云南大理地区恙虫病血清学诊断及病原体基因序列分析

目的 对云南大理地区恙虫病做血清学诊断及病原体基因序列分析。方法 采用间接免疫荧光方法(IFA)检测不明原因发热患者血清中的恙虫病东方体(Ot) IgG抗体,用巢式PCR(nPCR)法扩增患者血液中的Ot groEL与sta56基因并对扩增基因做序列分析。结果 19例不明原因发热病例中恙虫病患者8例,其中血清Karp型4例、Kato型3例、Karp和Kato混合型1例;3例确认恙虫病的血样本扩增到groEL基因片段, 其序列同源性为100%,基因序列做系统进化树显示该Ot与Karp和Kato株在同一分支上。从Karp和Kato混合血清型病例血样本扩得sta56基因片断,序列分析显示该Ot与台湾分离株在同一分支上。结论 这些云南大理的恙虫病患者由恙虫病东方体Karp或Kato血清型感染;个别患者可能Karp+Kato混合血清型感染,Karp+Kato混合血清型菌株可能在遗传进化上与某些台湾Ot株关系密切。

Serotyping and sequence analysis of Oreintia tsutsugamushi from patients with fever of unknown origin in Dali, Yunnan

To understand serotype and molecular character of Oreintia tsutsugamushi (Ot) in Dali of Yunnan Province, 19 blood samples from patients with fever of unknown origin were collected and sera were tested for antibody (Ab) of Ot with IFA. Partial groEL and sta56 segment of Ot were amplified separately by nested-polymerase chain reaction (nPCR) and sequenced. The homology with other known sequences was analyzed. The 4 out of 19 sera were positive for Ab of Ot Karp serotype, 3 for Ab of Ot Kato serotype and 1 for Ab of Ot Karp + Kato serotype. Ot groEL fragments were amplified successfully by nPCR in 3 cases of the 19 patients (detection rate, 15.79%), and their homology was all 100% through comparing groEL gene sequence. Phylogenetic analysis showed that the Ot Karp, AH-GD-OT-S6, ZJ-TT-OT-O21, UT332, and Kato strains were in the same branch. Of them, 481-507 bp for Ot sta56 fragments and 230 bp for DL1 specimen were amplified successfully by nPCR separately. DL1 and some Taiwan Ot strains such as KM05, HL04, TW45, KM17-2, and KM18 showed higher nucleotide homology, reaching up to 100%. These result indicated that in Dali of Yunnan, there were patients of Karp and Kato serotype tsutsugamushi disease. Also, there may be patient with Karp + Kato serotype, and the Ot is closely related to some Taiwan strains of Ot in genetic evolution relationship.