山东省首例实验室诊断人单核细胞埃立克体病调查

目的 报告山东省首例人单核细胞埃立克体病的发现、诊治经过及其实验室检测.方法 对该疑似病例开展流行病学调查并填写个案调查表,采集其血标本,套式-PCR技术检测血液中嗜吞噬细胞无形体和查菲埃立克体特异性16S rRNA基因.结果 患者为不明原因发热,伴WBC和PLT减少.嗜吞噬细胞无形体特异性核酸检测阴性,查菲埃立克体特异性核酸检测阳性.PCR扩增阳性产物进行测序并与GenBank中注册的已知序列进行比较分析,显示与查菲埃立克体的同源性＞99％.结论 山东省存在查菲埃立克体感染病例,进一步开展自然疫源地调查十分必要.     

Identification of Ehrlichia chaffeensis,Anaplasma phagocytophilum,and A. bovis in Haemaphysalis longicornis and Ixodes persulcatus ticks from Korea

A total of 1,467 tick (1,463 of Haemaphysalis longicornis, three of Ixodes persulcatus and one of I. turdus) collected from nine provinces of Korea were examined by TaqMan real-time PCR for the presence of Ehrlichia and Anaplasma species. One set of primers and a probe were designed for detection of all of the Ehrlichia and Anaplasma species. Template DNAs (total 803) were prepared either from pools of larvae, nymphs, adult males and females, or from the salivary gland and midgut of adult ticks. Only DNAs positive in TaqMan PCR were examined for A. phagocytophilum with nested PCR and for E. chaffeensis with PCR. Four A. phagocytophilum 16S rRNA gene PCR products were sequenced for comparison with sequences previously reported. Amplification of a 16S rRNA gene fragment of Ehrlichia and Anaplasma species was observed in 364 tick DNAs (45.3% of the total). Of these 364 positive ticks, species-specific PCRs confirmed that 35 H. longicornis and one I. persulcatus were positive for A. phagocytophilum and one I. persulcatus was positive in E. chaffeensis. Except for one (AB-GGHL, GenBank accession number [GAN] AF470698), three of the four 16S rRNA gene fragment sequences of the A. phagocytophilum-positive samples were similar or identical to the sequences of variants of A. phagocytophilum deposited in GenBank. The 16S rRNA gene fragment sequence of AB-GGHL was similar to that of Anaplasma (Ehrlichia) bovis 16S rRNA (GAN U03775). The identities of the Anaplasmataceae genus and species DNA in the 327 ticks that could not be confirmed infected with either E. chaffeensis, A. phagocytophilum, or A. bovis are not known. This study is the first to demonstrate the presence of E. chaffeensis, A. phagocytophilum and A. bovis in Korean ticks.     

Anaplasmosis in farmers and domestic animals in Anhui province,China

ObjectiveTo investigate the epidemiological status of anaplasmosis among farmers and domestic animals in Guangde County where the unusual nosocomial human to human transmission of human granulocytic anaplasmosis occurred in Anhui province and the index patient in this event lived, as well as Huaiyuan County and Mingguang City in Anhui province.MethodsFrom April to May in 2009, 596 farmers, 132 goats, 12 dogs and 6 cows was collected from Guangde, Mingguang and Huaiyuan counties and the IgG antibodies against A. phagocytophilum in farmers, goats, dogs and cattle was determined using an immunofluorescence assay (IFA). The A. phagocytophilum 16S rRNA gene was amplified from blood samples from domestic animals using nested PCR, and the genetic diversity of the 16S rRNA gene was analysed.ResultsThe percentage of farmers with IgG antibodies against A. phagocytophilum in the 3 survey counties was 33.7%, and the individual percentages for Guangde, Mingguang and Huaiyuan counties were 76.5%, 59.2% and 10.4%, respectively. The total seroprevalence in dogs, goats and cattle was 33.3%, 0.8% and 0%, respectively. The percentage of samples positive for amplification of the 16S rRNA gene of A. phagocytophilum in goats, dogs and cattle was 33.3%, 25.0% and 0%, respectively. Analysis of the genetic diversity of the 16S rRNA gene showed that there were two genotypes of A. phagocytophilum. Group A was endemic in Guangde County, which is located in the southeast of Anhui province, where the nosocomial human granulocytic outbreak of anaplasmosis in 2006 occurred. Group B was located north of Huaiyuan County.ConclusionsWe concluded the prevalence of A. phagocytophilum among farmers and domestic animals in Anhui province was demonstrated, and diagnosis and differential diagnosis of zoonotic “rickettsial” infections should be emphasized in clinical practice. A systemic ecological survey should be conducted to reduce the public health threat to humans and animals.     

立克次体的16SrRNA基因序列分析

目的　研究立克次体的１６ＳｒＲＮＡ基因序列的差异,为其分类和鉴定提供依据。方法　从基因数据库中选择不同的立克次体属代表种的１６ＳｒＲＮＡ基因序列,在计算机上用多序列排列程序对序列进行同源位排列和对比分析,以及依据序列的相似程度构建进化树。结果　立克次体的１６ＳｒＲＮＡ基因含有４ 个高变区,这些高变区的序列具有种或属的特异性,依据序列的相似程度构成的进化树可将立克次体目内成员分为柯克斯体、巴通体、立克次体、埃立克体等４ 个大基因群,每个大基因群含有若干个小基因群。结论　１６ＳｒＲＮＡ基因高变区的序列可用于设计立克次体种、属特异性ＰＣＲ引物和杂交探针;１６ＳｒＲＮＡ 基因序列的分析是目前立克次体分类和鉴定的最可靠依据     

Methicillin-resistant Staphylococcus aureus nasal carriage in neonates and children attending a pediatric outpatient clinics in Brazil

BackgroundIn Latin America, few studies have been carried out on methicillin-resistant Staphylococcus aureus carriage in the pediatric population. We conducted a survey of nasal S. aureus carriage in neonates and in children attending the pediatric outpatient clinics in a large Brazilian city with high antimicrobial consumption.MethodsPernasal swabs of neonates were collected upon admission and at discharge in four neonatal intensive care units and of children less than five years of age during outpatient visits. Methicillin-resistant S. aureus isolates were characterized for antibiotic susceptibility, mec gene presence, pulsed-field gel electrophoresis, spa type, SCCmec-type, multilocus sequence type, and presence of Panton-Valentine leukocidin genes.ResultsS. aureus was carried by 9.1% and 20.1% of the 701 neonates and of 2034 children attending the outpatient clinics, respectively; methicillin-resistant S. aureus carriage was detected in 0.6% and 0.2%, of the these populations, respectively. Healthcare-associated methicillin-resistant S. aureus strains found in neonates from neonatal intensive care units and outpatients were genetically related to the Brazilian (SCCmec-III, ST239) and to the Pediatric (SCCmec-IV, ST5) clones. Community-associated methicillin-resistant S. aureus was only detected in outpatients. None of the methicillin-resistant S. aureus strains contained the Panton-Valentine leukocidin gene. Methicillin-resistant S. aureus strains related to the Brazilian clone showed multidrug resistance pattern.ConclusionsDespite the high antibiotic pressure in our area, and the cross transmission of the healthcare-associated methicillin-resistant S. aureus clones between neonatal intensive care units and outpatients, the prevalence of methicillin-resistant S. aureus carriage is still low in our setting.     

吉林省林区蜱粒细胞无形体感染的调查

目的调查吉林省蜱粒细胞无形体感染。方法运用聚合酶链反应方法对吉林延边地区采集的蜱标本粒细胞无形体16S rRNA和gltA基因片段进行扩增及序列分析,将扩增序列与GenBank注册的基因序列进行比较,构建粒细胞无形体gltA基因进化树。结果共检测游离蜱427只,其中全沟硬蜱100只,森林革蜱327只,粒细胞无形体感染阳性率分别为4.00%和0.00%。寄生蜱感染阳性率2.9%。寄生蜱与游离蜱感染率差别无显著性。16S rRNA序列与我国已在GenBank注册的AF205140序列一致,与国外粒细胞无形体16S rRNA序列存在不同程度的差异,相似性为97%～99%;gltA基因与GenBank的粒细胞无形体gltA基因片段比较,相似性为87%～97%,推导的氨基酸序列相似性为84%～99%。结论我国吉林省林区存在蜱粒细胞无形体感染。     

分支杆菌临床分离株的16S-23S rRNA基因转录间隔区的序列分析

目的　建立鉴定分支杆菌分离株的 16S - 2 3SrDNA转录间隔区 (internaltranscribedspacer ,ITS)序列分析的方法 ,用该方法对临床分离株进行鉴定。方法　对从重庆某医院分离的 2 9株分支杆菌菌株分别进行了PCR扩增 ,得到它们的16S - 2 3SrDNAITS片段 ,并测定所得到片段的DNA序列。用DNA分析软件将所获得的序列与GenBank的分支杆菌序列相比较 ,计算种间相似性 ,并用序列构建分支杆菌菌株的系统发育树 ,对分离株进行鉴定。结果　在ITS序列分析中 ,有 10株的序列分别与结核分支杆菌复合群 (Mycobacteriumtuberculosiscomplex ,MTC)、戈登分支杆菌 ,脓肿分支杆菌的序列完全一致。依据完全一致的序列可以准确鉴定这些临床分离株为相应的种 ;4株 16SrDNA序列分析不能准确鉴定的分离株中其中 3株(M 4、M 5、M 12 )鉴定为戈登分支杆菌 ,1株 (M 2 3)鉴定为脓肿分支杆菌。部分分离株的的ITS序列在GenBank序列检索中无完全一致的匹配序列 ,这些不同的序列之间的相似程度为 2 7.1%～ 99.2 %。用临床分离株的ITS序列与其最相似的已知分支杆菌的ITS序列构建的系统发育树 ,菌株分群与 16SrDNA序列构建的系统发育树的分群基本一致。结论　分支杆菌的16S - 2 3SrDNAITS序列比 16SrDNA序列有更大的多样性 ,该序列分析可作为     

Implanted Port Catheter System Infection Caused by Methicillin-resistant Staphylococcus pseudintermedius ST71-SCCmec type III

Staphylococcus pseudintermedius is commonly associated with skin and soft tissue infections in dogs. However, infections caused by S. pseudintermedius are only rarely reported in humans, and this pathogen is frequently misidentified as S. aureus. We herein report a case of an implanted port catheter system infection caused by methicillin-resistant S. pseudintermedius (MRSP) in a patient with hepatocellular carcinoma. The patient was also a dog owner. S. pseudintermedius was first identified using the Vitek2 system (BioMérieux). Whole-genome sequencing revealed that this MRSP was a sequence type 71-carrying staphylococcal cassette chromosome mec type III (ST71-SCCmec III) isolate.     

内蒙古部分地区草原革蜱携带无形体检测与进化分析

目的 探究内蒙古地区草原革蜱无形体感染情况,鉴定蜱传无形体种属并进行系统进化分析。方法 基于无形体16S rRNA基因、groEL基因和MSP4基因合成特异引物,对内蒙古地区采集的蜱虫标本进行PCR扩增,将阳性样本进行测序和分析并构建系统发育进化树。结果 将采集的905只蜱虫经形态学鉴定后将168只饱血蜱与737只未饱血蜱分为220个蜱虫混合样本,经检测有41个无形体阳性样本均来自于单只饱血蜱样本,总阳性检出率为4.53%(41/905),其中呼伦贝尔地区阳性率为1.03%(3/292);赤峰地区阳性率为3.13%(17/543);鄂尔多斯地区阳性率为30%(21/70),Blast比对分析结果显示与绵羊无形体同源性高达99%以上。系统发育进化树和同源性比对分析结果显示内蒙古地区16S rRNA基因的序列与新疆绵羊无形体xjmns03(JN400674)同源性最高,为99.49%;groEL基因序列与苏丹青尼罗州绵羊无形体G-BN-40(MG383903)同源性最高,为98.77%;MSP4基因序列与与陕西绵羊无形体D/SX786(MN307493)同源性最高,为100.00%。结论 内蒙古地区存在蜱传绵羊无形体,鄂尔多斯地区阳性率最高,为避免蜱传疾病对动物及人类健康造成危害,应重点对鄂尔多斯地区进行科学防控。     

Detection of the mecA gene and identification of Staphylococcus directly from blood culture bottles by multiplex polymerase chain reaction

Introduction

Staphylococcus spp. – both S. aureus, including methicillin-resistant strains (MRSA) and coagulase negative staphylococci (CoNS) – are relevant agents of healthcare-associated infections. Therefore, the rapid recognition of MRSA and methicillin-resistant CoNS from blood stream infections is critically important for patient management. It is worth noting that inappropriate empiric therapy has been associated with higher in-hospital mortality.

Intra-species sequence variability in 28s rRNA gene of Oesophagostomum venulosum isolated from goats of West Bengal,India

ObjectiveTo identify genotypes of Oesophagostmum venulosum (O. venulosum) prevailing in West Bengal, India by comparing variation of nucleotide sequences among 28S rRNA.MethodsPCR amplification of partial segment of 28 S rRNA sequence and analysis of sequence amplified product by single strand conformation polymorphism (SSCP).ResultsTwo distinct conformers among male and female parasites were identified by PCR-SSCP analysis. Sequence analysis among conformers revealed the presence of five single nucleotide polymorphisms (SNP) in codon 64, 66, 86, 125 and 146. Secondary RNA prediction structure showed that out of 5 SNPs, 4 occurred at interior loop of RNA which confirmed evolutionary changes among isolates prevailing in this region.ConclusionsSNPs occured in different isolates of O. venulosum might influence critical changes in rRNA folding pattern which influence evolutionary changes among isolates.     

4种病原菌特异基因片段阳性蜱的16S rRNA基因克隆文库分析

目的 建立16S rRNA基因克隆文库分析蜱媒菌群的方法,观察该方法对蜱寄生病原菌的检测效率以及对细菌群落多样性分析和对病原菌的筛检能力.方法 用伯氏疏螺旋体、汉赛巴通体、嗜吞噬细胞无形体和查菲埃立克体4种病原菌特异基因设计引物,扩增蜱标本提取的核酸,以上述4种病原菌特异基因片段扩增均阳性的蜱核酸提取物做模板,用16S rRNA基因的通用引物进行PCR扩增、纯化、连接、克隆和测序,建立16S rRNA基因克隆文库,将测序结果与数据库进行比对,计算克隆文库Coverage值和Shannon-Wiener多样性指数.结果 测出103个有效序列,检出16种已知种属的细菌,其中8个是优势类型(克隆子数>5个);检测到伯氏疏螺旋体、汉赛巴通体和立克次体3种病原菌,但这3种病原菌均不是优势类型(克隆子数均<5个).Coverage值为96.11%,Shannon-Wiener多样性指数为2.40.克隆序列分析结果表明,蜱寄生细菌主要为α、γ变形菌纲,占56.25%(9/16).结论 16S rRNA基因序列分析可以对蜱标本进行菌群相对定量研究,可以同时检出多种病原菌,是一种较好的细菌菌群多样性分析和病原菌筛检方法.     

浙江磐安地区啮齿动物嗜吞噬细胞无形体感染研究

目的 调查浙江省磐安地区啮齿动物无形体属嗜吞噬细胞无形体的自然感染状况,了解其分子生物学特性。方法 在浙江省磐安地区低山田地里捕获多种啮齿动物230只(黄胸鼠、社鼠、褐家鼠、小林姬鼠、黄毛鼠、青毛鼠、大林姬鼠、白腹巨鼠、针毛鼠、黑线姬鼠和松鼠);解剖取肝脾;提取DNA,用巢式PCR扩增无形体属16S rRNA片段;PCR产物测序,并进行Blast 同源性比较,最后采用MEGA4.0软件与GenBank登录的其它无形体属种株进行系统发生分析。结果 230只啮齿动物中,1只黄胸鼠、1只褐家鼠和2只白腹巨鼠肝脾标本检出无形体属16S rRNA片段,磐安地区野鼠无形体属立克次体的自然感染率为1.7%。3份阳性标本PCR产物测序,序列比对分析表明,从褐家鼠同时检出分别与嗜吞噬细胞无形体和浙江无形体株ZJ05/2009完全一致的序列,黄胸鼠和1只白腹巨鼠的序列与浙江无形体株ZJ05/2009完全一致。结论 浙江省磐安地区野鼠检出致病性无形体属核酸,存在无形体属不同种株的复合感染感染。提示可能存在无形体病自然疫源地,需要进一步密切关注致病性无形体的人群感染情况。     

湖北省长角血蜱携带嗜吞噬细胞无形体的调查

目的调查湖北省长角血蜱是否携带嗜吞噬细胞无形体。方法运用聚合酶链反应方法对湖北随州地区采集的蜱标本进行嗜吞噬细胞无形体16S rRNA基因片段进行扩增和序列分析,将扩增序列与GenBank注册的基因序列进行比较。结果共检测游离蜱72只,蜱种鉴定均为长角血蜱,嗜吞噬细胞无形体最小感染率为4.22%;所检出的嗜吞噬细胞无形体16S rRNA基因与我国已在GenBank注册的24个相对应序列同源性100%。结论湖北省长角血蜱携带嗜吞噬细胞无形体。     

Cardiobacterium valvarum临床分离株的生物学特性研究及16S rRNA鉴定

目的 采用不同的方-法描述Cardiobacterium valvarum(C. valvarum)临床分离株的生物学特性,利用16S rRNA基因测序技术进行分子鉴定。 方-法 转种阳性血培养标本到血琼脂平板上进行细菌培育,革兰染色涂片镜检,用VITEK 2 Compact全自动微生物鉴定分析仪对临床分离株进行细菌鉴定,基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)对分离株的蛋白质进行高通量测定,E-test法对分离株作药敏试验。提取分离株的DNA,采用16S rRNA基因测序技术对PCR的产物进行测序,在NCBI的BLAST网站上与GenBank数据库上的序列做相似性比较,用MEGA7.0.26软件构建该分离株的系统进化树。结果 经细菌培养发现小而圆、光滑、不透明,灰色的菌落;经革兰染色后镜下见到小、两端圆形、革兰阴性的多形性杆菌;VITEK 2 Compact上机、MALDI-TOF-MS技术均未得到该分离株的鉴定结-果;16S rRNA基因测序技术测得该菌株基因全序列约为1 450 bp,与C. valvarum F0432的16S rRNA同源性为99.59%,鉴定为C. valvarum。结论该菌的形态、生化反应均无代表性,采用16S rRNA基因测序技术可以对C. valvarum进行鉴定,对该疾病的诊断具有重要意义。     

附红细胞体套式PCR方法的建立及其在交叉感染研究中的初步应用

目的建立一种快速、准确检测附红细胞体的套式PCR方法,并在交叉感染研究中初步应用。方法根据Gen-Bank收录的猪附红细胞体16S rRNA基因序列为例,设计两对特异引物,建立套式PCR诊断方法。结果所建立的套式PCR检测方法,具有极高的敏感性和特异性。结论将猪附红细胞体人工感染小白鼠、家兔、犬及鸡,利用所建立的套式PCR进行检测,证明了猪附红细胞体在3种实验动物之间存在交叉感染。     

18S ribosomal DNA based PCR diagnostic assay for Trichomonas vaginalis infection in symptomatic and asymptomatic women in India

ObjectiveTo identify the cases of trichomoniasis in symptomatic and asymptomatic Trichomonas vaginalis (T. vaginalis) infected patients by PCR amplification of hypervariable 18S rRNA gene and to assess the sensitivity of restriction fragment length polymorphism (RFLP) technique for their diagnosis.MethodsWe enrolled 498 women of child bearing age groups, with their pre-informed consent, attending OPD for their routine checkups and STI related problems. Trichomoniasis was diagnosed on the basis of wet mount preparations and PCR with a primer set targeting a well-conserved region in the 18S rRNA genes of T. vaginalis, respectively. Sequencing was done for differentiating the symptomatic and asymptomatic strains of axenic and clinical isolates.ResultsAfter PCR diagnosis T. vaginalis infection was detected in 17 (3.42%) out of 498 clinical isolates. Seventeen axenic and sixteen clinical strains of T. vaginalis tested were successfully detected by PCR yielding a single predicted product of 312 bp in gel electrophoresis followed by restriction digestion with restriction endonuclease HaeIII. After restriction digestion they gave two bands, one of 101 and the other of 211 bp, while there was negative response with DNA from Chlamydia trachomatis, Neisseria gonorrhoeae, Giardia lamblia, Toxoplasma gondii, Leishmania donovani and Entamoeba histolytica. An optimal analytical sensitivity and specificity of one T. vaginalis organism per PCR was achieved. Sequence of symptomatic and asymptomatic strains of axenic and clinical isolates are somewhat differentiated on the basis of point mutations in their 18S rRNA gene.ConclusionsOnly few factors are known to predict symptoms of T. vaginalis infection, although the majority of women are infected with trichomoniasis are reported. Therefore the application of sensitive PCR based diagnosis may be quite useful for routine diagnosis of T. vaginalis strains.     

牛源隐孢子虫上海分离株的巢式PCR鉴定

目的 用巢式PCR鉴定1株自然感染的上海牛源隐孢子虫。 方法 改良抗酸染色法检查含隐孢子虫卵囊的牛粪,油镜下观察卵囊形态,测量大小。以牛粪中的隐孢子虫卵囊DNA为模板,根据隐孢子虫18S rRNA序列设计2对引物,巢式PCR扩增目的片段,测序,同源性比对,并运用MEGA4.0软件构建系统发育树。 结果 上海牛源隐孢子虫分离株卵囊呈圆形或椭圆形,大小为（5.6±0.49）mm×（5.2±0.51）mm。扩增出的18S rRNA基因片段为812 bp。上海牛源隐孢子虫分离株的18S rRNA与巴西的牛隐孢子虫（Cryptospridium bovis,GenＢank登录号为151935628）序列一致性为100%,两者在种系发育树上为同一分支,亲缘关系最近;与中国青海、蒙古国、美国、突尼斯等地的牛隐孢子虫序列一致性均为99%。 结论 上海牛源隐孢子虫分离株为牛隐孢子虫（C. bovis）。