DIRECT CULTIVATION OF THE CAUSATIVE AGENT OF HUMAN GRANULOCYTIC EHRLICHIOSIS

Background. Human granulocytic ehrlichiosis is a potentially fatal tic k-borne infection that has recently been described, This acute febrile illness is characterized by myalgias, headache, thrombocytopenia, and elevated serum aminotransferase levels, The disease is difficult to d iagnose because the symptoms are nonspecific, intraleukocytic inclusio ns (morulae) may not be seen, and the serologic results are often init ially negative, Little is known about the causative agent because it h as never been cultivated. Methods. We studied three patients with symp toms and laboratory findings suggestive of human granulocytic ehrlichi osis, including unexplained fever after probable exposure to ticks, gr anulocytopenia, and thrombocytopenia. Peripheral blood was examined fo r ehrlichia microscopically and with use of the polymerase chain react ion (PCR), Blood was inoculated into cultures of HL60 cells (a line of human promyelocytic leukemia cells), and the cultures were monitored for infection by Giemsa staining and PCR. Results. Blood from the thre e patients, only one of whom had inclusions suggestive of ehrlichia in neutrophils, was positive for human granulocytic ehrlichiosis on PCR, Blood from all three patients was inoculated into HL60 cell cultures and caused infection, with intracellular organisms visualized as early as 5 days after inoculation and cell lysis occurring within 12 to 14 days, The identity of the cultured organisms was confirmed by immunofl uorescence microscopy, PCR analysis, and DNA sequencing, DNA from the infected cells was sequenced in regions of the 16S ribosomal gene repo rted to differ between the agent of human granulocytic ehrlichiosis an d closely related species, including Ehrlichia equi and E. phagocytoph ila, which cause infection in animals. The sequences from all three hu man isolates were identical and differed from the strain of E. equi st udied in having guanine rather than adenine at nucleotide 84. Conclusi ons. We describe the cultivation of the agent of human granulocytic eh rlichiosis in cell culture, The ability to isolate this organism shoul d lead to a better understanding of the biology, treatment, and epidem iology of this emerging infection. (C) 1996, Massachusetts Medical Soc iety.