Table 3. Diagnostic grading system to judge the certainty of the correct diagnosis of HME [26,28,29].

Diagnostic method	Description	Grade of diagnostic certainty	Case classification (provided illness clinically compatible with ehrlichiosis)	Comment
PCR	Detection of Ehrlichia spp. DNA in a clinical specimen via amplification of a specific target by polymerase chain reaction (PCR) assay	A+	Direct evidence, confirmed diagnosis	High level of evidence, especially in the first week of illness and before start of antibiotics, mostly done from whole blood specimens, also possible in solid tissue and bone marrow specimens
Culture	Isolation of Ehrlichia spp. from a clinical specimen in cell culture	A+	Direct evidence, confirmed diagnosis	High level of evidence, especially in the first week of illness and before start of antibiotics, difficult to carry out, time demanding
Immunostaining of biopsy/autopsy tissue	Demonstration of ehrlichial antigen in a biopsy/autopsy sample by immunohistochemical methods	A+	Direct evidence, confirmed diagnosis	High level of evidence, difficult to carry out, time demanding
Serology—IgG IFA, paired samples	Serological evidence of a fourfold rise in IgG-specific antibody titer to E. chaffeensis/E. canis antigens by indirect immunofluorescence assay (IFA) in paired serum samples (i.e. an acute phase sample [first week of infection] and a convalescent phase sample [2–4 weeks later])	A	Indirect evidence, confirmed diagnosis	High level of evidence, serological gold standard, cross-reaction with other rickettsial diseases possible
Blood smear or buffy coat preparation microscopy	Identification of intracellular morulae in monocytes (E. ewingii: granulocytes) by microscopic examination	B+	Direct evidence, probable diagnosis	Intermediate level of evidence, easy to carry out, examiner-dependent, likelihood of detection depends on level of Ehrlichia in blood; limited specificity as morulae of E. ewingii cannot by differentiated from morulae of A. phagozytophilum which also show a tropism for granulocytes
Serology—IgG IFA single sample or ELISA	Serological evidence of elevated IgG antibody reactive with E. chaffeensis/E. canis antigen by ELISA or IFA (CDC uses an IFA IgG cutoff of ≥1:64)	B	Indirect evidence, probable diagnosis	Intermediate level of evidence, no certain differentiation between acute and past infection possible, cross-reaction with other rickettsial diseases possible
Serology—IgM ELISA or IFA	Serological evidence of elevated IgM antibody reactive with E. chaffeensis/E. canis antigen by IFA, ELISA, or assays in other formats	C	Indirect evidence, possible diagnosis	Low level of evidence, IgM tests are not always specific and not useful as single means of diagnosis
Clinical diagnosis	Signs and symptoms compatible with ehrlichiosis	D	Clinical diagnosis	The lowest level of evidence for correct diagnosis

CDC, Centers for Disease Control and Prevention; DNA, deoxyribonucleic acid; ELISA, enzyme-linked immunosorbent assay; IFA, immunofluorescence assay; IgG, immunoglobulin G; IgM, immunoglobulin M; PCR, polymerase chain reaction.