Comparison of PCR Assays for Detection of the Agent of Human Granulocytic Ehrlichiosis, Anaplasma phagocytophilum

Abstract

Human granulocytic ehrlichiosis is an emerging infectious disease in the United States and Europe, and PCR methods have been shown to be effective for the diagnosis of acute infections. Numerous PCR assays and primer sets have been reported in the literature. The analytical sensitivities (limits of detection) of 13 published PCR primer sets were compared using DNA extracted from serial dilutions of Anaplasma phagocytophilum-infected HL-60 cells. The specificity of the assays that were able to detect <2.5 infected cells was tested by the use of template DNA extracted from Ehrlichia chaffeensis, Rickettsia rickettsii, and Bartonella henselae. The assays with the lowest limits of detection were shown to be a nested assay that amplifies the 16S rRNA gene (primer pairs ge3a-ge10 [primary] and ge9-ge3 [nested]; detects 0.25 infected cell), a direct assay that amplifies the major surface protein gene msp2 (primer pair msp2-3f-msp2-3r; detects 0.25 infected cell), and a direct assay that amplifies the 16S rRNA gene (primer pair ehr521-ehr790; detects 0.25 infected cell). The specificity and limit of detection of the MSP2 and 16S rRNA direct assays were further tested by use of A. phagocytophilum template DNA from both North America and Europe and from human, tick, white-footed mouse, equine, deer, bovine, and wood rat samples and of template DNA from closely related species (Anaplasma marginale, the white-tailed deer agent, and additional E. chaffeensis-positive samples). Three manufacturers' PCR kits were tested and showed distinct variations in the limit of detection, specificity, and nonspecific background amplification. The importance of these results for the molecular diagnosis of human granulocytic ehrlichiosis is discussed.Human ehrlichiosis is an acute, febrile illness that is caused by at least three known agents in the United States. Ehrlichia chaffeensis infects mononuclear white blood cells, while Ehrlichia ewingii and Anaplasma phagocytophilum (previously Ehrlichia phagocytophila, the human granulocytic ehrlichiosis [HGE] agent) infect granulocytic-lineage leukocytes (2, 6-9, 11). HGE was first described in 1994, and the number of confirmed cases has increased to the point where it is now the predominant form of ehrlichiosis (3,7,20; J. McQuiston, personal communication) and second to Lyme disease as the most common tick-borne infection in the United States. Tetracyclines are efficacious for the treatment of all forms of ehrlichiosis; however, the nonspecific symptoms of infection can make diagnosis problematic. Serological methods are most commonly used for diagnosis, but these assays are often negative in the early stages of an acute infection, as antibody will typically be absent during the first week of infection (1). Diagnosis prior to seroconversion has utilized molecular methods that are based on PCR amplification of the DNA of the agent, and numerous PCR-based assays and primer sets have been reported (7,13,16,19,(24)(25)(26)(27)29). The purpose of this study was to compare a selection of these ass...

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Coexistence of tick-borne pathogens in game animals and ticks in western Poland

“…Then, in order to sequence the obtained amplicons, the nested PCR with pairs of primers HS1, HS6 and HS43, HS45 complementary to sequences of the heat shock operon groESL of A. phagocytophilum was additionally applied ( Table 1). The composition of reaction mixture for PCR and the reaction conditions were the same as described by Massung and Slater (2003).…”