Table 2. Overview of laboratory methods available to diagnose HME and HGA and their advantages, disadvantages and application/use [26,27].

Diagnostic method	Advantages	Disadvantages	Application / use
Microscopy of blood smear or buffy coat preparation	Widely available	Limited sensitivity (demands expertise, depends on density of morulae); highest sensitivity during acute phase/first week of infection; limited specificity (does not allow conclusive species differentiation)	Used in the acute phase of infection when PCR is not available
PCR	High specificity (allows species differentiation); high sensitivity in the acute phase of infection; also suitable for biopsy/ autopsy samples	Decreased sensitivity beyond the acute phase/first week of infection and after administration of appropriate antibiotics	Used for diagnosis in the acute phase of infection; availability often limited to larger/reference laboratories
Serology (IFA, ELISA)	Enables retrospective diagnosis beyond the acute phase of infection	Not useful in acute phase of infection (due to delayed seroconversion); confirmation of diagnosis demands paired samples (acute and convalescent serum); limited specificity due to persisting antibodies after infection and cross-reactivity of assays with other rickettsial pathogens; decreased sensitivity after early administration of appropriate antibiotics	Paired serology by IFA is the serological gold standard but the result will only be available retrospectively; used for epidemiological studies
Immunostaining of biopsy / autopsy tissue	High specificity; can also be applied to biopsy/ autopsy samples	Availability limited to larger/reference laboratories; decreased sensitivity after administration of appropriate antibiotics	Useful for confirming the diagnosis in fatal cases where diagnostic levels of antibodies did not develop before death
Cell culture	High specificity	Low sensitivity; time and resource demanding; decreased sensitivity after administration of appropriate antibiotics	Diagnostic reference standard, but availability largely restricted to reference and research laboratories

IFA, immunofluorescence assay; ELISA, enzyme-linked immunosorbent assay, PCR, polymerase chain reaction.