首次实验室证实北京平谷地区恙虫病东方体暴发流行

目的了解北京市平谷地区是否存在恙虫病东方体感染。方法采用巢式聚合酶链反应（nPCR）,对平谷地区采集的30份临床标本进行恙虫病东方体热休克蛋白（groEL）基因和56×103蛋白扩增并测序分析。采用间接免疫荧光试验（IFA）对血清标本恙虫病东方体特异抗体检测。结果采用PCR共检测血液标本30份,均阳性（100%）;用IFA检测28份患者血清抗体,25份阳性（89.3%）;长片段PCR扩增,3份标本扩增阳性,阳性结果测序比对所有核苷酸序列相同,与Kawasaki的同源性为96%。结论首次从分子流行病学和血清流行病学的角度证实北京平谷地区存在恙虫病东方体。     

Orientia tsutsugamushi in eschars from scrub typhus patients

To verify the value of eschars for the diagnosis of scrub typhus and to characterize genotypes of Orientia tsutsugamushi in patients, we examined eschars and blood specimens of 7 patients from Shandong Province, People's Republic of China, for O. tsutsugamushi by polymerase chain reaction targeting the Sta56 gene. All 7 eschars and acute-phase blood samples were positive, while no specific DNA amplicons were obtained from the 7 convalescent-phase blood samples collected after antimicrobial drug therapy. The findings indicate that patients' eschars can be used for detection and genetic characterization of O. tsutsugamushi during the convalescent phase.     

套式-聚合酶链反应在山东省恙虫病检测中的应用

目的 :利用分子生物学手段对山东省部分地区的人和鼠进行调查 ,了解恙虫病在山东省的感染情况。方法 :采用套式 -聚合酶链反应 (nested- PCR)技术 ,对在山东省 6个市 (县、区 )捕获的野鼠或家鼠的肝、脾、肾组织标本 ,以及临床诊断为恙虫病的病人血标本进行检测。结果 :共检测鼠脏器标本 198份 ,阳性 8份 ,阳性率为 4 .2 4 % ;共检测临床诊断恙虫病病人血标本 14份 ,阳性 7份 ,阳性率为 5 0 %。结论 :首次在山东省黄河以北地区捕获的鼠体内检测到恙虫病东方体 ,其中两只鼠为褐家鼠 ,表明山东省广泛存在恙虫病的自然疫源地     

Serological and blood culture investigations of Nepalese fever patients

Serological testing of paired (i.e. admission and convalescent) sera from 103 fever patients in Kathmandu, Nepal, was performed to estimate the prevalence rates of scrub typhus, murine typhus, Leptospira and dengue virus antibodies and to determine their role in the cause of active infections. Blood cultures from 15 patients grew Salmonella enterica serovar Typhi, 8 grew S. Paratyphi A and 6 grew other bacteria. Diagnostic antibody levels were detected against murine typhus (27/103; 26%), scrub typhus (23/103; 22%), Leptospira (10/103; 10%) and dengue virus (8/103; 8%). Nineteen patients (18%) had diagnostically raised antibodies to more than one infectious agent. Seven S. Typhi (7/15; 47%) and two S. Paratyphi A (2/8; 25%) patients had significant scrub typhus, murine typhus, Leptospira or dengue virus IgM antibody titres. This study confirms the presence of leptospiral, rickettsial and dengue infections in Kathmandu as well as evidence for mixed infections with S. Typhi and Orientia tsutsugamushi or Rickettsia typhi. These infections should be kept in mind when considering the differential diagnoses of fever and empirical treatment options in Nepal. Many patients demonstrated static IgM antibody results between paired serum collections, suggesting recent rather than acutely active infections.     

用多聚酶链反应技术检测实验感染恙虫病立克次体的恙螨

作者首次采用多聚酶链反应技术检测感染恙虫病立克次体的媒介恙螨，包括成虫的幼虫。证实ＰＣＲ是一可用于恙虫病流行病学调查的新的敏感方法。     

Simple, rapid and sensitive detection of Orientia tsutsugamushi by loop-isothermal DNA amplification

We present a loop-mediated isothermal PCR assay (LAMP) targeting the groEL gene, which encodes the 60kDa heat shock protein of Orientia tsutsugamushi. Evaluation included testing of 63 samples of contemporary in vitro isolates, buffy coats and whole blood samples from patients with fever. Detection limits for LAMP were assessed by serial dilutions and quantitation by real-time PCR assay based on the same target gene: three copies/microl for linearized plasmids, 26 copies/microl for VERO cell culture isolates, 14 copies/microl for full blood samples and 41 copies/microl for clinical buffy coats. Based on a limited sample number, the LAMP assay is comparable in sensitivity with conventional nested PCR (56kDa gene), with limits of detection well below the range of known admission bacterial loads of patients with scrub typhus. This inexpensive method requires no sophisticated equipment or sample preparation, and may prove useful as a diagnostic assay in financially poor settings; however, it requires further prospective validation in the field setting.     

建立染料法荧光定量PCR检测恙虫病东方体

目的建立染料法荧光定量PCR检测恙虫病东方体。方法根据恙虫病东方体56kD外膜蛋白基因序列设计特异性引物,建立染料法(SYBR green I)实时荧光定量PCR,评估其灵敏性及特异性;并对恙虫病东方体鸡胚培养物、东方体感染小白鼠脏器标本以及恙虫病病人血样等多种样本进行检测。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(r2=0.99),灵敏性评估显示20μl体系单PCR反应管靶基因大于28个拷贝即可被有效检测,最低检出浓度为2拷贝/μl,比普通PCR检测方法提高1~2个数量级,并且具有较好的重复性;建立的SYBR I染料法具有良好的特异性,与立氏立克次体R株等其他5种立克次体,以及被检测的其他几种病原菌DNA模板均不发生特异性扩增;用建立的染料法对东方体鸡胚培养物、东方体感染动物脏器,以及采集的现场恙虫病病人血样共59份标本进行检测,其中57份检出阳性。用该定量PCR检测恙虫病东方体实验感染小鼠的血、脾脏、肾脏、肝脏标本,结果脾脏中东方体检出量最多,肝脏和肾脏次之,血中的恙虫病东方体量较低。结论本研究建立荧光定量PCR方法均具有很高的特异性和敏感性,适合各种样本中东方体的快速检测,可用于实验室的快速诊断。     

New Orientia tsutsugamushi strain from scrub typhus in Australia.

In a recent case of scrub typhus in Australia, Orientia tsutsugamushi isolated from the patient''s blood was tested by sequence analysis of the 16S rDNA gene. The sequence showed a strain of O. tsutsugamushi that was quite different from the classic Karp, Kato, and Gilliam strains. The new strain has been designated Litchfield.     

江苏省靖江市2011-2015年恙虫病流行特征及影响因素

目的 分析江苏省靖江市恙虫病的临床特点、流行病学特征及影响因素,为防治工作提供依据。方法 对2011-2015年靖江市恙虫病病例进行个案调查,结合聚合酶链式反应（polymerase chain reaction,PCR）核酸检测、酶联免疫法IgG抗体检测和储存宿主、传播媒介调查等,采用描述性分析、χ²检验等方法进行统计分析。结果 2011-2015年共报告恙虫病962例,年均发病率为28.80/10万,西片乡镇高发,发病时间集中于10~11月,农民占85.45%,男女性别比为1：1.35,84.89%的病例发病前3周有田间作业史。急性期病人恙虫病东方体核酸阳性率为72.70%,一般人群恙虫病东方体IgG抗体阳性率为9.64%。流行区鼠密度为10.97%,鼠型动物主要为黄胸鼠和臭鼩,螨种为小盾纤恙螨。结论 靖江市恙虫病流行呈快速增长趋势,临床症状典型,疫情为秋冬型,建议加强监测,在秋收季节前对40岁以上农民开展知识宣教与行为干预。     

Epidemiological and serological study of scrub typhus among Chinese military in the Pescadores islands of Taiwan.

An outbreak of 69 cases of scrub typhus occurred among Chinese military personnel stationed in the Pescadores Islands, Taiwan Province, Republic of China between May and November 1975. A retrospective epidemiological study of this outbreak indicated that military personnel over 40 were more likely to have scrub typhus than those under 40. High risk groups included the Garrison Force (home guard), anti-aircraft gunners and infantry and armoured units stationed at Hsing-jen. The onset of symptoms in 69% occurred within one year of residence in the Pescadores. The clinical course of scrub typhus and the serological response to infection were also studied. Eschar formation, fever, headache chills and lymph node enlargement were the predominant clinical manifestations noted. The indirect immunofluorescent antibody test (IFAT) demonstrated diagnostic (four-fold) rises in antibody titres to Rickettsia tsutsugamushi reference strains in 36 of 41 paired sera tested. 11 of 19 patients from whom only single sera were obtained had IFA titres presumptive of scrub typhus (greater than or equal to 1:160). Of 19 patients experiencing possible primary infections, 13 (68%) responded with antibodies directed against more than one reference strain of R. tsutsugamushi. These results suggest that several antigenically diverse strains of R. tsutsugamushi may be active in the Pescadores.     

Real-time multiplex PCR assay for detection and differentiation of rickettsiae and orientiae

The high incidence of rickettsial diseases in Southeast Asia necessitates rapid and accurate diagnostic tools for a broad range of rickettsial agents, including Orientia tsutsugamushi (scrub typhus) and Rickettsia typhi (murine typhus), but also spotted fever group infections, which are increasingly reported. We present an SYBR-Green-based, real-time multiplex PCR assay for rapid identification and differentiation of scrub typhus group, typhus group and spotted fever group rickettsiae using 47kDa, gltA and ompB gene targets. Detection limits for amplification of these genes in reference strains ranged from 24 copies/microl, 5 copies/microl and 1 copy/microl in multiplex and 2 copies/microl, 1 copy/microl and 1 copy/microl in single template format, respectively. Differentiation by melt-curve analysis led to distinct melt temperatures for each group-specific amplicon. The assay was subjected to 54 samples, of which all cell-culture and 75% of characterised clinical buffy coat samples were correctly identified. Real-time PCR has the advantage of reliably detecting and differentiating rickettsial and orientia cell-culture isolates in a single-template assay, compared with the more time-consuming and laborious immunofluorescence assay. However, further optimisation and validation on samples taken directly from patients to assess its clinical diagnostic utility is required.     

Changing risk of scrub typhus in relation to socioeconomic development in the Pescadores islands of Taiwan.

Long term changes in risk of Rickettsia tsutsugamushi infection among civilian residents of the Pescadores Islands of Taiwan were associated with changing social conditions. Age specific incidence rates of scrub typhus in the Pescadores before 1940 were highest among children under 5 years of age. Rickettsiae can be demonstrated in vectors, wild animals continue to be infected and scrub typhus occurs in military personnel. A serologic survey for antibody to R. tsutsugamushi was conducted during 1975 and 1977 and failed to show evidence of previous infection among children. Two events appear to be associated with the decreased incidence of rickettsial infection in the young: increased urbanization and increased enrollment in schools. Both changes accompanied socioeconomic development which took place in the islands during the past 50 years. Prevalence of antibody to R. tsutsugamushi continues to be equal in the sexes and is undoubtedly due to similar occupational exposure of both sexes in fields and farms where vectors are numerous. The absence of apparent morbidity due to scrub typhus among the civilian populace was attributed to the mild nature of the disease caused by Pescadores strains, misdiagnosis and a lack of obligatory reporting.     

Scrub typhus, Republic of Palau

Scrub typhus, caused by Orientia tsutsugamushi, is a severe febrile illness transmitted to humans by trombiculid mites, which normally feed on rodents. The first known outbreak of scrub typhus in Palau occurred in 2001 to 2003 among residents of the remote southwest islands. To determine the extent of scrub typhus distribution in Palau, we tested serum samples from humans and rodents for antibodies to O. tsutsugamushi. Of 212 Palau residents surveyed in 2003, 101 (47.6%) had immunoglobulin G (IgG) antibody titers >1:64, and 56 (26.4%) had concurrent IgG and IgM antibody titers >1:512 and 1:64, respectively. Of 635 banked serum samples collected from Palau residents in 1995, 34 (5.4%) had IgG antibody titers >1:64. Sera collected from rodents (Rattus norvegicus and R. rattus) in 2003 and 2005 were tested, and 18 (28.6%) of 63 had IgG antibody titers >1:64. These findings suggest that scrub typhus is endemic in Palau.     

河南省首次恙虫病暴发疫情的实验室证实

目的调查以发热、头痛、淋巴结肿大、皮疹为主要症状的不明疾病病因。方法对可疑病人进行流行病学调查,血用血清学、免疫学、套式PCR方法检测病人血清抗体滴度、血清型及立克次体特异性基因片段。结果对采集到20例病人进行血清外裴OXk凝集反应,阳性率达100%,11份二次血清OXk效价呈4倍升高;恙虫立克次体IFA检测lgM和lgG抗体,20份均为阳性,最高达到1:2560;恙虫病血清学分型G illiam 4例,Karp 6例,Kato 8例。套式PCR检测病人血结果3份扩增出R t特异性DNA片段。结论首次证实了河南省恙虫病暴发疫情。     

实时荧光定量PCR检测普氏立克次体

目的 采用新型TaqMan—MGB探针建立检测普氏立克次体的实时荧光定量PCR方法。方法 根据普氏立克次体外膜蛋白B的基因（ompB）序列设计引物和探针，以克隆的ompB基因片段作DNA模板，在荧光定量PCR检测仪（ABI7900型）上建立实时荧光定量检测方法。结果 建立的定量标准曲线的循环阈值（Q）与模板拷贝数呈良好的线性关系（r=0．999）；与巢式PCR相比较，荧光定量PCR检测敏感性是其100倍。用荧光定量PCR检测莫氏立克次体及其他相关立克次体和细菌DNA，检出结果均为阴性。用荧光定量PCR检测普氏立克次体感染的豚鼠血标本，某些样本检测为阳性，而用巢式PCR检测的结果均为阴性。结论 研究中建立的检测普氏立克次体实时荧光定量PCR具有很高的特异性和敏感性，适合于快速检测样本中微量普氏立克次体DNA，可用作临床实验室快速确诊流行性斑疹伤寒。     

我国秋季型恙虫病地方特点及流行状况分析

恙虫病是由恙虫病东方体引起的自然疫源性疾病,根据其流行的季节特征大致分为夏季型、秋季型和冬季型3种类型,各种类型的恙虫病流行特征存在较大的差异。本文根据我国文献报道秋季型恙虫病疫情资料进行分析,归纳总结了我国秋季恙虫病的一些主要的流行特征,为制定秋季型恙虫病预防控制措施提供参考和依据。     

Endemic scrub typhus-like illness, Chile

We report a case of scrub typhus in a 54-year-old man who was bitten by several terrestrial leeches during a trip to Chiloé Island in southern Chile in 2006. A molecular sample, identified as related to Orientia tsutsugamushi based on the sequence of the16S rRNA gene, was obtained from a biopsy specimen of the eschar on the patient's leg. Serologic analysis showed immunoglobulin G conversion against O. tsutsugamushi whole cell antigen. This case and its associated molecular analyses suggest that an Orientia-like agent is present in the Western Hemisphere that can produce scrub typhus-like illness. The molecular analysis suggests that the infectious agent is closely related, although not identical, to members of the Orientia sp. from Asia.     

云南省保山市一起地方性斑疹伤寒暴发的调查

目的 了解云南省保山市一起地方性斑疹伤寒暴发的流行病学特征.方法 收集患者的流行病学资料.应用外斐反应和间接免疫荧光方法同时检测患者血清中莫氏立克次体、恙虫病东方体IgG抗体.用PCR检测鼠类脾脏标本中莫氏立克次体、普氏立克次体gltA基因,恙虫病东方体56kDa蛋白基因,斑点热群立克次体ompA基因,埃立克体16S rRNA基因和无形体16SrRNA基因.结果 2009年7-8月保山市隆阳区该起地方性斑疹伤寒累计发病58例,其中48例为临床诊断病例,10例为实验室诊断病例.3例地方性斑疹伤寒实验室诊断病例存在Karp型恙虫病东方体感染.PCR检测黄胸鼠脾脏85份,其中莫氏立克次体gltA片段阳性3份(阳性率为3.5%),其序列与莫氏立克次体Wilmington株(GenBank U59714.1)同源性为100%;普氏立克次体、恙虫病东方体、斑点热群立克次体、无形体和埃立克体均为阴性.结论 经流行病学、临床资料和实验室检测证实,保山市隆阳区该起疫情为地方性斑疹伤寒.从当地优势鼠种黄胸鼠中检测到莫氏立克次体核酸及序列,表明当地存在地方性斑疹伤寒疫源地.

Abstract：

Objective To understand the epidemiologic characteristics of endemic typhus in Baoshan city. Methods Epidemiological data were collected and characteristics were analyzed. IgG antibody(Ab) of Rickettsia mooseri and Orientia tsutsuganushi in serum of patients were tested using both Weil-Felix and IFA method. The Rickettsia mooseri gltA gene, Rickettsia prowazekii gltA gene,Orientia tsutsugamushi 56 kDa protein gene, SFGR ompA gene, Ehrlichia sp. 16S rRNA gene and Anaplasma sp. 16S rRNA gene in spleen of mice were examined by PCR. Results Fifty- eight endemic typhus cases were found in Longyang district of Baoshan city, during July to August, 2009.Among them, 48 cases were confirmed by clinical diagnosis and 10 cases by laboratory tests. The Ab of Orientia tsutsugamushi Karp serotype was detected in 3 cases from laboratory diagnosis. The spleen samples from 85 Rattns flavipectus were tested using PCR. Of them, 3 samples for Rickettsia mooseri gltA gene showed positive (positive rate was 3.5% ), and the homology of 3 Rickettsia mooseri and Rickettsia mooseri Wilmington strain (GenBank U59714.1) was 100% through comparing gene sequence. The results of PCR for detecting Rickettsia prowazekii, Orientia tsutsugamushi, SFGR,Anaplasma sp. and Ehrlichia. sp were all negative. Conclusion The outbreak of endemic typhus was confirmed in Longyang district of Baoshan city through epidemiological data, clinical diagnosis and laboratory tests. Rickettsia mooseri DNA was detected in the dominant Raw flavipectus, suggesting that endemic typhus did exist in the local areas.     

Scrub typhus in the Republic of Palau, Micronesia

In October 2001, an outbreak of febrile illness began in the southwest islands group of the Republic of Palau. Through October 2003, a total of 15 southwest islanders experienced fever >39.5 degrees C and abdominal distress, both lasting >7 days. Orientia tsutsugamushi, the agent of scrub typhus, was subsequently identified as the cause.     

山东费县秋冬型恙虫病临床流行病学与病原学研究

为了解山东地区姜虫病流行情况，对费县部分重疫区姜虫病患者进行了个案调查及病原学分离。结果表明，费县姜虫病具有典型的四大特征，即溃疡或焦痂９１．０１％（１７２／１８９）、皮疹９５．７７％（１８１／１８９）、发烧９８．９４％（１８７／１８９）、淋巴结肿大８４．６６％（１６０／１８９）；以农民为主，流行高峰在１０月中下旬。对恙虫病人分布的概率模式分析，表明当地恙虫病分布在时间、空间上均有聚集性。患者早期血清抗体阳性率７５．４２％。床边接种小鼠３２组，其中１２组分离到恙虫病立克次体（Ｒｔ）。小白鼠一般接种后５～１０天发病，解剖可见肝脾明显肿大。血清学鉴定，除部分毒株未定型外，其余均与Ｒｔ标准株Ｇｉｌｉａｍ型呈阳性反应，这与当地恙虫病人血清抗体型别一致。