Goodman J L, Nelson C, Vitale B, Madigan J E, Dumler J S, Kurtti T J, Munderloh U G
Department of Medicine, University of Minnesota School of Medicine, Minneapolis, USA.
N Engl J Med. 1996 Jan 25;334(4):209-15. doi: 10.1056/NEJM199601253340401.
Human granulocytic ehrlichiosis is a potentially fatal tick-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 diagnose because the symptoms are non-specific, intraleukocytic inclusions (morulae) may not be seen, and the serologic results are often initially negative. Little is known about the causative agent because it has never been cultivated.
We studied three patients with symptoms and laboratory findings suggestive of human granulocytic ehrlichiosis, including unexplained fever after probable exposure to ticks, granulocytopenia, and thrombocytopenia. Peripheral blood was examined for ehrlichia microscopically and with use of the polymerase chain reaction (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.
Blood from the three 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 immunofluorescence microscopy, PCR analysis, and DNA sequencing. DNA from the infected cells was sequenced in regions of the 16S ribosomal gene reported to differ between the agent of human granulocytic ehrlichiosis and closely related species, including Ehrlichia equi and E. phagocytophila which cause infection in animals. The sequences from all three human isolates were identical and differed from the strain of E. equi studied in having guanine rather than adenine at nucleotide 84.
We describe the cultivation of the agent of human granulocytic ehrlichiosis in cell culture. The ability to isolate this organism should lead to a better understanding of the biology, treatment, and epidemiology of this emerging infection.
人粒细胞埃立克体病是一种最近被描述的潜在致命的蜱传感染。这种急性发热性疾病的特征为肌痛、头痛、血小板减少和血清转氨酶水平升高。该疾病难以诊断，因为其症状不具特异性，可能看不到白细胞内包涵体（桑葚体），而且血清学结果最初往往为阴性。由于病原体从未被培养过，所以对其了解甚少。
我们研究了三名有症状且实验室检查结果提示人粒细胞埃立克体病的患者，这些症状和检查结果包括在可能接触蜱后出现无法解释的发热、粒细胞减少和血小板减少。对外周血进行显微镜检查及聚合酶链反应（PCR）检测埃立克体。将血液接种到HL60细胞（一种人早幼粒细胞白血病细胞系）培养物中，并通过吉姆萨染色和PCR监测培养物是否感染。
三名患者的血液，其中只有一名患者的中性粒细胞中有提示埃立克体的包涵体，PCR检测显示人粒细胞埃立克体病呈阳性。三名患者的血液均接种到HL60细胞培养物中并引发感染，接种后最早5天可见细胞内微生物，12至14天内发生细胞裂解。通过免疫荧光显微镜检查、PCR分析和DNA测序确认了培养微生物的身份。对感染细胞的DNA在据报道人粒细胞埃立克体病原体与密切相关物种（包括引起动物感染的马埃立克体和嗜吞噬细胞埃立克体）之间存在差异的16S核糖体基因区域进行测序。所有三个人类分离株的序列相同，与所研究的马埃立克体菌株的区别在于核苷酸84处为鸟嘌呤而非腺嘌呤。
我们描述了在细胞培养中培养人粒细胞埃立克体病原体的方法。分离这种微生物的能力应有助于更好地了解这种新出现感染的生物学特性、治疗方法和流行病学。
