河北新发斑点热及人粒细胞无形体病实验室调查分析

Molecular characteristics of emerging spotted fever group Rickettsiae and Anaplasma phagocytophilum in Hebei province, China

目的对河北省新发蜱传斑点热群立克次体及人粒细胞无形体进行分子流行病学特征分析。方法对2009-2012年在河北省辛集、迁安及定州市收集的101份临床可疑立克次体病病例急性期血液DNA样本,采用2套巢氏PCR法分别扩增斑点热群立克次体热休克蛋白基因(groEL)和人粒细胞无形体16S rRNA基因并测序,序列采用NCBI网站进行Blast分析后,选择不同地区、不同宿主来源相应基因,使用DNAStar Meg Align同源分析。结果 10.9%(11/101)的病例groEL扩增阳性,同源分析当地斑点热群立克次体groEL基因分为2个序列型,尽管有限片段无法区别立克次体种,但PCR阳性病例血清与我国常见黑龙江立克次体、西伯利亚立克次体、海南斑点热群立克次体及蚤传斑点热立克次体无抗原抗体反应,提示可能为新发斑点热。人粒细胞无形体16S rRNA基因扩增阳性率为8.9%(9/101),且病例急性期血清人粒细胞无形体Ig M抗体全部阳性,其中3例急性期与恢复期血清发生Ig G抗体转换。16S rRNA基因测序成功的9个序列(341 bp)100%同源,并且与该地区无形体病例分离株CZ-HGA-2 100%同源。结论河北地区可能存在新发蜱传人粒细胞无形体病例及新发斑点热。加强立克次体实验室诊断及鉴别诊断、进一步开展病原学、相关媒介及宿主流行病学调查均具有重要的临床及公共卫生意义。

Objective To better understand the molecular epidemiological characteristics of emerging spotted fever group Rickettsiae and Anaplasma phagocytophilum identified in Hebei province. Methods One hundred and one clinical probable cases of typhus were collected from Xinji city, Qianan city and Dingzhou city, Hebei province during 2009-2012 and 101 acute stage and 17 convalescence blood samples were collected. Sera separated from blood were used for detecting the IgM and IgG antibodies against Rickettsiae prowazekii, R. typhi, R. felis, Bartonella henselae, Orientia tsutsugamushi, R. heilongjiangensis, R. sibirica, Ehrlichia chaffeensis, Coxiella burnetii, Chinese spotted fever group rickettsia Hainan-1 and Chinese A. phagocytophilum strain CZ-HGA-2 and LZ-HGA-3, respectively. Two sets of nested PCR, which targeting the A. phagocytophilum 16S rRNA gene and spotted fever group rickettsiae groEL gene respectively, were conducted using the acute stage blood DNAs of patients as temples. Sequences were analyzed by DNAStar MegAlign. Results PCR positive rates were 10.9%（ 11/101 ） for amplifying groEL gene and two genetic groups of spotted fever group rickettsiae were identified. Although the sequence analysis failed to differentia the emerging spotted fever group rickettsiae from traditional ones because of limited PCR fragments, the sera from the patients with positive PCR did not reactive with the R. heilong^iangensis, R. sibirica, R. felis, and Chinese spotted fever group ricketsia Hainan-1, which indicated that the emerging spotted fever group rickettsiae might be prevalence in these areas. PCR positive rates of A. phagocytophilum were 8.9% （9/101）and all of the 9 patients with positive PCR were found to be positive of the IgM antibodies against A. phagocytophilum and 3 patients had IgG seroconversation （4-fold increase of IgG antibody titer）. The 9 sequences of the 16S rRNA genes ofA. phagocytophilum were not only 100% identity with each other but 100% identity with the local A. phagocytophilum strain CZ-HGA-2. Conclusion Emerging tick-borne rickettsiae were highly prevalence in Hebei province and further etiological investigations were urgently needed.