Human Granulocytic Ehrlichiosis Agent Research Articles

Detection of four Borrelia burgdorferi genospecies and first report of human granulocytic ehrlichiosis agent in Ixodes ricinus ticks collected in central Italy.

The presence of Borrelia burgdorferi s.l. and of Ehrlichia phagocytophila group was sought by PCR in Ixodes ricinus collected in a protected area of central Italy. Nymphs (n = 1475, gathered in 295 pools of 5 nymphs each) and adult ticks (n = 28) were examined. B. burgdorferi s.l. was detected in 13.8% of the nymph pools; of these, 63.4% were infected by B. valaisiana, 26.8% by B. afzelii, 7.3% by B. garinii, and 2.5% by B. burgdorferi s.s. Only a single adult male tick proved to host B. afzelii. The agent of human granulocytic ehrlichiosis (HGE) was detected in 2.7% of the nymph pools. Two HGE agent-positive nymph pools were also found to be positive for B. garinii and for B. afzelii, respectively. This is the first report from central Italy of the finding of the HGE agent in ticks.

Transcript Heterogeneity of the p44 Multigene Family in a Human Granulocytic Ehrlichiosis Agent Transmitted by Ticks

Transcript Heterogeneity of the <i>p44</i> Multigene Family in a Human Granulocytic Ehrlichiosis Agent Transmitted by Ticks

Evidence for modulated immune response to Anaplasma phagocytophila sensu lato in cats with FIV-induced immunosuppression

Human granulocytic ehrlichiosis (HGE) is an emerging infectious disease in which some patients experience unusual opportunistic infections. In this study, cats infected with the HGE agent, Anaplasma phagocytophila s.l., had clinical granulocytic ehrlichiosis (GE), anti-nuclear antibodies and increased IFN-γ mRNA. In FIV-immunosuppressed cats with GE, there was upregulated IL-10 transcription but not IFN-γ. Cats with FIV had poor response to vaccines, regardless of GE status. This preliminary report demonstrates that cats with FIV-infection make a good model of ehrlichiosis in an immunocompromised host, and that viral immunosuppression may not increase the severity of ehrlichiosis but may attenuate immune responses to the pathogen.

Seroprevalence of antibodies that react with Anaplasma phagocytophila, the agent of human granulocytic ehrlichiosis, in different populations in Westchester County, New York.

We determined the frequencies of antibodies to Anaplasma phagocytophila, the agent of human granulocytic ehrlichiosis (HGE), in different groups of adults and children from Westchester County, New York. The groups included 159 adult blood donors and 215 children who were seronegative for Borrelia burgdorferi antibodies, 118 adult patients and 57 children who were seropositive for B. burgdorferi antibodies, and 42 adult patients with culture-confirmed erythema migrans. Eighteen (11.3%) of the blood donors and 11 (5.1%) of the B. burgdorferi-seronegative children were found to have A. phagocytophila antibodies by indirect immunofluorescent-antibody assay (IFA). Nine of 42 (21.4%) patients with culture-confirmed erythema migrans tested at the baseline visit, 42 of 118 (35.6%) adults, and 3 of 57 (5.3%) children whose sera were reactive for B. burgdorferi antibodies also tested positive for A. phagocytophila antibodies. The geometric mean titer ranged from 219 to 315 for all groups, and the differences in titers among the groups were not statistically significant. Only one-third of the healthy blood donors reactive by IFA were confirmed to be positive by immunoblotting. We conclude that a significant proportion of adults and children without clinical evidence of HGE will test positive for A. phagocytophila antibodies when the conventional cutoff titer of 80 is used in the IFA. This information must be considered in interpretation of test results.

Serologic Evidence of Human Granulocytic Ehrlichiosis, Greece

To the Editor: Human granulocytic ehrlichiosis (HGE), a tickborne infectious disease, was first described in 1994 (1). Several cases have been reported in the United States; reports of acute cases in Europe have been rare, although European serosurveys of the prevalence of antibodies to the HGE agent have been conducted (2–4). No similar serosurvey has been conducted in Greece, although Ixodes ricinus, thought to be the principal tick vector in Europe (5), is present in northern Greece (6). Lyme disease, which is transmitted by the same tick, has never been reported, and the seroprevalence of Lyme borreliosis in Greece is very low (7). We examined sera of 300 persons (100 men and 200 women) ages 15–78 years (mean age ± standard deviation 52.7±18.0 years), which were collected at six county hospitals in northern Greece and sent to our laboratory from April to October 2000. The participants were mostly farmers, all of whom lived in rural areas of northern Greece. All participants were healthy and had been hospitalized for routine blood tests. Each patient completed a questionnaire about medical history. The selected patients had no known history of rickettsiosis and reported no febrile or influenza-like illness during the past 6 months. Each participant provided oral consent for the serum to be used for detecting antibodies against several infectious agents related to zoonoses. The following information was recorded for each participant: age, sex, occupation, and area of residence. Serum samples were tested by indirect immunofluorescence (IFA) with commercially available antigen (Focus Technologies, Cypress, California), which uses HGE-1–infected HL60 cells. Titers >64 were considered positive. All sera were also tested for Rickettsia conorii, R. typhi, Coxiella burneti, and Ehrlichia chaffeensis by IFA and for Borrelia burgdorferi by enzyme-linked immunosorbent assay and Western blot. Sera that reacted positively to more than one of these agents were excluded. Biostatistical analysis was performed by using the statistical package SPSS for Windows 10.0.1 (Standard version, SPSS Inc., Chicago, IL). The overall prevalence of antibodies to the HGE agent was 7.3% (8.0% for men and 7.0% for women). No statistically significant differences were observed in the prevalence of antibodies in the six prefecture hospitals. Participants had no statistically significant differences in sex or age. Antibody titers to HGE were low (of 22 positive sera, 12 had titers >64 and 10 had titers >128). Several serosurveys of the prevalence of antibodies to the HGE agent have been conducted across Europe in both healthy persons and patients with suspected or confirmed Lyme borreliosis (2,3,8). Since cases of B. burgdorferi infection are rare or nonexistent in Greece and the seroprevalence of Lyme borreliosis is very low, we selected as participants 300 healthy farmers who lived in rural areas. These persons compose a group at high risk for exposure to tick bites and therefore to I. ricinus. Our prevalence is higher than those observed in Bulgaria (2.9%) and Germany (1.9%) (2,3). This finding could be attributed to the fact that the prevalence in these countries was based on blood donors, unlike our survey. However, our prevalence is substantially lower than that in Slovenia, where 15.4% of the examined population had detectable antibodies to the HGE agent and several cases of HE have been confirmed (4). Our observation that no significant differences occurred in the prevalence of antibodies to the HGE agent in the six prefectures studied could be explained by the fact that these districts are small, with little variation in environmental and climatic conditions. Even though the antibody titers to the HGE agent were low in our survey, they suggest infection at an undetermined time (9). Seven of our sera were antibody positive to both the HGE agent and at least one other rickettsial agent or B. burgdorferi. This fact, which has been observed elsewhere (9), may result from coinfection or crossreaction. These sera were excluded. Our data suggest the possibility that HGE cases exist in Greece. Since such cases have been not been reported to date, they are likely underdiagnosed. Further research is needed to clarify the presence of the HGE agent in Greece.

Coinfection of Ixodes ricinus (Acari: Ixodidae) in northern Poland with the agents of Lyme borreliosis (LB) and human granulocytic ehrlichiosis (HGE)

Adult Ixodes ricinus ticks were collected from Pomerania province, northern Poland, to determine the presence of infection with agents of human granulocytic ehrlichiosis (HGE) and Lyme borreliosis by using the polymerase chain reaction (PCR). Of the 424 ticks 19.2% and 11.6% contained ehrlichiae and spirochetes, respectively. Frequency of single infection with the HGE agent was 63/424 while frequency of single infection with Borrelia burgdorferi sensu lato was 28/424. As many as 21/424 ticks (5%) contained both pathogens. This finding supports suggestions that both HGE agent and B. burgdorferi s. 1. perpetuate in the same foci and frequently co-infect the same tick vector thereby increasing the risk of humans acquiring mixed infection.

Reassessment of a midwestern Lyme disease focus for Borrelia burgdorferi and the human granulocytic ehrlichiosis agent.

Previous studies from the late 1980s defined the risk of human Lyme disease by determining the prevalence of Borrelia burgdorferi infection in Ixodes scapularis ticks and Peromyscus sp. mice captured from areas around La Crosse, Wis. High percentages of B. burgdorferi-infected I. scapularis ticks and P. leucopus mice were common in areas located north of Interstate 90 but were not detected in areas south of this major east-west thoroughfare. In this study, we reevaluated the extent of B. burgdorferi infection. High percentages of mice captured from sites north of the interstate were still infected with B. burgdorferi. In addition, B. burgdorferi was recovered from 12 (67%) of 18 mice captured from a site well south of the highway. However, none of 104 mice or 713 I. scapularis ticks captured from the study sites were infected with Ehrlichia spp. The results confirmed the continued high risk for humans to contract infection with B. burgdorferi and the significant southward expansion of the area in which Lyme disease is endemic. In contrast, the risk of acquiring human granulocytic ehrlichiosis remains minimal despite the abundance of appropriate vector ticks and reservoir rodents.

HGE antibodies in sera of patients with TBE in the Czech Republic

Altogether 112 sera from 67 patients hospitalised for tick-borne encephalitis (TBE) were tested for the presence of antibodies against human granulocytic ehrlichiosis (HGE). Indirect immunofluorescence assay and western blotting were used for the screening and confirmation respectively. A laboratory culture of HGE agent in HL-60 cells served as an antigen supply. Commercially available human sera were used as positive controls; the respective IgG and IgM cut-offs of 1/80 and 1/40 were applied. Six patients (8.9%) exhibited specific IgG antibodies against HGE at or above the cut-off level, while no serum sample was shown to be IgM-reactive at the cut-off. The mean age of the subset of positive patients was 42 (± 16.7) years which exceeded that of the total TBE set averaging 37.5 (± 17.3) years. One patient, a woman aged 32, exhibited an elevated HGE titre of 1/80 on admission 15 days after TBE onset, that still slightly increased up to 1/160 by the end of the following week. Corresponding WB-pattern showed significant MSP, p30, and p28 bands. These findings suggest a case of concurrent TBE with HGE. Overall, this study demonstrates that TBE/HGE co-infections could be encountered in central Europe.

Seroprevalence of human granulocytic ehrlichiosis infection in Belgium.

In order to determine the prevalence of human granulocytic ehrlichiosis (HGE) in Belgium, the sera of 216 patients previously diagnosed with Borrelia burgdorferi infection were analysed for possible coinfection with the agent of HGE. For this purpose, an indirect immunofluorescence assay was applied, and positive results were confirmed by Western blot using a 44-kilodalton recombinant protein (rP44) specific for the agent of HGE. Sixteen of the 216 (7.4%) sera tested were positive for the HGE agent using indirect immunofluorescence assay, and seven (3%) of them were confirmed positive by Western blot. These data suggest the agent for HGE is present in Belgium and may cause coinfection in patients infected with Borrelia burgdorferi, as has been reported in the USA and elsewhere in Europe. This is the first report documenting the identification of this agent in Belgium.

Novel potential reservoirs for Borrelia sp. and the agent of human granulocytic ehrlichiosis in Colorado.

Previous work demonstrated that Ixodes spinipalpis ticks maintained an enzootic cycle of Borrelia bissettii and the agent of human granulocytic ehrlichiosis (aoHGE) within woodrats (Neotoma mexicana) and deer mice (Peromyscus maniculatus) in northern Colorado (USA). Because I. spinipalpis is the only known vector of B. bissettii and aoHGE in Colorado, this study was designed to determine the reservoir status of other hosts of I. spinipalpis in five distinct ecological zones along the front range and foothills of Colorado. One hundred and twelve rodents of nine species were examined and 11 (10%) were polymerase chain reaction (PCR) positive for aoHGE; 37 (33%) were culture positive for B. bissettii, and five (4%) were coinfected with both organisms based on PCR and culture. Of these, three chipmunk species (Tamias minimus, T. quadrivittatus, and T. umbrinus) were culture positive for B. bissettii, with a single T. minimus coinfected with B. bissettii and aoHGE. In addition, one golden-mantled ground squirrel (Spermophilus lateralis) was positive for both B. bissettii and aoHGE. This is the first report of a golden-mantled ground squirrel harboring either B. bissettii or aoHGE and the initial observation that chipmunks may be a reservoir for B. bissettii in Colorado.

Antibodies to granulocytic ehrlichiae in cattle from Connecticut.

Enzyme-linked immunosorbent assays (ELISA) with a purified recombinant 44-kDa protein and indirect fluorescent antibody (IFA) staining methods incorporating whole-cell antigens of the human granulocytic ehrlichiosis (HGE) agent were used to detect antibodies to Ehrlichia phagocytophila genogroup organisms in cattle sera. The cattle lived in tick-infested areas of Connecticut, USA and were healthy at the times blood samples were collected in 1990, 1999 and 2000. Of the 339 serum samples analysed, 40 (12%) and 15 (4%) were positive by ELISA and IFA, respectively. Western immunoblots of a subset of sera verified antibody reactivity of six serum samples, positive by ELISA with titres of 640-2,560, to a protein with a molecular mass of c. 44 kDa. Although seroprevalence rates were low, cattle were exposed to the HGE agent at different sites and should be monitored for anaemia, leukopenia or thrombocytopenia, especially if there is evidence of unexplained decreased milk production. Different serological testing methods should be used to detect immunoglobulins.

Characterization and transcriptional analysis of gene clusters for a type IV secretion machinery in human granulocytic and monocytic ehrlichiosis agents.

Anaplasma (Ehrlichia) phagocytophila and Ehrlichia chaffeensis, the etiologic agents of granulocytic and monocytic ehrlichioses, respectively, are obligatory intracellular bacteria that cause febrile systemic illness in humans. We identified and characterized clusters of genes for a type IV secretion machinery in these two bacteria, and analyzed their gene expression in cell culture and mammalian hosts. Eight virB and virD genes were found in each bacterial genome, and all of the genes were transcribed in cell culture. Although the gene order and orientation were similar to those found in other bacteria, the eight virB and virD genes were clustered at two separate loci in each genome. Five of the genes (virB8, virB9, virB10, virB11, and virD4) were located downstream from a ribA gene. These five genes in both A. phagocytophila and E. chaffeensis were polycistronically transcribed and controlled through at least two tandem promoters located upstream of the virB8 gene in human leukemia cell lines. The virB9 gene of A. phagocytophila was transcriptionally active in peripheral blood leukocytes from human ehrlichiosis patients and experimentally infected animals. Three of the remaining genes (virB3, virB4, and virB6) of both A. phagocytophila and E. chaffeensis were arranged downstream from a sodB gene and cotranscribed with the sodB gene through one or more sodB promoters in human leukocytes. This suggests that transcription of the three virB genes in these two Anaplasma and Ehrlichia spp. is regulated by factors that influence the sodB gene expression. This unique regulation of gene expression for the type IV secretion system may be associated with intracellular survival and replication of Anaplasma and Ehrlichia spp. in granulocytes or monocytes.

Kinetics of CD11b/CD18 Up-Regulation During Infection with the Agent of Human Granulocytic Ehrlichiosis in Mice

The agent of human granulocytic ehrlichiosis (aoHGE) is a tick-borne, obligate intracellular, granulocytotropic bacterium able to infect numerous host species. Given its unique niche and the leukopenia often noted with infection, we investigated the effect of acute aoHGE infection on neutrophil activation by evaluating surface expression of the β2 integrin CD11b/CD18 in a mouse model using FACS analysis. Infection resulted in neutrophil activation with up-regulation of CD11b/CD18 in multiple strains of mice, however, hematologic analysis showed no apparent role for CD11b/CD18 in mediating peripheral leukopenia. Because IFN-γ is an important cytokine during granulocytic ehrlichiosis and is known to activate leukocytes, we investigated the potential role of IFN-γ in CD11b/CD18 up-regulation. Neutrophils from IFN-γ knock-out mice became activated during aoHGE infection, however, the kinetics of activation differed from wild-type mice. In addition, activation correlated directly with the presence of bacteria because neutrophils with large intracytoplasmic morula also expressed higher levels of CD11b/CD18. CD11b/CD18 seemed to be critical to early bacterial clearance and killing in vivo because infection of mice with targeted genetic disruption of CD11b/CD18 resulted in an initial increase in bacterial burden compared with wild-type mice. Similarly, in vitro culture of neutrophils from infected CD11b/CD18 knock-out mice resulted in a marked increase in bacterial proliferation compared with congenic controls. The data support crucial roles of CD11b/CD18 and IFN-γ–mediated cell activation as mechanisms that limit bacterial replication.

Prevalence of the agent of human granulocytic ehrlichiosis in Ixodes scapularis (Acari: Ixodidae) in the coastal southeastern United States.

Human granulocytic ehrlichiosis (HGE) is an emerging tick-borne disease recently recognized in the United States. The blacklegged tick, Ixodes scapularis Say, is the principle vector in the eastern United States. The disease has been commonly reported in the northeastern and upper midwestern states; however, suitable vectors and reservoir hosts exist in the southeast. To assay the prevalence of the HGE agent in vector ticks, we screened 818 individual I. scapularis from 15 locations in South Carolina, Georgia, and Florida using nested polymerase chain reaction, which targets the HGE agent 16S rRNA gene. Prevalence among locations ranged from 0 to 20%. The overall average prevalence of 15 sites was 1.6% (n = 818). Verification by sequencing the 16S rDNA from the positive samples showed 99.8-100% nucleotide identities with the sequences of the HGE agent in GenBank. These results were supported by the phylogenetic analysis using 16S rDNA sequences.

Effects of Anaplasma phagocytophila on NADPH oxidase components in human neutrophils and HL-60 cells.

The human granulocytic ehrlichiosis agent, Anaplasma phagocytophila, resides and multiplies exclusively in cytoplasmic vacuoles of granulocytes. A. phagocytophila rapidly inhibits the superoxide anion (O(2)(-)) generation by human neutrophils in response to various stimuli. To determine the inhibitory mechanism, the influence of A. phagocytophila on protein levels and localization of components of the NADPH oxidase were examined. A. phagocytophila decreased levels of p22(phox), but not gp91(phox), p47(phox), p67(phox), or P40(phox) reactive with each component-specific antibody in human peripheral blood neutrophils and HL-60 cells. Double immunofluorescence labeling revealed that p47(phox), p67(phox), Rac2, and p22(phox) did not colocalize with A. phagocytophila inclusions in neutrophils or HL-60 cells, and p22(phox) levels were also reduced. A. phagocytophila did not prevent either membrane translocation of cytoplasmic p47(phox) and p67(phox) or phosphorylation of p47(phox) upon stimulation by phorbol myristate acetate. The inhibitory signals for O(2)(-) generation was independent of several signals required for A. phagocytophila internalization. These results suggest that rapid alteration in p22(phox) induced by binding of A. phagocytophila to neutrophils is involved in the inhibition of O(2)(-) generation. Absence of colocalization of NADPH oxidase components with the inclusion further protects A. phagocytophila from oxidative damage.

Transcript heterogeneity of the p44 multigene family in a human granulocytic ehrlichiosis agent transmitted by ticks.

Human granulocytic ehrlichiosis (HGE) is an emerging tick-borne zoonosis caused by a strain of Anaplasma phagocytophila called the HGE agent, an obligatory intracellular bacterium. The agent expresses immunodominant 44-kDa outer membrane proteins (P44s) encoded by a multigene family. The present study established an experimental process for transmission of the HGE agent from infected mice (a reservoir model) to nymphal Ixodes scapularis ticks (a biological vector) and subsequently to horses (a patient model) by the adult infected ticks. Overall, a total of 20 different p44 transcripts were detected in the mammals, ticks, and cell cultures. Among them, a transcript from a p44-18 gene was major at acute stage in mice and horses but minor in ticks. Both mRNA and protein produced from the p44-18 gene were detected in the HGE agent cultivated in HL-60 cells at 37 degrees C, but their expression levels decreased in the organisms cultivated at 24 degrees C, suggesting that temperature is one of the factors that influence the expression of members of the p44 multigene family. Several additional p44 transcripts that were not detected in the mammals at the acute stage of infection were detected in ticks. Phylogenetic analysis of the 20 different p44 transcripts revealed that the major transcripts found in mammals and ticks were distinct, suggesting a difference in surface properties between populations of the HGE agent in different host environments. The present study provides new information for understanding the role of the p44 multigene family in transmission of the HGE agent between mammals and ticks.

The prevalence of Borrelia burgdorfieri (Spirochaetales: spirochaetaceae) and the agent of human granulocytic ehrlichiosis (Rickettsiaceae: Ehrlichieae) in Ixodes scapularis (Acari:Ixodidae) collected during 1998 and 1999 from Minnesota.

We tested 103 adult Ixodes scapularis Say from 12 counties in Minnesota for the presence of Borrelia burgdorferi and the causative agent of human granulocytic ehrlichiosis (HGE), using polymerase chain reaction (PCR). A total of 17 ticks (16.5%) was positive for B. burgdoiferi using nested PCR for the flagellin gene. or both PCR for the ospA gene and nested PCR for the flagellin gene. A total of four ticks (3.8%) was positive for the agent of HGE using nested PCR for 16S rDNA. Counties in Minnesota with established and recently reported populations of I. scapularis both had ticks infected with B. burgdorferi. The agent of HGE was only detected in counties with established I. scapularis populations.

Serologic evidence of infection with granulocytic ehrlichiae in black bears in Pennsylvania.

Serum samples from 381 black bears (Ursus americanus) killed in Pennsylvania (USA) on 24 November 1997 were analyzed for antibodies reactive to the agent of human granulocytic ehrlichiosis (HGE; Ehrlichia sp.) by indirect immunofluorescence assay. Antibody reactivity to HGE antigen was detected in 21% (81/381) of the samples collected. Reactive samples were reported from 56% (14/25) of the counties where bear samples were collected. Endpoint antibody titer ranged from 1:8 to 1:16, 192, with a geometric mean titer of 1:582. There was no significant difference in antibody prevalence between male and female bears (P < 0.01). However, adult bears were significantly more likely to have reactive antibodies than juvenile bears (P < 0.01). Attempts to amplify and detect granulocytic ehrlichial DNA from corresponding bear blood clots (n = 181) through nested polymerase chain reaction assays were unsuccessful. Further studies are needed for identification of the pathogen-responsible for induction of HGE-reactive. This is the first description of antibodies reactive to the HGE agent in black bears and suggests these mammals are infected with the agent of HGE or an antigenically related ehrlichial species.

EXPERIMENTAL INFECTION OF DUSKY-FOOTED WOOD RATS (NEOTOMA FUSCIPES)WITH EHRLICHIA PHAGOCYTOPHILASENSU LATO

Dusky-footed wood rats, Neotoma fuscipes, have been implicated in the natural maintenance of Ehrlichia phagocytophila sensu lato, the agent of human granulocytic ehrlichiosis (HGE), in northern California based on high seroprevalence and amplification of E. phagocytophila s.l. DNA from wood rat blood. In order to further assess granulocytic ehrlichiosis in wood rats, we examined wild-caught wood rats for infection and then performed experimental intra-peritoneal infections with E. phagocytophila s.l. in horse or wood rat blood, and tested animals for 120 days by polymerase chain reaction (PCR) and serology. Of 15 wood rats collected from northern California, three were antibody and PCR-positive for E. phagocytophila s.l. at the time of capture. The naturally infected wood rats remained PCR-positive for a mean of 52 days (+/- 7 SD). Experimental i.p. passage of E. phagocytophila s.l. in wood rat blood was successful in three of four wood rats and the mean duration of PCR-positivity was 75 days (+/- 21.2 SD). Experimental infection with E. phagocytophila s.l. in horse blood succeeded in all four of the recipients and the mean duration of PCR-positivity of 81 days (+/- 17.5 SD). No infected individual appeared to be ill based on feeding behavior, activity, and hydration status. These data confirm that wood rats are susceptible to E. phagocytophila s.l., may develop prolonged infection without clinical ehrlichiosis, and may play a role in maintaining E. phagocytophila s.l. in nature.

The serological response of patients infected with the agent of human granulocytic ehrlichiosis.

To characterize the serological response in humans to human granulocytic ehrlichiosis (HGE), we prospectively observed 152 patients for as long as 42 months. HGE was confirmed by detection of morulae in blood smears, polymerase chain reaction, blood culture, or a combination of these tests for 94 patients (62.3%), and 92 (97.8%) of the patients had specific serum antibodies thereafter. One hundred twenty-six (99.2%) of 127 patients tested at 1 month were seropositive (89 of 127 patients had seroconversion), and 150 (98.7%) of the 152 patients had become seropositive by 6 months. Eleven patients (7.3%) remained seropositive at 42 months. Neither antibiotic therapy initiated during the first week of illness nor preexisting immunosuppressive conditions abrogated a serological response. Indirect fluorescent antibody testing of acute-phase and convalescent-phase serum samples is a sensitive tool for laboratory confirmation of HGE.