity (9).

The sensitivity of detecting PCR-amplified HGE agent DNA in patient blood increased significantly when the epank1 primers were used. The epank1 primers amplified as few as 10 HGE agent epank1 genes. Additionally, epank1 primers amplified HGE agent DNA in an additional 10 patients who had been diagnosed with HGE by an IFA seroconversion but who were previously PCR negative with the HGE agent 16S rRNA gene primers. The amplification of HGE agent DNA with the epank1 primers for 91% of patients who were previously PCR positive with the HGE agent 16S rRNA gene primers also affirms a high PCR sensitivity. The exact explanation for increased sensitivity is not known but may be the result of targeting a multicopy gene.

The specificity of PCR with the epank1 primers is additionally high. In a previous study, only antibodies reactive with the E. phagocytophila genogroup reacted with the recombinant Epank1 protein, indicating that this protein antigen is unique to this group (Caturegli et al., submitted); correspondingly, only DNA from isolates of the E. phagocytophila genogroup could be amplified by PCR with the epank1 primers. Sequence analysis of the amplified region of epank1 revealed nucleic acid sequences of E. equi and HGE agent isolates that were nearly identical. Additionally, epank1 primers did not amplify HGE agent DNA from any of the patients who were previously HGE agent 16S PCR negative and seronegative. Therefore, specificity remained 100% when the epank1 primers were used for the PCR diagnosis of HGE, and the sensitivity increased to 95% compared with 48% with the 16S rRNA gene primers in the combined patient cohort.

Although the nucleic acid sequences in the PCR-amplified region of epank1 are nearly identical among the different E. equi and HGE agent isolates, some sequence variation is present among E. phagocytophila isolates (Caturegli et al., submitted). Sequence variation for epank1 between Swedish and Spanish E. phagocytophila strains was noted, and variation between the European E. phagocytophila strains and the North American strains is even greater. Amino acid sequence analysis of the Epank1 protein revealed similar variations among isolates. These variations among isolates and strains may explain why HGE agent DNA was amplified from the blood of one HGE patient from group A when alternate epank1 primers were used but not when the LA1 and LA6 epank1 primers were used (data not shown).

We have shown that PCR amplification of HGE agent DNA in infected patient blood with primers based upon epank1, which is unique for the E. phagocytophila group ehrlichiae, is both sensitive and specific. However, variation in epank1 sequences among strains may cause occasional false-negative results among North American patients. Regardless, the use of a genogroup-restricted gene with multiple copies in the genome as a target for PCR resulted in an improved clinical sensitivity with these archived blood samples. A pitfall of this strategy is the potentially larger degree of genetic heterogeneity in species- or group-restricted genes that may allow for decreases in sensitivity. It remains to be determined whether epank1 will be an appropriately sensitive target for PCR diagnosis in European patients with HGE. Further characterization of the heterogeneity in epank1 may reveal more-optimal regions that are conserved among the E. phagocytophila-group species for the design of primers to further improve the PCR diagnosis of HGE.

Currently, an optimal algorithm for laboratory confirmation of human ehrlichioses is not established. Definitive confirmation during active disease requires the use of PCR or culture. Two approaches for PCR confirmation include assays directed at specific etiologic agent identification and amplification of nucleic acids by using broad-range, "genus-specific" primers (2, 3, 7). Each approach is confounded by the potential for missed diagnoses and increased cost. Until issues of sensitivity, specificity, and cost regarding broad-range PCR for clinical applications are addressed, directed investigation using validated assays that identify specific etiologic agents is a proven approach.