Correlation between genotypes of Orientia tsutsugamushi and clinical characteristics of patients with scrub typhus in Guangzhou,China

Objective:To explore the correlation between genotypes of Orientia(O.)tsutsugamushi and clinical characteristics of scrub typhus patients.Methods:Clinical data of patients with scrub typhus admitted to different types of medical institutions in Guangzhou from September 2012 to December 2016 were collected using medical records.Demographic data,clinical manifestations,as well as hematological and biochemical indicators of patients infected with different genotypes were analyzed and compared.Results:A total of 192 patients were included in this study,including 121 patients with Karp genotype of O.tsutsugamushi infection(63.0%),36 patients with Gilliam genotype(19.0%),23 patients with Kato genotype(12.0%),and 12 patients with TA763 genotype(6.0%)infection.The median value of albumin in patients with Karp genotype infection was significantly lower than that of Gilliam-infected patients(P=0.032).Patients with Karp genotype infection had a significantly longer hospital stay(9 days)than those with Gilliam genotype(7 days)(P=0.009)and Kato genotype infection(6 days)(P=0.005).Karp-infected patients also represented for the largest number of patients with complicated organ involvement(88/133,66.2%).Furthermore,Karp-infected patients had higher risk of developing multiple organ dysfunction syndrome(18.2%)and requiring intensive care unit treatment(15.9%).Besides,patients with Gilliam genotype(8 days)and TA763 genotype infection(7.5 days)had shorter fever duration than those with Karp genotype(9 days)and Kato genotype(9 days)infection,respectively.Conclusions:Genotypes of Orientia tsutsugamushi were associated with varying clinical manifestations,organ involvement,and treatment outcomes,suggesting that genotypes ranged in virulence.

Comparison of a rapid diagnostic test and microimmunofluorescence assay for detecting antibody to Orientia tsutsugamushi in scrub typhus patients in China

Objective:To evaluate the detection of IgM and IgG antibodies to Orientia tsutsugamushi(O. tsutsugamushi) by rapid diagnostic test(RDT) and microimmunofluorescence assay(ml FA). Methods:RDT using a mixture of recombinant 56-kDa proteins of O.tsutsugamushi and mIFA assay were performed on 20 patients from Fujian and 13 patients from Yunnan Province,and 82 sera samples from healthy farmers in Anhui Province and Beijing City in 2009.Comparison of the RDT and mIFA assay was performed by using X test and the P level of 【0.05 was considered to be significance.Results:Among these 82 normal sera samples,the specificity of RDT was 100%for both IgM and IgG tests.In 33 samples from patients with scrub typhus,5 cases were positively detected earlier by RDT than by mIFA in IgM test,and 2 cases were positive in IgG test.Sensitivities of RDT were 93.9%and 90.9%for IgM and IgG,respectively.The sensitivity of combination lest of IgM and IgG was 100%.Geometric mean titer diluted sera from confirmed cases by IFA and RDT assay were 1:37 vs.1:113(P【0.001) in IgM test and 1:99 vs.1:279 (P【0.05) in IgG test.Conclusions:RDT is more sensitivite than mIFA in the early diagnosis of scrub typhus and it is particularly applicable in rural areas.

Prediction of T cell and B cell epitopes of the 22-, 47-, 56-, and 58-kDa proteins of Orientia tsutsugamushi

Objective:To predict B cell and T cell epitopes of 22-kDa,47-kDa,56-kDa and 58-kDa proteins.Methods:The sequences of 22-kDa,47-kDa,56-kDa and 58-kDa proteins which were derived from Orientia tsutsugamushi were analyzed by SOPMA,DNAstar,Bcepred,ABCpred,NetMHC,NetMHCⅡand IEDB.The 58-kDa tertiary structure model was built by MODELLER9.17.Results:The 22-kDa B-cell epitopes were located at positions 194-200,20-26 and 143-154,whereas the T-cell epitopes were located at positions 154-174,95-107,17-25 and 57-65.The 47-kD a protein B-cell epitopes were at positions 413-434,150-161 and 283-322,whereas the T-cell epitopes were located at positions 129-147,259-267,412-420 and 80-88.The 56-kDa protein B-cell epitopes were at positions 167-173,410-419 and 101-108,whereas the T-cell epitopes were located at positions 88-104,429-439,232-240 and 194-202.The 58-kDa protein B-cell epitopes were at positions 312-317,540-548 and 35-55,whereas the T-cell epitopes were located at positions 415-434,66-84 and 214-230.Conclusions:We identified candidate epitopes of 22-kDa,47-kDa,56-kDa and 58-kDa proteins from Orientia tsutsugamushi.In the case of 58-kDa,the dominant antigen is displayed on tertiary structure by homology modeling.Our findings will help target additional recombinant antigens with strong specificity,high sensitivity,and stable expression and will aid in their isolation and purification.

Utility of next-generation sequencing for the etiological diagnosis of Orientia tsutsugamushi infection

Background Scrub typhus,an acute febrile disease caused by Orientia tsutsugamushi,is transmitted to humans through infected chigger mites.We present a case of scrub typhus in a previously healthy man from Shandong Province diagnosed using next-generation sequencing(NGS)and PCR and review recent literature on NGS for scrub typhus diagnosis.Methods NGS was utilized for testing whole blood collected on admission.Confirmatory testing was done by detecting IgM and IgG antibodies to Orientia in acute and convalescent sera by ELISA.Orientia 47-kDa protein gene TaqMan and standard PCR of the 56-kDa protein gene and Sanger sequencing were performed on eschar scab DNA.Results The NGS diagnosis was confirmed by 47-kDa protein gene TaqMan and sequencing of a fragment of the O.tsutsugamushi 56-kDa protein gene from the eschar scab.Analysis of this sequence and the NGS data indicated O.tsutsugamushi strain Cheeloo2020 is a novel genotype.Mapping of the NGS data against the O.tsutsugamushi Gilliam strain genome sequence identified 304 reads with high similarity.Conclusions NGS is not only useful for multiplex diagnosis of scrub typhus,but also provides insight into the genetic diversity of O.tsutsugamushi.The common failure to submit sequences to databases makes it difficult to determine the minimal quantity and quality of NGS data being used for the positive identification of Orientia DNA in clinical specimens.

Genotype identification of Orientia tsutsugamushi isolated from Nan Peng Lie Islands in China

OBJECTIVE: To identify genotype of eight strains of Orientia tsutsugamushi (O. tsutsugamushi) isolated from Nan Peng Lie Islands in China and establish tsutsugamushi disease nature foci for this region. METHODS: The nested polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) were used. Three primers were selected from the DNA sequence of the gene encoding type-specific 56-kDa protein of the Karp strain. The positive products were digested by Hine II and Pst I, meanwhile profiles specific to each strain were analyzed. RESULTS: Three genotypes of O. tsutsugamushi including Karp, Kato and a new strain existed on Nan Peng Lie Islands. CONCLUSION: Nan Peng Lie Islands is tsutsugamushi disease nature foci.

Longitudinally extensive transverse myelitis associated with scrub typhus:A case report

Rationale:Longitudinally extensive transverse myelitis(LETM)is a rare inflammatory demyelinating disease predominantly associated with autoimmune and inflammatory disorders.Scrub typhus can cause a variety of neurological complications,which may result in poor recovery and long-term disability.This report reveals a rare linkage between LETM and scrub typhus.Patient concerns:A 24-year-old female presented with a history of fever for 10 days associated with progressive quadriparesis.Physical examination revealed bilateral lower limb weakness(grade 0/5)and bilateral upper limb weakness(grade 2/5)with reduced sensation below the T3 dermatome.Diagnosis:Scrub typhus presenting with longitudinally extensive transverse myelitis.Interventions:Steroid pulse therapy with antibiotics supported by early intensive rehabilitation.Outcomes:Complete recovery was achieved.Lessons:This case report highlights the importance of considering scrub typhus as a potential cause of LETM in endemic regions,particularly in patients presenting with febrile illness and neurological symptoms.

Omadacycline in the treatment of scrub typhus:Three case reports

BACKGROUND Scrub typhus is a naturally occurring acute infectious disease that is primarily transmitted through the bites of chiggers or larval mites infected by Orientia tsutsugamushi(O.tsutsugamushi).Omadacycline,a novel tetracycline,exhibits potent antibacterial efficacy against both typical bacteria and atypical pathogens.However,omadacycline application in the treatment of scrub typhus remains limited.CASE SUMMARY In the present work,we report several cases of scrub typhus,with the main clinical symptoms being fever,the formation of eschars or ulcers,local or systemic lymphadenopathy,headache,myalgia and rash.Blood samples were collected before omadacycline was administered,and O.tsutsugamushi infection was confirmed through targeted next-generation sequencing(tNGS).After two days of treatment,the patients’symptoms,including fever,were alleviated,with no adverse drug reactions.CONCLUSION tNGS is an effective method for diagnosing scrub typhus.Omadacycline can be considered an alternative option for antiinfective therapy in patients with O.tsutsugamushi infections.

恙虫病临床诊疗专家共识

恙虫病是由恙虫病东方体感染引起的一种自然疫源性疾病,流行于东亚和东南亚地区,主要表现为发热、焦痂或溃疡、淋巴结肿大及皮疹。恙虫病早期症状不典型,容易误诊和漏诊。中国微生物学会人兽共患病病原学专业委员会和中国医药生物技术协会生物诊断技术分会相关专家,根据领域相关研究进展和诊疗经验,并参考国内外相关文献与指南,结合我国临床实践与咨询论证,形成本共识。

恙虫病东方体混合重组抗原应用于胶体金免疫层析试纸条的制备及效价检测

胶体金免疫层析试纸条具有携带方便、操作简单且敏感性、特异性高等特点,近年来开始广泛应用于疾病的诊断。为了实现对恙虫病的快速检测,笔者利用双抗原夹心法,以胶体金标记恙虫病东方体56 kDa混合重组蛋白,56 kDa混合重组蛋白和兔抗恙虫病东方体多克隆抗体分别喷在检测线及质控线上,制备了用于检测恙虫病东方体抗体的胶体金免疫层析试纸条。本研究制备的胶体金试纸条检测限为61 ng/mL,和疟疾、伤寒、血吸虫、出血热以及斑点热等阳性血清无交叉反应,特异性达90%(54/60),检测的敏感性为91.96%(103/112),在4℃下可稳定保存6个月。本试纸条能够应用于人体血清中恙虫病东方体总抗体的检测。

成都市4种鼠传病原体的带毒率调查

目的 了解四川省成都市鼠类携带4种常见病原体的情况,为鼠传疾病的防控提供科学依据。方法 2021年9月至2022年8月采用夹夜法在成都市居民区、重点行业和农贸市场等生境捕获鼠类。采用实时荧光定量PCR法检测汉坦病毒、新型布尼亚病毒、钩端螺旋体和恙虫病东方体4种病原体。结果 全市7个区县共捕获鼠类207只,总捕获率为1%。其中褐家鼠150只,占72.46%;黄胸鼠57只,占27.54%。4种病原体检出率分别为汉坦病毒3.86%,钩端螺旋体1.45%,新型布尼亚病毒0.48%,恙虫病东方体尚未检测出。结论 需对重点行业开展灭鼠工作,防止鼠传疾病的发生与流行。

A review of the epidemic and clinical study on scrub typhus in China(2010-2020)

Scrub typhus is an acute febrile vector-borne zoonotic disease caused by the obligate intracellular growth bacterium Orientia tsutsugamushi(Ot).Mites are the primary vectors and rodents play a pivotal role in the transmission of scrub typhus.Due to the climate warming,increased human activity and other factors,cases of scrub typhus have been increased sharply during the past decade in China,especially in the northern China.To understand the incidence trend,epidemic pattern,clinical sign,diagnosis and therapy of this diseases as well as genotype evolution of Orientia tsutsugamushi,we summarized and analyzed the current knowledge of scrub typhus in China from 2010 to 2020.The data indicated that the dominate genotypes of scrub typhus in China were Karp,Kato and Gilliam.Although the disease was distributed national wide,Yunnan,Guangzhou and Fujian showed the highest incidence rate.The main vector of scrub typhus in southwest,middle east and southeast of China appeared a geographic preference respectively.Seasonal timing,age and occupation were the key factors that relate to the peak incidence of scrub typhus.Notably,farmer was the occupation with the highest risk of Ot infection.Further study on the epidemic characteristics,risk factors,diagnosis and treatments of scrub typhus will be of benefit to a comprehensive guideline for prevention and control of this ancient disease.

中国恙虫病流行及临床研究进展(2010-2020)

恙虫病是由恙虫病东方体(Orientia tsutsugamushi,Ot)感染引起的急性发热性疾病。主要通过恙螨幼虫叮咬传播,啮齿类动物在恙虫病传播过程中发挥重要作用。随着气候变化和人类活动范围的扩大等原因,我国恙虫病感染病例数和流行区域呈明显增加和北扩趋势。本文基于文献总结分析了2010~2020年我国各地区恙虫病发病情况、流行病学特征、临床研究等进展。结果表明Kato、Karp、Gilliam型为我国恙虫病东方体主要流行基因型;恙虫病在我国各地区均有分布,其中云南、广州、福建等地是高发区;传播媒介种类呈明显地理差异;本病发病高峰期与季节温度、人群年龄、职业等相关。对我国恙虫病流行趋势变化、人群感染风险、环境影响等因素的研究,将为优化媒介防控措施、完善临床诊治方案、提高突发疫情的应急处置能力及精准防控策略的制定提供科学的指导。

安徽三界地区啮齿动物感染恙虫病东方体调查

目的调查安徽三界地区啮齿动物自然感染恙虫病东方体分离株,并确定其基因型别。方法流行季节在野外用鼠笼捕鼠并鉴定鼠种、携螨率;巢式PCR对鼠脏器进行恙虫病东方体56 kD基因片段检测;将鼠脏器接种小白鼠进行病原体分离,PCR扩增分离株Ot 56 kD基因片段,并将目的片段进行测序,基因序列同源性及进化分析。结果捕获啮齿动物数及其携螨率,黑线姬鼠60%(15/25);褐家鼠45.5%(5/11);东方田鼠和鼩鼱各2只,均阴性。40只野鼠脏器有3只恙虫病东方体PCR扩增阳性,阳性率为7.5%;从黑线姬鼠标本中分离到1株恙虫病东方体Sanjie,56 kD基因片段测定该株与台湾TT0711a和NT0511b分离株同源性最高(98%),系统发生树分析也显示与Kawasaki型共处于同一分支。结论安徽三界地区存在鼠类恙虫病东方体自然感染,黑线姬鼠为其主要宿主,当地恙虫病东方体属Kawasaki基因型。

应用快速免疫层析法检测恙虫病特异性IgM、IgG及总抗体

目的 建立快速免疫层析法检测恙虫病特异性IgM、IgG及总抗体 ,并对其敏感性和特异性进行评价。 方法 以胶体金标记纯化的基因工程重组抗原 ,在玻璃纤维上预包被金标记的纯化抗原 (Au -Ag)和鼠IgG ,在硝酸纤维素膜上检测线处包被羊抗人 μ链、羊抗人IgG或重组抗原 ;在对照线处包被羊抗鼠IgG ,组装成检测试纸。在试纸条的玻璃纤维上加待检血清 ,再滴加 2滴反应液。观察金标抗原释放后NC膜上检测线和对照线的反应情况 ,检测线和对照线同时出现红色条带判为阳性 ,仅对照线出现红色条带判为阴性 ,对照线未出现条带为无效试验。结果 间接免疫荧光法IFA检测IgG和金标免疫层析法检测IgM、IgG、总抗体的敏感性分别为 88 1% ,92 3% ,90 9%和 98 6 % ;特异性分别为 96 0 % ,94 7% ,94 7%和93 3%。结论 金标免疫层析法可替代传统方法用于检测恙虫病东方体特异性抗体。

恙虫病的免疫机制研究进展

恙虫病(scrub typhus)是经恙螨叮咬感染恙虫病东方体(Orientia tsutsugamushi)引起的一种自然疫源性疾病,在日本、东亚、澳大利亚北部、太平洋地区西部和西南部、印度洋地区广泛分布,发热、皮疹、焦痂、淋巴结肿大为其主要临床表现。恙虫病东方体主要感染人的内皮细胞,也可感染树突细胞、巨噬细胞、分叶核白细胞、淋巴细胞,并在细胞内专性寄生。现对恙虫病东方体致病机理的研究进展进行简要综述,以期为恙虫病的治疗提供新的思路。

实时荧光定量PCR检测恙虫病东方体

目的建立检测恙虫病东方体的实时荧光定量PCR(quantitative real-ti me PCR)方法。方法根据恙虫病东方体56kD外膜蛋白基因序列设计引物和探针,以克隆的56kD基因片段作DNA模板,建立实时荧光定量PCR检测方法。结果建立的荧光定量PCR标准曲线的循环阈值(Ct)与模板拷贝数呈良好的线性关系(r=0.999)。荧光定量PCR检测恙虫病东方体的灵敏度约为套式PCR的100倍,并且具有良好的重复性。用该定量PCR检测其它相关立克次体和病原菌DNA样本,检出结果均为0。用该定量PCR检测恙虫病东方体实验感染小鼠的血、脾脏、肺脏、肝脏标本,结果脾脏中东方体出现最早和检出量最多,肝脏和肺脏次之。血中的恙虫病东方体量较低。结论本研究建立的荧光定量PCR方法具有很高的特异性和敏感性,可用于东方体感染早期血样本的快速检测作恙虫病感染早期诊断,并且可以定量分析评价恙虫病东方体感染的程度。

小盾纤恙螨经卵传递病原体研究

目的 探索快速取得大量小盾纤恙螨未食子代幼虫的方法 ,以适应研究恙螨经卵传递病原体的需要。方法 将小黑板放置在肾综合征出血热 (HFRS)及恙虫病疫区草地上 ,10min后取回计算螨数并鉴定螨种。该法可收集到大量小盾纤恙螨未食子代幼虫。 2 0～ 3 0只幼虫为 1批叮刺 1只小白鼠乳鼠以分离汉坦病毒 (HV)或叮刺 1只小白鼠以分离恙虫病东方体 (Ot)。结果 2 4批恙螨幼虫叮刺乳鼠 ,分离到 5株HV ;6只小白鼠被恙螨幼虫叮刺 ,分离到 2株Ot。试验中所用幼虫未曾叮刺过 ,但鼠被这些幼虫叮刺后能感染上HV和Ot ,表明这些螨所带病原体系由母代螨经卵传递而来。结论 小黑板可采集到疫区草地上大量小盾纤恙螨未食幼虫 ,为研究恙螨经卵传递病原体提供了一种快速、简便、经济的方法。研究结果证明小盾纤恙螨可经卵传递HV和Ot,可作为HFRS和恙虫病的传播媒介。

45年恙螨与媒介恙螨传播恙虫病的基础研究

报告作者等 45年来恙螨以及媒介恙螨传播恙虫病基础研究结果 ,主要包括 :①恙螨调查研究 :种类的发现 ,恙虫病东方体的分离和检测 ,媒介恙螨孳生地及其恙虫病流行 ;②恙螨生活史和实验生态研究 :恙螨培育成功与生活史的实验 ,恙螨实验生态和恙虫病流行的预测预报 ;③恙螨传病机制及其控制恙虫病流行研究 :恙虫病东方体阳性恙螨模型的建立及其东方体在恙螨体内的动态 ,恙螨体内东方体垂直传递和传病潜力 ,以及防灭恙螨与控制恙虫病的流行。

山东省泰安市鼠类自然感染恙虫病东方体调查

目的了解山东省泰安地区鼠类自然感染恙虫病东方体(Ot)的情况。方法应用巢式PCR对山东省泰安地区采集的鼠类标本Ot-Sta56kDa基因片段进行检测,对阳性标本进行序列分析。结果在泰安地区秋冬季共捕获鼠类68只,阳性率为5.88%;不同鼠种间阳性率差异无统计学意义(Fisherχ2=4.714,P=0.062)。其中3只鼠胚胎标本均未检出Ot。序列同源性分析显示,2只鼠脾标本(08P,53P)的核苷酸序列与Sdu-1型的核苷酸序列同源性最高,为99.77%;其次,与Taguchi和Oishi型的同源性均为95.98%,与Kanda和Kawasaki型的同源性均为95.74%。而另2只鼠类标本(55P,56P)的核苷酸序列与TA686型的同源性最高,分别为86.27%和77.11%。系统发生树分析也显示,08P和53P与Sdu-1型在同一分支,它们又与Kawasaki、Kanda、Taguchi和Oishi型共处于同一分支;而55P和56P与TA686型在同一分支。结论山东省泰安地区鼠类存在Ot自然感染。该地区Ot的基因型较为复杂,不仅存在与临沂地区相同的Ot型,该型与日本的Kawasaki型相似;同时还存在与泰国的TA686型相似的Ot型。

基于GroEL基因的实时荧光定量PCR检测恙虫病东方体方法的建立及评价

目的建立一种敏感、特异的实时荧光定量PCR方法,用于检测临床标本中的恙虫病东方体。方法根据恙虫病东方体GroEL蛋白基因序列设计引物和探针,建立实时荧光定量PCR检测方法。并从灵敏度、特异度、重复性及临床标本的检测能力等方面对本方法进行综合评价。结果建立的实时荧光定量PCR方法具有良好的特异性,可检测出恙虫病东方体Gilliam、Karp、Kato和Kawasaki株。建立的标准曲线循环阈值(Ct)与模板拷贝数呈现良好的线性关系(r=0.99),灵敏度分析显示样品最低检出浓度为21.8拷贝/μL。批内及批间重复性实验变异系数均小于1.5%,显示本方法具有良好的重复性。对本实验室保存的82份临床标本进行检测,并与常规巢式PCR方法比较,本方法检测出26份阳性标本中的25份,检出率为96.15%。结论本研究建立的实时荧光定量PCR方法具有较高的敏感性、特异性和稳定性,可用于临床病人及暴发疫情的实验室快速检测。