Serological Responses to Ehrlichia equi, Ehrlichia chaffeensis, and Borrelia burgdorferi in Patients from New York State

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1 JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1997, p Vol. 35, No /97/$ Copyright 1997, American Society for Microbiology Serological Responses to Ehrlichia equi, Ehrlichia chaffeensis, and Borrelia burgdorferi in Patients from New York State SUSAN J. WONG, 1 * GERALDINE S. BRADY, 2 AND J. STEPHEN DUMLER 3 Wadsworth Center 1 and Bureau of Communicable Disease Control, 2 New York State Department of Health, Albany, New York 12201, and Ehrlichia Research Laboratory, Division of Medical Microbiology, Department of Pathology, Johns Hopkins Hospital, Baltimore, Maryland Received 7 November 1996/Returned for modification 7 January 1997/Accepted 28 May 1997 Serological testing at the New York State Department of Health for human granulocytic ehrlichiosis in the residents of Westchester County, N.Y., was performed with specimens from 176 patients by the indirect fluorescent-antibody (IFA) technique with Ehrlichia equi MRK-infected neutrophils. To understand whether human monocytotropic ehrlichiosis also occurs in this northeastern geographic region, specimens were also tested for antibodies to Ehrlichia chaffeensis Arkansas. Screening tests and immunoblots for Lyme disease (Borrelia burgdorferi infection) were also performed. Thirty-two patients had antibodies only to E. equi and 21 patients had antibodies to both E. equi and E. chaffeensis, whereas 12 patients had only E. chaffeensis antibodies by the IFA technique. The remaining patients did not have antibodies to either ehrlichia. Eighteen serum samples from 13 of these patients were coded and sent to the Ehrlichia Research Laboratory (Baltimore, Md.) for repeat analysis by the IFA test and for E. equi and E. chaffeensis immunoblots. Immunoblot analysis for E. equi in samples with positive IFA test results confirmed the results for eight of the nine specimens. Immunoblot analyses for E. chaffeensis were negative for all 18 serum samples. Borrelia-reactive antibodies were found in sera both from patients with granulocytic ehrlichiosis and from patients with monocytotropic ehrlichiosis from New York State. Our results suggest that E. equi antigen is an appropriate substrate for identifying human granulocytic ehrlichiosis. E. chaffeensis antigen lacks appropriate sensitivity to serve as a surrogate substrate for the detection of human granulocytic ehrlichiosis and should be used solely for the diagnosis of human monocytotropic ehrlichiosis. Heat shock proteins may, in some cases, cause cross-reactivity between B. burgdorferi and ehrlichiae. Human granulocytic ehrlichiosis (HGE) was identified in the upper Midwest United States in 1990 and was reported in 1994 (3). The Ehrlichia equi-like agent of HGE is transmitted by the black-legged tick (Ixodes scapularis; also called the deer tick), which may also transmit Borrelia burgdorferi and Babesia microti (19, 20, 23, 27, 32). Moreover, this tick is abundant in the lower Hudson River Valley, an area where Lyme disease is highly endemic (34). In contrast, the Lone Star tick, Amblyomma americanum, the vector for Ehrlichia chaffeensis, is abundant in areas of Long Island, N.Y., and the south, southcentral, and mid-atlantic regions of the United States (24). Prior to July 1995, the Centers for Disease Control and Prevention (CDC), Atlanta, Ga., was testing for antibodies only to E. chaffeensis. As a result, some New York State (NYS) residents with clinical ehrlichiosis had been diagnosed with human monocytotropic ehrlichiosis (HME) on the basis of E. chaffeensis serologic tests at CDC. Laboratory investigations of ehrlichiosis by the NYS Department of Health (DOH) were initiated in July 1995 (7). Physicians in Westchester County, N.Y., were asked to identify patients with clinically suspected cases of ehrlichiosis. All sera from patients with suspected ehrlichiosis submitted to the Wadsworth Center were tested for the presence of antibodies to E. equi, E. chaffeensis, and B. burgdorferi. HGE is being intensely studied (13, 15, 18, 31, 33). The spectrum of illness varies from mild to severe disease that results in hospitalization and admission to an intensive care * Corresponding author. Mailing address: Wadsworth Center, New York State Department of Health, P.O. Box 22002, Albany, NY Phone: (518) Fax: (518) unit (4); however, the extent of morbidity and mortality from E. equi-like-agent infection in the Northeast is not yet well defined (1, 8, 34 36). HGE has been described as relatively mild in a study at a single NYS medical center, while there is an association with acute respiratory distress syndrome in residents of Westchester County and Long Island, N.Y., and fatal infection associated with opportunistic pathogens has been described in a patient from Connecticut (4, 8, 16, 34). Currently, diagnosis is mostly based on clinical suspicion with retrospective serologic confirmation, even though little is known about the sensitivity or specificity of existing serologic tests and the effects that early therapy may have on subsequent antibody production (1, 10, 12). Our study aimed to compare serological indirect fluorescent-antibody (IFA) test results between two laboratories performing a high volume of serologic tests for ehrlichiosis in order to (i) identify whether both HGE and HME were acquired in NYS, (ii) determine whether serologic cross-reaction between the two ehrlichial species is detectable in clinical samples, and (iii) identify whether there is serologic evidence of coinfection with both Ehrlichia species. Further evaluation of sera by Ehrlichia immunoblotting was used to define the specificity of anti-ehrlichia serologic reactions of sera from patients in this region. The possibility of coinfection, concurrent infection, or cross-reaction between an Ehrlichia species and B. burgdorferi was also investigated. MATERIALS AND METHODS Case finding and definitions. Physicians in NYS were requested to report confirmed cases of ehrlichiosis with accompanying clinical information. Free serologic testing for HGE and HME was provided by the Wadsworth Center. Physicians also completed a questionnaire about signs, symptoms, and past laboratory evidence of B. burgdorferi infection in these patients. A patient with an 2198

2 VOL. 35, 1997 EHRLICHIA AND BORRELIA ANTIBODIES IN NEW YORK STATE 2199 acute febrile illness, headache, malaise, and/or myalgia and a single elevated titer of 80 for E. equi was defined as having a probable case of HGE. A symptomatic patient was considered to have a confirmed case of HGE when there was a fourfold rise in titer to E. equi between acute- and convalescent-phase sera. A few cases of HGE were defined by the presence of E. equi DNA in blood, as detected by PCR as described previously (1, 7). Cases of HME were defined as described previously (33). Blood specimens for serologic testing. Serum from patients suspected of having ehrlichiosis on the basis of clinical manifestation, leukopenia, thrombocytopenia, and elevated serum hepatic transaminase levels was requested. Blood was drawn at the first visit to a physician or as soon as ehrlichiosis was considered in the differential diagnosis. First sera were defined as samples obtained within the first 2 weeks of illness. Follow-up sera were requested 2 or more weeks after the first serum sample was obtained or 2 weeks after the onset of symptoms if no acute-phase specimen was available. First serum specimens were tested upon arrival in the laboratory and then again paired with the follow-up sera. When received, aliquots of serum were prepared and were frozen at 65 C. Sera from patients outside of NYS with confirmed cases of HME. Paired serum specimens from 20 patients with well-characterized cases of HME from nine states, primarily in the southern United States, were provided by CDC to assist in answering questions about cross-reactivity between E. chaffeensis and the HGE agent and about coinfection and cross-reaction between E. chaffeensis and B. burgdorferi in NYS residents. Patients from whom these sera were obtained resided in Arkansas, California, Georgia, Maryland, Massachusetts, Missouri, New Jersey, Tennessee, and Virginia. IFA tests for E. equi MRK and E. chaffeensis Arkansas. Serological tests at NYS DOH were performed as described previously (3, 7, 9, 11). E. equi MRK antigen slides were purchased from J. Madigan, University of California, Davis. These slides consisted of peripheral blood neutrophils prepared from the blood of a horse acutely infected with E. equi. The slides were maintained frozen at 55 C until used. E. chaffeensis Arkansas-infected DH82 canine histiocytes (provided by J. Dawson, CDC) were used to prepare antigen slides as described previously (9). Fluorescein-isothiocyanate-conjugated goat anti-human immunoglobulin (immunoglobulin G [IgG], IgA, and IgM) was purchased from Kirkegaard & Perry Laboratories, Gaithersburg, Md., and was used at a 1:50 dilution. Dilutions of serum for screening by both IFA tests were 1:80. At the Wadsworth Center, the titers of routine clinical specimens were determined to 640 or to the endpoint for selected sera. Serologic tests at the Ehrlichia Research Laboratory (ERL) of one of the authors (J.S.D.) were performed with antigens prepared on-site as described previously with E. equi MRK-infected equine leukocytes and E. chaffeensis Arkansas-infected DH82 cells (11). Immunoblots of Ehrlichia spp. Immunoblots with E. equi MRK and E. chaffeensis Arkansas antigens were performed as described previously (10, 11). E. chaffeensis was propagated in DH82 cells, and E. equi was prepared from peripheral blood leukocytes of an experimentally infected horse. In brief, ehrlichiae were purified from lysed infected cells by Renografin density gradient centrifugation, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotransfer of separated antigens onto nitrocellulose. Uninfected equine neutrophil and uninfected DH82 cell lysates were used as controls for each serum sample examined. Nitrocellulose transfer papers were cut into 3- to 4-mm strips and were treated with a 1:80 dilution of patient and control sera. Bound antibody was detected by sequential reaction with biotinylated goat anti-human IgG-IgA-IgM, followed by streptavidin-alkaline phosphatase and 5-bromo-4- chloro-3-indolylphosphate toluidinum nitroblue tetrazolium. Controls included convalescent-phase sera from patients with HGE or HME or sera from individuals with no history of ehrlichial infections. In pilot studies, the sensitivity of the E. chaffeensis immunoblot was proven when antibodies to either the 28- or the 29-kDa antigen were detected in eight of nine convalescent-phase serum samples from patients with HME who had IFA test titers of between 80 and 2,560. E. equi immunoblots were considered positive if a 44-kDa band was detected. B. burgdorferi antibody screening and immunoblots. Lyme disease screening tests for B. burgdorferi antibodies were performed by fluorescent immunoassay with a whole-cell sonicate of the B31 strain B. burgdorferi (ATCC 2521) and the VIDAS Lyme screen (17). The cutoff for a positive VIDAS Lyme screen was a test value of 1.00, while samples with test values of between 0.75 and 1.00 were considered equivocal. The test value is the ratio of the fluorescence of the sample to the fluorescence of a standard. Confirmatory immunoblotting was performed with HBH-8 antigen as described previously (28). Criteria for positive IgG and IgM blots were those published by CDC and the Association of State and Territorial Public Health Laboratory Directors (2). Bands with the molecular sizes described for immunoblot diagnosis of Lyme disease were defined as highly B. burgdorferi specific; these included bands of 18, 23, 28, 30, 39, 45, 58, 66, and 93 kda. The 41-kDa band (flagellin) shows extensive cross-reactivity with treponemes, but it is also the most frequently identified antibody in sera from patients with Lyme disease (2). Testing algorithm. All sera submitted to the Wadsworth Center laboratory for serologic tests for ehrlichiosis were tested by the IFA test for antibodies to E. equi and E. chaffeensis. These sera were also tested for antibodies to B. burgdorferi in two or more Lyme disease screening immunoassays. Sera with equivocal or positive Lyme disease screening test results were analyzed by a B. burgdorferi immunoblot assay with a polyvalent (anti-igg, IgA, IgM) conjugate. The banding pattern from the polyvalent immunoblot was analyzed to determine whether IgG and/or IgM immunoblots were necessary (28). Eighteen serum samples from patients in NYS suspected of having ehrlichiosis on the basis of clinical signs and symptoms were tested for antibodies reactive with Ehrlichia species and B. burgdorferi. Five sets of paired serum samples with increasing antibody titers were selected. Two serum samples with no antibodies to Ehrlichia species, two serum samples with E. equi antibody titers greater than E. chaffeensis antibody titers, one serum sample reactive to E. chaffeensis only, and one serum sample with equal titers to E. equi and E. chaffeensis were chosen. These sera were selected to represent a spectrum of Borrelia antibody levels. These 18 serum samples were then coded and shipped to ERL for repeat IFA serology and immunoblot analyses for E. equi and E. chaffeensis. RESULTS IFA test results with sera from patients from the lower Hudson River Valley suspected of having HGE. Antibodies to both E. equi and E. chaffeensis were detected by the IFA technique in the sera of suspected ehrlichiosis patients residing in NYS. Fifty-three confirmed or probable HGE cases were identified at the Wadsworth Center from analysis of specimens taken from 176 patients during 1994 and There were slightly fewer confirmed cases of HGE than probable cases of HGE. Thirty-two patients (18.2%) had antibodies to E. equi only, whereas 21 patients (11.9%) demonstrated serologic reactivity to both E. equi and E. chaffeensis. In these dually reactive sera, antibody levels were generally much higher to E. equi than to E. chaffeensis, suggesting that E. equi was the etiologic agent (Table 1). Twelve patients (6.8%) had titers of 80 to E. chaffeensis only. Antibody titers to E. equi ranged from 80 to 20,480. Of the 53 patients with symptoms of HGE and antibody titers of 80 against E. equi, 17 (32.1%) met the confirmed case definition of HGE, 23 (43.4%) met the probable case definition, and the classifications for the remaining 13 (29.5%) were uncertain due to missing or incomplete data. Two symptomatic patients met a case definition of HME by a fourfold change in antibody titer to E. chaffeensis. Ten patients had antibody titers to E. chaffeensis of 160 or greater, and clinical data were available for five of these patients, allowing them to be defined as having probable cases of HME. Clinical and epidemiologic data for HGE patients providing sera. Twenty-eight (53%) of 53 patients with HGE were male. The median age was 48 years, with an age range of 9 to 90 years. Fever (87%), myalgia (53%), malaise (47%), and headache (76%) were the most common symptoms noted. The reported mean temperature was 39.4 C (range, 38.8 to 41.1 C). Thirty (76.9%) of 39 patients had leukopenia (less than 5,000 leukocytes/ l). Thrombocytopenia (less than 150,000 platelets/ l) was documented in 29 (76.3%) of 38 patients tested. Twenty-three (60.5%) of 38 patients had both leukopenia and thrombocytopenia. Ten (18.9%) patients reported a previous history of Lyme disease, 17 (32.1%) patients did not have a history of Lyme disease, while no information about Lyme disease was reported for the remaining 26 (50%) patients. Among those patients with a reported history of Lyme disease, no erythema migrans, Bell s palsy, or other symptom of early or late Lyme borreliosis were noted. Lyme disease screening tests for patients with HGE. Antibodies to B. burgdorferi detected in sera from patients with confirmed or probable cases of HGE were compared to B. burgdorferi antibodies detected in sera from patients whose sera were tested but which were negative for E. equi antibodies. Of 53 patients with confirmed or probable cases of HGE, Lyme disease serology was performed with sera from 49 individuals. Nineteen (35.8%) of the patients with HGE had positive serology and 16 (30.2%) had equivocal screening test results for Lyme disease.

3 2200 WONG ET AL. J. CLIN. MICROBIOL. TABLE 1. Serologic results for Ehrlichia spp. and B. burgdorferi from two laboratories a Patient (serum sample) E. equi (44-kDa protein) Immunoblot result (ERL) E. chaffeensis (28- to 29-kD6 protein) B. burgdorferi immunoblot results for HGE patients. Eight HGE patients had positive IgM B. burgdorferi immunoblots. Two patients had positive IgG blots, and two patients had positive polyvalent blots without further IgG or IgM testing. Overall, 12 (22.6%) of the patients with HGE had positive immunoblots for B. burgdorferi. The polyvalent- and IgG-positive immunoblots were obtained only for sera that scored reactive on screening tests. One reactive IgM blot was obtained for a serum sample with an equivocal Lyme disease screening test result and one was obtained for a serum sample with a negative Lyme disease screening test result. This Lyme disease screening test-negative serum sample was tested by immunoblotting because a follow-up specimen was positive. Polyvalent immunoblots for B. burgdorferi antibodies were performed with sera from 35 of the patients with HGE, and then IgG immunoblots were performed with the first serum sample received from 17 patients and IgM immunoblots were performed with the first serum sample received from 16 patients. Convalescent-phase sera were obtained from 25 of the 35 patients with HGE for whom polyvalent immunoblots were performed with their acute-phase specimens. Sufficient serum for B. burgdorferi immunoblots was available for follow-up from only 10 of 17 patients. The frequency of antibody reactivity to B. burgdorferi proteins of different molecular sizes is presented in Table 2. The polyvalent, IgG, and IgM blots were analyzed independently. Follow-up sera were obtained a median of 26 days (range, 10 to 92 days) after the first specimens were obtained. The decision to analyze both serum samples from a given patient together (Table 2) allowed a larger data pool when results of blots for Lyme disease for patients with HGE were compared with the results obtained with sera submitted for E. equi serologic testing but that were negative for E. equi antibodies. Antibodies to B. burgdorferi proteins occurred far less frequently in the sera of patients without HGE (Table 3) than in the sera of patients with HGE (Table 2). Antibodies to B. burgdorferi antigens of 37, 39, 41, 75, and 93 kda were most frequently observed in IgG immunoblots from HGE patients, while IgM immunoblots detected the 23-kDa (OspC) protein most often (58%). Antibodies to the 75-kDa Titer by IFA test E. equi E. chaffeensis NYS ERL NYS ERL VIDAS Lyme disease screening test result (interpretation) a 1 Neg , (pos.) 2 (weak) Neg (neg.) 3 Neg. Neg (neg.) 4 Neg. Neg (neg.) 5 (very weak) Neg (pos.) 6 Neg. Neg (neg.) 7 Neg. Neg (neg.) 8 Neg. Neg (pos.) 9 (first) Neg. Neg (pos.) 9 (conval.) Neg , (pos.) 10 (first) Neg. Neg (neg.) 10 (conval.) Neg , (neg.) 11 (first) Neg. Neg , (neg.) 11 (conval.) Neg , (neg.) 12 (first) Neg. Neg (neg.) 12 (conval.) very weak) Neg , (equiv.) 13 (first) Neg. Neg (neg.) 13 (conval.) Neg. 80 1, (equiv.) a Neg., negative;, positive; Pos., positive; first, first serum sample from patient; conval., convalescent-phase serum sample from patient. b Lyme disease (B. burgdorferi) screening test result for sera from NYS. heat shock protein were second only to 41-kDa (flagellin) antibodies in both polyvalent and IgG immunoblots (Table 2). Ehrlichia species and B. burgdorferi serology reported by the Wadsworth Center and ERL. Table 1 presents the IFA test results for 18 serum samples tested at both the Wadsworth Center and ERL for E. equi and E. chaffeensis antibodies, immunoblot results for these two Ehrlichia species from ERL, and B. burgdorferi screening test serology from the Wadsworth Center. All seven serum samples determined at the Wadsworth Center to be negative for both E. equi and E. chaffeensis were also negative at ERL. Most (9 of 10) serum samples positive for E. equi at the Wadsworth Center were also positive at ERL. The E. equi immunoblots correlated with positive E. equi serology by the IFA test except in the case of specimen 11a (an acute-phase specimen), which was strongly positive by the IFA test but negative by immunoblotting. All serum specimens were negative for E. chaffeensis by immunoblotting, and only one serum sample (of six serum samples positive at NYS DOH) was positive at ERL, with a titer of 80 to E. chaffeensis. Representative immunoblots for E. equi and E. chaffeensis are presented in Fig. 1. The three serum samples with the highest E. chaffeensis titers at the Wadsworth Center (serum samples 5, 8, and 11; convalescent phase) had strongly positive (serum samples 5 and 8) or negative (serum sample 11c) Lyme disease screening test results, with at least several bands detected on polyvalent immunoblots for B. burgdorferi (Fig. 2). The Ehrlichia species immunoblot data demonstrate that the sera dually reactive (for E. equi and E. chaffeensis) by the IFA test came from patients with E. equi infection. Clinical, epidemiologic, and laboratory data for patients with positive Ehrlichia immunoblots. Table 4 presents the available laboratory values, county of residence, and clinical presentation of each of the 13 patients in the Ehrlichia immunoblot study. Fever, thrombocytopenia, and leukopenia were frequent findings. When elevated serum hepatic transaminase levels were noted, low platelet counts were also found. No erythema migrans was reported. The clinical presentation and laboratory findings were not suggestive of Lyme disease.

4 VOL. 35, 1997 EHRLICHIA AND BORRELIA ANTIBODIES IN NEW YORK STATE 2201 TABLE 2. Frequency of antibodies to B. burgdorferi antigens in sera from NYS patients with HGE by immunoblotting Molecular size (kda) of protein band B. burgdorferi immunoblots of the 18 serum samples in the Ehrlichia immunoblot study. Figure 2 indicates that 12 of 18 serum samples had bands in polyvalent B. burgdorferi immunoblots. For eight serum samples bands were detected in IgG immunoblots, whereas for six serum samples bands were detected in IgM immunoblots. A previous history of Lyme disease was documented for only three patients (patients 1, 9, and 13) of the seven patients with positive IgC, and/or IgM immunoblot results. Interestingly, the patients with the highest Lyme disease screening test results and the strongest bands in the immunoblots (patients 5 and 8) did not have a history of Lyme disease. Patient 5 probably had Lyme disease in the past on the No. (%) of sera that reacted a Polyvalent conjugate IgG conjugate IgM conjugate First Conval. Total First Conval. Total First Conval. Total (25) (22) (31) (15) (19) (0) (48) (44) (27) (13) (11) (19) (20) (11) (15) (7) (7) (0) (13) (15) (15) (78) (74) (54) (10) (22) (4) (13) (26) (27) (13) (7) (8) (13) (11) (0) (12) (15) (0) (3) (7) (0) (32) (19) (58) (8) (15) (0) (5) (7) (0) Total a First, first serum sample that reacted; Conval., convalescent-phase serum samples that reacted; Total, sum of number of first and convalescent-phase serum samples that reacted; percent, percentage of first and convalescent-phase serum samples that reacted. TABLE 3. B. burgdorferi immunoblot antibody frequency in sera submitted from patients negative for HGE Molecular size (kda) of protein band No. (%) of blots showing reactivity to the following: Polyvalent antibody IgG IgM (7) 1 (2) 5 (9) 84 2 (1) 3 (5) 0 (0) (7) 2 (3) 0 (0) 66 6 (4) 0 (0) 3 (5) 61 4 (3) 1 (2) 2 (4) 58 2 (1) 0 (0) 1 (2) 45 6 (4) 2 (3) 1 (2) (38) 6 (10) 10 (18) 39 7 (5) 4 (7) 1 (2) 37 6 (4) 5 (9) 4 (7) 34 0 (0) 1 (2) 2 (4) 31 1 (1) 1 (2) 0 (0) 30 2 (1) 2 (3) 0 (0) 28 1 (1) 1 (2) 0 (0) (8) 8 (14) 10 (18) 21 1 (1) 2 (3) 0 (0) 18 1 (1) 2 (3) 0 (0) Total no basis of the strength and number of bands detected, and patient 8 likely had a recent B. burgdorferi infection on the basis of the presence of 23- and 41-kDa antibodies. Comparison of patient sera from CDC and sera from NYS residents with HME. Since July 1995, sera from 21 patients (from 1994 to 1996) have been identified at the Wadsworth Center to be reactive to E. chaffeensis only with titers of 80. Clinical and epidemiological data were available for 11 individuals. Fever was present in all 11 patients. The mean platelet count was / l (range, to / l), and the mean leukocyte count was / l (range, to / l). Malaise, myalgia, and headache were frequent, occurring in 82% of the patients. Three of these individuals had symptoms and met a definition of a case of HME on the basis of a fourfold change in E. chaffeensis antibody titer or positive PCR. Eight individuals had symptoms and met a definition of a probable case of HME, and clinical data for 10 other individuals with positive E. chaffeensis serology at a titer of 80 were not available. The mean VIDAS Lyme screening test value for 29 serum samples from the 21 patients with possible cases of HME was 1.19 (range, 0.07 to 4.60). Figure 3 presents representative Borrelia immunoblots chosen from among the sera from patients with HME in NYS. Patients 14 and 15 had negative immunoblots for B. burgdorferi infection, although patient 15 demonstrated some antibodies to crossreactive bacterial proteins. Patients 16 to 18 had strong bands at 23 and 41 kda (OspC and flagellin, respectively). IgM antibodies to the 23-kDa protein are particularly intense compared to those of IgG. Many of the IgG antibody specificities to higher-molecular-mass proteins were detected. These protein bands at 58, 61, 63, and 75 kda are in the range of the molecular sizes of bacterial heat shock proteins. Since sera from approximately one-third of patients in NYS with confirmed and probable cases of HGE showed reactivity to E. chaffeensis, we examined the reactivities of 46 serum samples from patients confirmed by CDC to have human ehrlichiosis (HME) for reactivity to E. equi and B. burgdorferi. These sera were from individuals in nine states covering a wide

5 2202 WONG ET AL. J. CLIN. MICROBIOL. FIG. 1. Immunoblots of human sera that reacted with both E. equi and E. chaffeensis antigens by the IFA test. (A) E. equi immunoblots. Patient serum was reacted with paired strips containing E. equi antigen (left) and control uninfected horse leukocyte lysate (right). Each pair of antigen strips was reacted with serum from the following subjects: patient 1 (lane 1), patient 2 (lane 2), patient 9 (acute-phase serum) (lane 3), patient 9 (convalescent-phase serum) (lane 4), a patient convalescing from HGE (positive control) (lane 5), and a healthy human subject with no history of HGE or HME (negative control) (lane 6). Note the presence of bands at the approximate molecular size of 44 kda in lanes 1, 2 (arrowhead), and 4, representing the E. equi, E. phagocytophila, and HGE agent group-specific protein antigen. (B) E. chaffeensis immunoblots. Each patient serum sample was reacted with E. chaffeensis antigen (left) and control uninfected DH82 cell lysate (right). All lanes were reacted with the same serum samples described above for panel A except for the control in lane 5, which was reacted with a positive control serum sample from a patient convalescing from HME. Note the dark bands at approximately 28 and 29 kda in lane 5 (positive control), which represent the E. canis and E. chaffeensis group-specific antigens (arrows). Numbers to the left of each panel are in kilodaltons. Patient no. geographic area within the range of A. americanum and included many from areas where the prevalence of Ixodes ticktransmitted disease is very low (5, 21). Only one of these serum samples (from New Jersey) was reactive to E. equi. VIDAS Lyme disease screening tests were predominantly negative, with a mean test value of 0.36 (range, 0.00 to 1.45) for 46 serum samples. Three serum samples which were equivocal (from Arkansas, Missouri, and Tennessee) and the one positive serum sample (from Arkansas) were evaluated by immunoblotting (Fig. 4). No B. burgdorferi-specific antibodies were seen. Only bands at 61 and 63 kda were detected. Note that the convalescent-phase sera from all four patients had more immunoblot reactivity than the first serum sample. In three patients the increased reactivity was related to IgG. In one patient the antibody which developed was IgM. DISCUSSION When NYS implemented serologic testing for the human ehrlichioses, it became apparent that HGE was not as uncommon as was previously believed. By E. equi serology, HGE has now been identified in several states from California to New England in areas where I. scapularis ticks frequently transmit B. burgdorferi to humans (18, 31, 33). These studies indicate TABLE 4. Clinical, laboratory, and epidemiologic data for NYS patients whose sera were included in the Ehrlichia immunoblot subset a Age (yr) Sex Res Fever PLT (10 3 / l) WBC (10 3 / l) Malaise Myalgia Headache Other 1 45 F W Y NA Y Y 2 56 M W Y NA Y N 3 NA M NA NA NA NA NA NA NA Rash, vasculitis 4 NA M NA NA NA NA NA NA NA 5 83 F W Y NA NA NA 6 62 F W Y NA Y Y 7 62 M W Y Y Y NA 8 52 F D Y NA Y Y 9 56 F W Y NA Y NA 1LFT M W Y N Y Y M W Y NA NA Y 1LFT M W Y NA Y NA F W Y Y N Y a Abbreviations: Res, residence; PLT, platelet count; WBC, white blood cell count; W, Westchester County, N.Y.; NA, data missing; D, Dutchess County, N.Y.; 1LFT, elevated serum alanine transaminase or aspartate transaminase activity; F, female; M, male; Y, yes; N, no.

6 VOL. 35, 1997 EHRLICHIA AND BORRELIA ANTIBODIES IN NEW YORK STATE 2203 FIG. 2. B. burgdorferi immunoblots of NYS patient sera included in the Ehrlichia immunoblot study. Sera were tested with polyvalent (lanes P), IgG (lanes G), and IgM (lanes M) antibody-enzyme conjugates. (A) The sera were from seven different individuals. Serum from patient 2 is not shown since the VIDAS screening test was negative and immunoblot analysis was not performed. (B) For patients 9, 10, 11, 12, and 13, acute- and convalescent-phase sera were sent coded for Ehrlichia immunoblots at ERL. Appropriate polyvalent-positive, IgG-positive, and IgM-positive sera and negative control sera were run with each set of clinical specimens tested on different days. The control strips are not shown. The numbers to the left of each panel are in kilodaltons. that E. equi MRK-infected horse neutrophils were appropriate for screening patients suspected of having ehrlichiosis and that HGE is the predominant form of ehrlichiosis in NYS. Moreover, because of low-level cross-reactivity between the HGE agent and E. chaffeensis, CDC data on the geographic extent of HME outside the geographic range of A. americanum need to be reassessed. Many positive serologic results for E. chaffeensis in the Northeast may be interpreted as cross-reactions with E. equi, since titers to the latter agent in patients from these areas are usually much higher (1, 34). However, our findings do not support the use of E. chaffeensis as a surrogate antigen for ehrlichiosis serology in residents in the Northeast. Thus, if A. americanum is present, both HGE and HME serologies should be performed. Our data suggest that the cross-reactivity to E. chaffeensis is FIG. 3. Representative B. burgdorferi immunoblots reacted with sera from NYS HME patients. Serum from patient 14 had an E. chaffeensis IFA test titer of 80 and a positive Lyme screening test result, but no bands were detected in acute- and convalescent-phase sera on B. burgdorferi immunoblots. Serum from patient 15 had an E. chaffeensis titer of 80 and was positive by PCR at both ERL and NYS DOH, and later, specimens had titers to E. chaffeensis of 640 by the IFA test. Note the lack of any B. burgdorferi-specific antibodies in this serum sample taken during the acute phase of HME. Serum from patient 16 had an E. chaffeensis titer of 640 and a positive B. burgdorferi IgM immunoblot, although no previous history of Lyme disease was documented. Serum from patient 17 had an E. chaffeensis titer of 320 but the patient had no previous history of Lyme disease. An anti-23-kda protein (OspC) IgM and an anti-41-kda protein (flagellin) IgG suggest recent Lyme disease. Patient 18 had an E. chaffeensis titer of 160 and no history of previous Lyme disease. Positive and negative controls were performed with all clinical specimens, but control strips are not shown. P, polyvalent (anti-igg, anti-iga, and anti-igm); G, anti-igg; M, anti-igm. Numbers to the left are in kilodaltons. stimulated by the HGE agent, but not the converse. Only one-third of the serum samples from HGE patients in NYS had low-titer antibodies to E. chaffeensis. Cross-reactions were more frequently seen in sera with high HGE antibody titers. Most patients in 1995 received a tetracycline antibiotic (doxycycline) as therapy, and antibody responses (titers) were lower than those in the previous year. Antibodies that recognized E. chaffeensis antigens may have been below the detection limit of the IFA assay. A. americanum ticks are infrequently found in Westchester County, so dual infection of this tick with both ehrlichial agents is unlikely (24). The travel histories of the patients outside of Westchester County were only infrequently documented on the epidemiology questionnaires; nevertheless, the accuracies of these responses have not been verified. Analysis of the well-characterized sera from HME patients from nine states produced only 1 serum sample among 46 serum samples tested with reactivity to E. equi. That serum sample was from a patient from New Jersey; the titer to E. equi was higher than that to E. chaffeensis, and the serum sample was probably misclassified serologically. The data further suggest geographic variation between ehrlichiae, since a lower level of serologic cross-reactivity between E. equi and E. chaffeensis has been reported in the upper Midwest compared with that in the Northeast (1, 34). The antigenicities and pathogenicities of bacterial strains are known to vary among geographic regions (4, 5). It is reasonable to expect further geographic variation in Ehrlichia strains that could contribute to the serologic cross-

7 2204 WONG ET AL. J. CLIN. MICROBIOL. FIG. 4. Representative B. burgdorferi immunoblots from non-nys HME patients. Reactions with acute- and convalescent-phase sera are shown for the four patients with equivocal and positive VIDAS Lyme disease screening test results. Patients 1 to 3 had equivocal Lyme disease screening test results. The second specimen from patient 4 had a positive Lyme disease screening test result. Only antibodies to the 61- and 63-kDa B. burgdorferi proteins are seen. No B. burgdorferi-specific antibodies are detected. Patients 1 to 3 generated anti-61-kda IgG and anti-63-kda IgG antibodies. Patient 4 developed anti-61-kda IgM antibodies. Positive and negative control immunoblots are not shown. P (polyvalent), anti-igg, anti-iga, and anti-igm; G, anti-igg; M, anti-igm. Numbers on the left are in kilodaltons. reactions between Ehrlichia spp. and positive serologic tests for Lyme disease (1). Bacterial infections frequently generate cross-reactive antibodies to bacterial heat shock proteins (25). Such antibody responses in patients with undocumented Lyme disease could account for some of the positive E. chaffeensis IFA test serology noted at the Wadsworth Center (29). Convalescent-phase sera from both HME patients in NYS and HME patients not in NYS showed increases in IgG reactivity to B. burgdorferi heat shock (61- and 63-kDa) proteins. Heat shock protein antibodies were second only to flagellin antibodies in the sera of NYS residents with confirmed or probable HGE when tested by Lyme disease immunoblotting (Table 2). Likewise, when sera from patients with other rickettsial and non-hge ehrlichial infections were tested by E. equi immunoblotting, most cross-reactions developed against proteins in the molecular size range of 58 to 75 kda, the typical size range for heat shock proteins (11). Moreover, E. chaffeensis is known to express an immunoreactive protein homologous to the Escherichia coli GroEL protein (29). Potential explanations for positive Lyme disease serology in patients with HGE include B. burgdorferi-specific or cross-reactive antibodies induced by the HGE infection or stimulation of a memory response to previous B. burgdorferi infection induced by cross-reactive ehrlichial antigens (6). Since the anti-23-kda antibodies of the sera from HGE patients in NYS were present at the highest frequency in the IgM immunoblots, the immunoblot banding patterns suggest a first encounter of the immune system with this immunogen, not a memory response (14, 21, 26). The report by Mitchell et al. (22) indicates that 15.8% (3 of 19) of HGE patients have serologic evidence of coinfection with another organism, either B. burgdorferi or B. microti (22). This is similar to the 20% positivity rate for B. burgdorferi immunoblots found in our series of 49 HGE patients and seems plausible. However, the definitive determination of coinfection will rely on the demonstration of concurrent B. burgdorferi and HGE agent infection by cultivation or PCR methods (37). HME, acquired locally, is a disease of the residents of NYS that occurs less frequently than HGE. The Lyme disease serology performed with sera from patients with HME not in NYS demonstrates that E. chaffeensis infection does not induce B. burgdorferi-specific antibodies. Although we have not confirmed B. burgdorferi coinfection in HME patients in NYS by B. burgdorferi PCR or isolation, these data suggest that when serum antibodies to E. chaffeensis are present in NYS residents, as detected by the IFA test, and when B. burgdorferispecific antibodies are of the IgM class, the patient has probably acquired both agents via two different but recent tick bites. Another pattern seen in patients in NYS with positive serology to E. chaffeensis is B. burgdorferi IgG antibody to proteins with higher molecular sizes. Some patients in NYS have been found to seroconvert between first and follow-up specimens on both the HME IFA and the Lyme disease screening tests and immunoblots. These B. burgdorferi immunoblots reveal an IgG response that resembles memory immunity to bacterial heat shock proteins. These patients were probably infected with E. chaffeensis that induced antibodies reactive with bacterial heat shock proteins, including those in B. burgdorferi. Wormser and colleagues (36, 37) have recently reported findings similar to ours for the first 10 HGE patients from Westchester County. We propose that the criteria of CDC and the Association of State and Territorial Public Health Laboratory Directors for interpreting Lyme disease serology may be appropriate in geographic regions where HGE is rare (2). In regions where E. equi and B. burgdorferi are found in the same ticks, Lyme disease serology may be misinterpreted unless the laboratory obtains several serum samples over the course of the illness and tests them for both HGE and Lyme disease. The laboratory test result must be interpreted in the context of a clinical history regarding signs and symptoms of ehrlichiosis and Lyme disease. Moreover, it will be critical for physicians to be aware that some patients with serologic reactions to B. burgdorferi need to be evaluated for ehrlichiosis (36, 37). To appropriately evaluate the significance of serologic studies for HGE, HME, and Lyme disease, it will be mandatory that skilled serologists use the appropriate antigens and carefully interpret the results (30). The inability to discern serologic cross-reactions among these pathogens could further confound the compilation of accurate information required as an aid for clinical diagnosis. 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