Ehrlichia first attracted attention in 1935 as pathogens of dogs [3] and shortly thereafter as potential zoonotic agents. An infection similar to mononucleosis was reported in western Japan [4], and the ehrlichial cause of Sennetsu fever was subsequently confirmed by subinoculating mice with patient blood and observing the characteristic inclusions (morulae) within mononuclear cells. The index case of human ehrlichiosis in North America was discovered in a tick-exposed Arkansas resident in 1986 [5]. Morulae were seen within the patient's monocytes, and his serum reacted against antigens of E. canis, the agent of tropical canine pancytopenia. In 1990, a new species, closely related to E. canis, was cultivated from a febrile Arkansas patient and designated E. chaffeensis [6, 7]. About 400 illnesses caused by this agent have since been confirmed in 30 states. Nine of these cases were fatal.

Since 1990, morulae have been discovered in the granulocytes of 12 febrile patients from northern Minnesota and Wisconsin [8]. The serum of these patients did not react with E. chaffeensis antigen. In addition, an analysis of sequences obtained from a polymerase chain reaction (PCR) assay using universal eubacterial 16S ribosomal DNA (rDNA) primers indicated that the pathogen was closely related to, if not conspecific with, E. phagocytophila and E. equi [9], agents of ovine tick-borne fever and equine ehrlichiosis, respectively.

Two of the 12 reported cases were fatal. This infection has been designated human granulocytic ehrlichiosis because morulae are restricted to polymorphonuclear leukocytes. The public health importance, especially the geographic distribution, of human granulocytic ehrlichiosis remains undescribed because of the novelty of the disease and because confirmatory diagnostic tests are not widely available. We describe the definitive identification of this ehrlichia organism from a patient in New England, a discovery that suggests that human granulocytic ehrlichiosis may infect residents of certain sites outside the upper midwestern United States.

Case Report

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A 68-year-old woman presented to the Nantucket Cottage Hospital in Massachusetts on 1 November 1994 with a 5-day history of fever, confusion, headache, and fatigue. She had been in her usual state of good general health and had no significant medical history other than a previous tick bite in June 1994. She took no medications routinely and had no allergies.

The patient had removed an unidentified tick from her left flank approximately 5 days before symptoms developed. The tick had not been saved. She had noted no rashes or other signs or symptoms after the tick bite. At presentation, the patient appeared flushed and was somewhat confused as to the time and place, but was otherwise alert and neurologically intact. Her oral temperature was 40 °C, her pulse rate was 88 beats/min, and her blood pressure was 110/60 mm Hg. Physical examination showed no evidence of rash or cellulitis at the site of the tick bite and no other remarkable findings. No organomegaly was noted.

An automated complete blood count showed an abnormal leukocyte count (3200 x 10⁹/L) and platelet count (40 000 x 10⁹/L). A manual differential count showed 32% segmented neutrophils, 27% bands, 34% lymphocytes, 6% monocytes, and 1% metamyelocytes. The hematocrit was 0.38. A chemical analysis of the blood showed low potassium and calcium levels and slightly elevated urea nitrogen levels (22 mg/dL). Liver enzyme levels were elevated: alanine aminotransferase level, 0.07 µkat/L; aspartate aminotransferase level, 0.12 µkat/L; and lactate dehydrogenase level, 7.80 µkat/L. Infection with Babesia microti was suspected, and a Giemsa-stained thin peripheral blood smear was carefully scrutinized. No intraerythrocytic organisms were noted, but an inclusion was noted in a band neutrophil. The patient was suspected of having Lyme disease or ehrlichiosis, and doxycycline (100 mg orally twice a day for 10 days) was started pending confirmatory analysis at the Harvard School of Public Health. The patient defervesced within 24 hours and had an uneventful recovery.

An anticoagulated venous blood sample (ethylenediaminetetracetate, 10 mM) obtained at presentation and kept refrigerated for 2 days was examined for evidence of ehrlichiae. Buffy-coat smears stained with the Wolbach Giemsa variant for rickettsiae [10] showed rare inclusions (12 on 5 slides) that were morphologically consistent with ehrlichial morulae within bands and metamyelocytes (Figure 1). The patient's serum contained borderline IgM (titer, 1:64) against E. chaffeensis antigen by the indirect fluorescent antibody test [6] but did not contain IgG. No reactivity was noted to antigens of Babesia microti (indirect fluorescent antibody test [11]) or Borrelia burgdorferi (immunoblot [12]).

View larger version (162K): [in this window] [in a new window]   Figure 1. Morula (arrow) within a metamyelocyte from the patient's blood (buffy-coat smear, Wolbach Giemsa stain; original magnification, x 1250).

In a room in which intact Ehrlichia species or their DNA had never been manipulated, an aliquot of the anticoagulated blood was pipetted into a sterile cryotube using a barrier-filtered micropipettor. This sample was sent to the Centers for Disease Control and Prevention for analysis by PCR and automated cycle sequencing. By using a modification of the previously described nested PCR procedure with ehrlichia-specific primers [13] for the 16S rRNA gene, an 1158-base pair amplification product was obtained and sequenced. The sequence of this amplification product (GenBank accession number U23038) did not differ from the sequence of the 16S rRNA gene amplified from the persons in Minnesota and Wisconsin who had diagnoses of human granulocytic ehrlichiosis [9].

Convalescent serum samples obtained on 17 November 1994 and 10 January 1995 continued to show borderline IgM reactivity to E. chaffeensis antigen. Specific IgG to E. chaffeensis was first detected in the 10 January sample (titer, 1:128). Immunoglobulin G reactivity to antigen of E. equi, which has been used as a surrogate for the agent of human granulocytic ehrlichiosis, was not seen in the serum sample obtained at the original presentation (1 November). However, IgG reactivity was present at a titer of 1:64 in the 17 November sample and at a titer of 1:256 in the 10 January sample. No reactivity to antigens of Rickettsia rickettsii, R. typhi, or Coxiella burnetii was seen in the acute and convalescent serum samples (indirect fluorescent antibody test). Seroconversion for Lyme disease or babesiosis was not observed.

Discussion

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An elderly tick-exposed patient from Nantucket, Massachusetts, presenting with a febrile illness and nonspecific symptoms, was initially suspected of having Lyme disease or babesiosis. Laboratory test results indicated both thrombocytopenia and leukopenia, which we use as markers for B. microti infection (unpublished data), and a Giemsa-stained thin blood smear was carefully scrutinized. A suspicious inclusion detected within a granulocyte led to a careful search for ehrlichiae, and classic morulae were detected by examining buffy-coat smears. Ehrlichial DNA was amplified and sequenced, and the identity of the agent was confirmed by comparing this sequence with those amplified from patients from the upper midwestern United States who were accessioned in GenBank.

Our report describes the first definitive identification of this pathogen in a New England resident. A case of human ehrlichiosis was reported from nearby Cape Cod in 1992 [14]; in retrospect, this case may be considered to have been caused by the agent of human granulocytic ehrlichiosis [15]. Serum from that patient was reported to strongly react (IgG titer >1:512) to antigens of E. chaffeensis. In contrast, serum samples from the original Minnesota and Wisconsin cases of human granulocytic ehrlichiosis have no reported reactivity with E. chaffeensis [8]. However, convalescent serum samples from several cases of human granulocytic ehrlichiosis that have recently been diagnosed and confirmed by molecular analysis in Minnesota apparently react with antigens of E. chaffeensis (Persing DH, Mayo Foundation. Personal communication). The degree of cross-reactivity between E. chaffeensis and the agent of human granulocytic ehrlichiosis may eventually be described as more cases of human granulocytic ehrlichiosis are analyzed. However, the presence of granulocytic morulae and the fact that the agent has the same molecular identity as the pathogen identified in the upper midwestern United States establish the diagnosis of human granulocytic ehrlichiosis in our patient. Whether the agent constitutes an antigenic variant remains to be determined.

The agent of human granulocytic ehrlichiosis may frequently infect residents of sites where deer ticks are abundant but may be diagnosed as a rashless Lyme disease. Human granulocytic ehrlichiosis may be successfully treated with tetracyclines, and a response to treatment might affirm a presumptive diagnosis of Lyme disease. In 1991, we found that 11% of serum samples obtained from residents of a well-studied Lyme-disease focus in Cape Cod reacted with antigen of E. chaffeensis [16], and analysis of serum samples from 100 febrile patients presenting to the Nantucket Cottage Hospital during the summer of 1992 showed seropositivity in 8 samples (unpublished observation). Because we now believe that serum from certain patients with human granulocytic ehrlichiosis may cross-react with antigens of E. chaffeensis, these serologically detected cases probably represent human granulocytic ehrlichiosis. The date of symptom onset in our patient suggests that the vector is the deer tick Ixodes dammini [17], which is the only human-biting tick active on Nantucket during the fall months (unpublished observation). We have recently shown the tick's competence as an experimental vector for the agent of human granulocytic ehrlichiosis (Telford SR III, Dawson JE, Katavolos P, Warner CK, Kolbert CP, Persing DH. Perpetuation of the agent of human granulocytic ehrlichiosis in a deer tick-rodent cycle. Unpublished manuscript) and have documented a 10% prevalence of infection in host-seeking adult I. dammini from Wisconsin [18] and Nantucket. Clinicians should thus consider the diagnosis of human granulocytic ehrlichiosis in any patient with a febrile illness after the documented engorgement of this aggressive tick, in addition to a diagnosis of Lyme disease or babesiosis.