河南省1例输入性内脏利什曼病的实验室诊断

目的　分析河南省1例输入性内脏利什曼病病例,探讨内脏利什曼病的实验室诊断方法。方法　分析患者的流行病学资料和临床资料,镜检观察骨髓涂片中杜氏利什曼原虫无鞭毛体;rK39试纸条检测血清中利什曼原虫抗体;用两对引物K13A?K13B和 LITSR?L5.8S分别扩增利什曼原虫动基体DNA和核糖体DNA内转录间隔区基因片段。结果　 该患者曾去过内脏利什曼病流行区,有不规则发热、脾肿大、全血细胞减少、白蛋白/球蛋白比例倒置等症状,骨髓涂片查见杜氏利什曼原虫无鞭毛体,rK39试纸条检测阳性,两对引物K13A?K13B和 LITSR?L5.8S分别扩增出87 bp和285 bp的片段。两片段序列与杜氏利什曼原虫相应序列的相似性分别为94%和100%。结论　结合患者的流行病学资料和临床表现以及实验室检测结果,确诊该病例为内脏利什曼病病例,病原体为杜氏利什曼原虫。     

洛阳市1起输入性皮肤利什曼病疫情流行病学 筛查报告

目的　了解中国石油天然气第一建设公司乌兹别克斯坦务工归国员工中利什曼原虫感染情况，并及时采取相关防控措施，防止疫情扩大。方法　设计调查问卷采集筛查对象相关信息，由内科医生触诊肝、脾及浅表淋巴结，皮肤科医生检查皮肤经常暴露处皮肤损害情况，对筛查对象行B超检查肝脾大小，采集筛查对象血清，应用内脏利什曼病快速检测试纸条（rK39）检测，对于筛查中发现的有皮肤损害者，采集皮损处组织镜检利什曼原虫，病原学检测阳性组织用分子生物学方法鉴定虫种。结果　共181人接受筛查，内科检查中6人颈部可触及肿大淋巴结，皮肤科检查中12人有皮肤损害表现，B超检查提示5人肝脾肿大，rK39试纸条检测血清显示3人结果呈阳性。共采集2人份典型皮损处组织，其中1份组织病原学检测发现利什曼原虫；提取前鞭毛体DNA，用利什曼原虫属特异性引物K13A/K13B和L5.8S/LITSR分别扩增出120 bp和350 bp的片段，片段序列与硕大利什曼原虫相应序列（GenBank登录号：EU370906.1、FN677342.1）同源性分别为90%和98%。结论　筛查发现6例皮肤利什曼病患者，其中2例尚未痊愈。1例未痊愈者确诊为由硕大利什曼原虫感染引起的输入性皮肤利什曼病。     

洛阳市1起输入性皮肤利什曼病疫情流行病学 筛查报告

目的　了解中国石油天然气第一建设公司乌兹别克斯坦务工归国员工中利什曼原虫感染情况,并及时采取相关防控措施,防止疫情扩大。方法　设计调查问卷采集筛查对象相关信息,由内科医生触诊肝、脾及浅表淋巴结,皮肤科医生检查皮肤经常暴露处皮肤损害情况,对筛查对象行B超检查肝脾大小,采集筛查对象血清,应用内脏利什曼病快速检测试纸条（rK39）检测,对于筛查中发现的有皮肤损害者,采集皮损处组织镜检利什曼原虫,病原学检测阳性组织用分子生物学方法鉴定虫种。结果　共181人接受筛查,内科检查中6人颈部可触及肿大淋巴结,皮肤科检查中12人有皮肤损害表现,B超检查提示5人肝脾肿大,rK39试纸条检测血清显示3人结果呈阳性。共采集2人份典型皮损处组织,其中1份组织病原学检测发现利什曼原虫;提取前鞭毛体DNA,用利什曼原虫属特异性引物K13A/K13B和L5.8S/LITSR分别扩增出120 bp和350 bp的片段,片段序列与硕大利什曼原虫相应序列（GenBank登录号：EU370906.1、FN677342.1）同源性分别为90%和98%。结论　筛查发现6例皮肤利什曼病患者,其中2例尚未痊愈。1例未痊愈者确诊为由硕大利什曼原虫感染引起的输入性皮肤利什曼病。     

两例输入性皮肤利什曼病的诊断与病原体鉴定

目的对两例疑似皮肤利什曼病进行病原学诊断,并应用分子生物学方法对虫种进行鉴定。方法两例皮肤病患者,分别曾在阿尔及利亚(病例1)和沙特阿拉伯(病例2)务工,表皮均有多个面积较大的溃疡。取皮损处组织涂片、染色、镜检,皮损处组织液置NNN培养基培养,查找原虫。取含利什曼原虫的培养液,离心收集原虫,用2对利什曼原虫种特异性引物ITS1-ITS2和K13A-K13B分别扩增利什曼原虫核糖体DNA内转录间隔区和动基体DNA的基因片段,扩增产物进行测序和Blast序列分析。结果病例1患者的皮损组织涂片镜检未查见利什曼原虫,皮损处组织液经NNN培养基培养10 d后查见前鞭毛体;病例2患者的皮损组织涂片镜检发现利什曼原虫无鞭毛体,皮损处组织液培养8 d后查见前鞭毛体。引物ITS1-ITS2从分离于2例患者的利什曼原虫均扩增出约330 bp的片段,与硕大利什曼原虫(Leishmania major)相应序列同源性均为100%;引物K13A-K13B均扩增出约120 bp的片段,与硕大利什曼原虫相应序列同源性均为96%。4个序列GenBank登录号为JF831924~JF831927。结论两例皮肤病患者均确诊为输入性皮肤利什曼病,...     

甘肃省文县流行区人群婴儿利什曼原虫无症状感染现状

目的 分析甘肃省文县内脏利什曼病流行区人群利什曼原虫无症状感染现状，评价PCR、ELISA和rK39免疫层析试条法检测利什曼原虫无症状感染的潜能。 方法 2004年10月在甘肃文县对269例无内脏利什曼病现症及病史的人群采取随机取样法采集静脉血，分别用RV1?鄄RV2和K13A-K13B两组PCR引物检测血样中的利什曼原虫特异DNA，以利什曼原虫可溶性抗原为包被抗原的ELISA法和rK39免疫层析试条法分别检测利什曼原虫特异性抗体，并比较几种检测方法的敏感性。 结果 PCR、ELISA和rK39免疫层析试条法检测人群利什曼原虫无症状感染的阳性率分别为30.9%（83/269)、24.2%（65/269)和0(0/269)。 结论 甘肃省文县内脏利什曼病流行区人群存在大量利什曼原虫无症状感染者，PCR是检测无症状感染较敏感、特异的方法。     

杜氏利什曼原虫特异性kDNA片段扩增及用于内脏利什曼病诊断的研究

本文报道了用杜氏利什曼原虫特异的一对寡核苷酸引物Ⅰ和Ⅱ进行PCR,扩增微环kDNA分子上一种特异性kDNA片段,进行杜氏利什曼原虫虫种鉴定和病原体检测。敏感性分析表明用此法能够检测到的最低模板DNA需要量为1fg,检测健康犬全血稀释的利什曼原虫前鞭毛体,最低可达2个/ml。扩增六种不同的利什曼原虫kDNA样品,在L.donovani四川人株、四川犬株和L.infantum出现阳性产物,其长度和设计扩增长度一致,扩增产物和地高辛标记的重组质粒探针pLK2斑点杂交结果表明为利什曼原虫kDNA序列。应用此对引物,检测8份内脏利什曼病患者骨髓样品和4份血清样品,经琼脂糖凝胶电泳,Southern杂交证实,分别有7例和2例阳性,显示出可喜的应用前景。     

河南省1例输入性皮肤利什曼病的诊断与分析

患者,男,2017年6月到乌兹别克斯坦务工,7月2日发现左胸臂部有2个小红疹,回国后于10月15日前后发现红疹变大,有少量脓液。用利什曼病快速检测试纸条检测患者血清利什曼原虫抗体结果为阴性。取皮损处组织镜检未查见利什曼原虫无鞭毛体。取皮损处组织置NNN培养基培养,培养至第8天镜检查见大量前鞭毛体。提取前鞭毛体DNA,用利什曼原虫属特异性引物K13A/K13B和L5.8S/LITSR分别扩增出120和350 bp的片段,片段序列与硕大利什曼原虫相应序列(Gen Bank登录号:EU370906.1和FN677342.1)的同源性分别为90%和98%。确诊患者为输入性皮肤利什曼病,病原体为硕大利什曼原虫。患者经过葡萄糖酸锑钠注射液治疗2个疗程(总剂量7.2 g,每个疗程6 d,两个疗程间隔1周),病情得到控制,现已康复。     

基于利什曼原虫K26重组抗原检测黑热病特异抗体的免疫层析试条方法的建立与效果评价

目的 建立基于利什曼原虫K26重组抗原(Recombinant K26, rK26)检测内脏利什曼病特异抗体的胶体金免疫层析试条方法，并评价效果。方法 用胶体金标记链球菌G蛋白(Streptococcal Protein G, SPG),并将其吸附于交联释放垫上；将利什曼原虫K26重组抗原作为包被抗原包被于硝酸纤维素膜适当位置，制成检测特异抗体的免疫层析试条。用该试条检测病原学确诊的内脏利什曼病、其它寄生虫病以及健康者血清，以评价其检测的敏感性和特异性。同时用rK39(Recombinant K39, rK39)试条进行平行检测。结果 rK26试条法和rK39试条法检测内脏利什曼病患者血清的敏感性分别为91.82%(101/110)和93.64%(103/110);与10份疟疾患者血清、10份日本血吸虫病患者血清、10份细粒棘球蚴病患者血清、5份弓形虫病患者血清、5份并殖吸虫病患者血清和5份华支睾吸虫病患者血清均无交叉反应，40份健康者血清也均为阴性，rK26试条法和rK39试条法的总特异性均为100.00%。rK26试条法和rK39试条法阳性检出率之间的差异无统计学意义(χ^...     

杜氏利什曼原虫无鞭毛体蛋白基因真核表达重组质粒构建

目的 构建杜氏利什曼原虫无鞭毛体蛋白（amastin）编码基因的真核表达重组质粒pcDNA3.1-amastin。方法 提取杜氏利什曼原虫基因组DNA进行PCR扩增,将扩增出的无鞭毛体蛋白基因片段导入质粒载体pcDNA3.1（＋）,构建真核表达重组质粒pcDNA3.1-amastin。结果 扩增出大小约550bp的无鞭毛体蛋白基因;重组质粒pcDNA3.1-amastin经鉴定正确。结论 成功构建杜氏利什曼原虫无鞭毛体蛋白基因真核表达重组质粒pcDNA3.1-amastin。     

一例输入性皮肤利什曼病病原体的鉴定

对广州一例未知病原体感染引起的皮肤病患者血样进行免疫学和分子生物学鉴定,以确定其病原体。取静脉血,用Leish r K39 dipstick金标试纸检测利什曼原虫抗体情况。取皮损处组织,75%乙醇固定后,提取基因组DNA。用两对利什曼原虫种特异性引物LITSR-L5.8S和NAGTL1-NAGTL4分别PCR扩增利什曼原虫核糖体DNA内转录间隔区1和N-乙酰氨基葡萄糖苷转移酶的基因片段,扩增产物进行测序和BLAST序列分析。Leish r K39 dipstick金标试纸检测结果呈弱阳性。PCR结果显示,引物LITSR-L5.8S和NAGTL1-NAGTL4分别扩增出约404 bp和1 405 bp的片段,两片段序列与硕大利什曼原虫(Leishmania major)相应序列的相似性均为99.7%。其序列Gen Bank登录号分别为KU975160和KX150476。确诊该皮肤病患者为输入性皮肤利什曼病病例,病原体为硕大利什曼原虫。     

Molecular characterization of a kinesin-related antigen of Leishmania chagasi that detects specific antibody in African and American visceral leishmaniasis.

We report the cloning of a Leishmania chagasi antigen gene and an evaluation of leishmaniasis patient antibody responses to the recombinant protein, rK39. rK39 contains a 39-amino acid repeat that is part of a 230-kDa protein predominant in L. chagasi tissue amastigotes. Sequence analyses showed this protein, LcKin, to be related to the kinesin superfamily of motor proteins. Southern blot analyses demonstrated LcKin-related sequences in seven species of Leishmania, with conservation of the repeat between L. chagasi and Leishmania donovani. Serological evaluation revealed that 98% (56 of 57) of Brazilian and 100% (52 of 52) of Sudanese visceral leishmaniasis patients have high antibody levels to the rK39 repeat. Detectable anti-K39 antibody was virtually absent in cutaneous and mucosal leishmaniasis patients and in individuals infected with Trypanosoma cruzi. The data show that rK39 may replace crude parasite antigens as a basis for serological diagnosis of visceral leishmaniasis.     

Visceral leishmaniasis in immunocompromised hosts

Visceral leishmaniasis is infrequently reported in immunocompromised hosts; hence, the clinical manifestations and outcome of the disease in these patients are unknown. In a series of 10 patients with visceral leishmaniasis complicating renal transplantation (three), hematologic neoplasms (two), systemic lupus erythematosus (two), or infection with human immunodeficiency virus (three), typical hallmarks of kalaazar such as enlargement of spleen or hyperglobulinemia were absent in three and six patients, respectively. Extensive visceral involvement was noted by biopsies or autopsies in four patients. Diagnosis was made during evaluation for fever of unknown origin. Myriads of amastigotes were seen in bone marrow smears. Measurement of antibodies against Leishmania donovani was positive in each patient tested. Ultimately, three patients died, and chronic infections refractory to treatment developed in two other patients. Visceral leishmaniasis is a potentially fatal infection in immunocompromised hosts. Current antiparasitic therapy frequently fails to eradicate L. donovani from infected tissues.     

First case of indigenous visceral leishmaniasis from central India

Visceral leishmaniasis is endemic in the eastern states of India, but central India remains free of leishmaniais. This report describes the first indigenous case of visceral leishmaniasis in a seven-year-old girl from central India. The child presented with fever for 10 days and was diagnosed by bone marrow examination, serology using rKE16 and rK39 antigens, and a polymerase chain reaction specific for the kinesin gene. Sequencing of the immunodominant region of the kinesin gene of the parasite showed four tandem repeats, each 117 basepairs. The first tandem repeat of this strain had 97% homology with the corresponding first tandem repeat of the Leishmania donovani KE16 strain and 92% homology with the L. chagasi BA-2 strain. The second, third, and fourth tandem repeats had 97%, 98%, and 99% homology, respectively, with the L. donovani KE16 strain, and 89%, 96%, and 92% homology, respectively, with the L. chagasi BA-2 strain. This case shows that more than one genetic variant of L. donovani is circulating in various parts of India.     

检测特异抗体胶体金免疫层析试条诊断内脏利什曼病患者现场评价

目的 在现场评价检测内脏利什曼病特异抗体的胶体金免疫层析试条诊断内脏利什曼病患者的效果。方法 2013年采集动物源型内脏利什曼病流行区(四川省、甘肃省)和人源型内脏利什曼病流行区(新疆喀什市)疾病预防控制中心门诊就诊的病人血样和骨髓样本,用镜检法、内脏利什曼病试条和rK39试条进行平行测试,以镜检法为金标准,比较两种试条法检测的敏感性和特异性有无差异。结果 内脏利什曼病试条和rK39试条检测镜检确诊的97例内脏利什曼病患者的敏感性分别为98.87%(96/97),97.94%(95/97),二者无统计学差异(χ²=0.34,P>0.05)。两种试条检测非内脏利什曼病病例145例, 均有2例假阳性反应,特异性为98.62(143/145)。对来自动物源型内脏利什曼病流行区和人源型内脏利什曼病流行区的内脏利什曼病患者分别统计阳性率,结果显示内脏利什曼病试条和rK39试条法检测动物源型和人源型流行区的内脏利什曼病患者血样之间阳性率的差异均无统计学意义(χ²=0.22 /0.01,P>0.05)。两种试条法检测动物源型和人源型内脏利什曼病流行区非内脏利什曼病患者血样之间特异性的差异也均无统计学意义(χ²=0.29,P>0.05)。结论 快速检测内脏利什曼病的胶体金免疫层析试条适用于检测动物源型内脏利什曼病流行区患者血样中的特异性抗体。     

A comparative study of the effectiveness of diagnostic tests for visceral leishmaniasis

We compared the validity of pancytopenia, the formol-gel test (FGT), the indirect fluorescence antibody test (IFAT), the direct agglutination test (DAT), and the rK39 dipstick test as diagnostic criteria for visceral leishmaniasis (VL) in Nepal. Between September 2000 and January 2002, 310 clinical suspects had a bone marrow aspirate, and if negative, a spleen aspirate smear examined for Leishmania donovani. Sensitivity and specificity of all tests were determined compared with parasitology and by latent class analysis (LCA). Compared with parasitology, the sensitivities of the other tests were as follows: pancytopenia = 16.3% (95% confidence interval [CI] = 11.3-22.5%), FGT = 39.9% (95% CI = 32.7-47.4%), IFAT = 28.4% (95% CI = 22.0-35.5%), DAT = 95.1% (95% CI = 90.8-97.7%), and the rK39 dipstick test = 87.4% (95% CI = 81.7-91.9%). Sensitivity estimates obtained by LCA were similar, but specificity estimates were substantially higher (DAT = 93.7% versus 77.8%; rK39 dipstick test = 93.1% versus 77.0%). The DAT or the rK39 dipstick test can replace parasitology as the basis of a decision to treat VL in Nepalese peripheral health services.     

Atypical lesions as a sign of cutaneous dissemination of visceral leishmaniasis in a human immunodeficiency virus-positive patient simultaneously infected by two viscerotropic Leishmania species

Leishmaniasis is considered an emerging opportunistic disease in human immunodeficiency virus (HIV)-infected patients who have considerably variable clinical presentation. We report a patient with visceral leishmaniasis who had unexpected clinical aspects (atypical cutaneous lesions appearing after long-term evidence of visceral parasites). The patient had hepatoesplenomegaly in the absence of fever, but was otherwise generally healthy. The HIV viral load was low despite severe immunossupression (low lymphocyte proliferation and low level of interferon-γ, concomitant with a high lymphocyte activation status). Surprisingly, two Leishmania strains were isolated from his bone marrow (typical L. infantum sequence MON-1, type A) and skin (L. donovani MON-2 sequence); this second strain had not been previously identified in Brazil. The association of visceral leishmaniasis and HIV/acquired immunodeficiency syndrome is a largely unknown disease, particularly in areas in which leishmaniasis is not endemic. Such atypical cases indicate that this disease can be undiagnosed in clinical settings.     

Chronic diarrhea and malabsorption caused by Leishmania donovani.

Chronic diarrhea and malabsorption are uncommon in immunocompetent patients with visceral leishmaniasis. We report two immunocompetent patients with visceral leishmaniasis where the predominant presentation was chronic diarrhea. One of them had clinically overt malabsorption and duodenal mucosa was loaded with Leishmania donovani bodies. The other patient had diffuse colonic aphthous and discrete ulcerations and Leishmania donovani bodies were seen in the crush smears of the colonic mucosa. With amphotericin B, there was reversal of malabsorption and healing of colonic ulcers.