rK39免疫层析试条诊断人体内脏利什曼病和感染犬

目的 评价rk39免疫层析试条对内脏利什曼病诊断和流行病学调查中的应用价值.方法 用rk39试条检测就诊者或临床医生考虑为黑热病的患者或进行过病原学检查的患者,并在黑热病疫区现场检测家犬.结果 用rk39试条检测具有流行病学史就诊者358人,黑热病抗体阳性244例,阳性检出率68.16％.接受rk39试条与病原学2种方法检查的207人中,均为阳性者131例,2者阳性符合率为100％;对单-显示rk39试条阳性71例,用锑剂治疗均治愈,证实均为黑热病.rk39试条检查疫区家犬443只,检出阳性犬43只,阳性率为9.71％,阳性犬以5岁以下犬龄多见.结论 与传统诊断内脏利什曼病方法相比较,rK39试条具有更快速、操作简便、敏感和特异性高以及低损伤性,可用于流行区的内脏利什曼病的诊断和筛选;rK39试条可在潜伏期中发现黑热病患者和无症状感染犬,对黑热病患者的诊断和感染犬的检出具有很高的价值.     

重组抗原(rK39)试纸条诊断黑热病

目的　了解rK39试纸条用于临床黑热病现症患者检测的效果。方法　以rK39试纸条结果与病原学结果比较或以rK39试纸条阳性结果与特效药物治疗结果比较,判断rK39试纸条检测黑热病的可靠性。结果　rK39试纸条和病原学 2种方法平行检查 49人,共查出阳性 36人,rK39试纸条阳性 35人 (97 .22% ),病原学检查阳性 21人(58.33% )。21例病原学阳性者中,rK39试纸条显示阳性 20例,假阴性为 4 76%。单一显示rK39试纸条阳性的 29例患者均用葡萄糖酸锑钠治疗有效,证实都是黑热病。在rK39试纸条检测的 72人中,通过病原学检查和疗效观察结果印证共有黑热病 51例,rK39试纸条显示阳性 49人 (96. 07% )。结论　应用rK39试纸条诊断黑热病现症患者的阳性检出率可达 96. 07%,与病原学方法相比,检出率明显提高(P<0. 01)。     

新发现的内脏利什曼病流行区新疆民丰县Ⅰ. 流行病学调查

目的 对新疆民丰县一个新发现的内脏利什曼病（黑热病）流行区进行现场流行病学调查。 方法 2007年6月采用回顾性调查和现况调查相结合,对民丰县安迪尔乡所有居民进行逐户入户调查。内容包括家庭成员中20年来的健康状况、既往有无疑似内脏利什曼病史（包括已过世者）,并对15岁以下儿童进行体查（触诊肝、脾）;对部分居民进行利什曼素皮内试验和rk39免疫层析试条检测。 结果 共调查居民313人,未见现症病例。既往有疑似内脏利什曼病史者60例,其中13例已过世。171人进行利什曼素皮内试验检测,阳性率为99.4%（170/171）,其中有疑似内脏利什曼病病史者的阳性率为96.6%（28/29）。197人进行rk39免疫层析试条检测,阳性率为10.2%（20/197）,其中有疑似内脏利什曼病史者的阳性率为19.4%（6/31）,追溯诊断为既往内脏利什曼病病例。 结论 新疆民丰县为内脏利什曼病流行区。     

新发现的内脏利什曼病流行区新疆民丰县Ⅲ:临床确诊首例患者所在点的流行病学调查

目的 调查新疆民丰县安迪尔牧场首例临床确诊的内脏利什曼病患者所在地该病的流行与分布情况. 方法 2007年11月采用回顾性调查和现况涮查相结合的方法,对民丰县安迪尔牧场开西木库勒村3组所有居民进行逐户入户调查,内容包括家庭成员近20年来的健康状况、既往有无疑似内脏利什曼病病史(包括已过世者),对15岁以下儿章进行杏体(触诊肝、脾),对部分居民进行利什曼素皮内试验和rk39免疫层析试条榆测. 结果调查居民共计407人,未发现现症病例.既往有疑似内脏利什曼病病史者12例,其中自愈者9例,3例已过世.利什曼素皮内试验榆测354人,阳性率为51.13%(181/354);有疑似内脏利什曼病病史者的阳性率为66.67%(4/6);无疑似内脏利什曼病病史者的阳性率为50.86%(177/348).接受rk39免疫层析试条检测的有6人,阳性率为16.67%(1/6),阳性者为2例疑似内脏利什曼病病史者之一. 结论根据所获得的流行病学资料,可确定当地为荒漠型内脏利什曼病流行区.     

新疆库尔勒市哈满沟煤矿内脏利什曼病流行现况调查

目的 调查新疆库尔勒市塔什店镇哈满沟煤矿内脏利什曼病的流行现况。 方法 2008年6月采用回顾性调查与现况调查相结合, 对哈满沟矿区所有居民进行逐户入室调查。内容包括既往有无疑似内脏利什曼病病史, 部分居民进行利什曼素皮内试验, 并对15岁以下儿童皮内试验阴性者进行体检（肝、脾触诊）和rk39免疫层析试条检测, 具备内脏利什曼病症状和体征、试条阳性者进行骨髓穿刺, 镜检是否感染利什曼原虫无鞭毛体。在居民点及附近野外捕集白蛉, 解剖鉴定白蛉, 并检查前鞭毛体有无自然感染。 结果 接受利什曼素皮内试验185人, 阳性39例, 阳性率为21.1%（39/185）。在当地居住不足6年的成年人和在当地出生的5岁以下儿童共45人, 均为阴性;在当地居住6年以上者阳性率为27.9%（39/140）。15岁以下儿童皮内试验阴性者（81人）接受rk39免疫层析试条检测, 阳性1例, 经骨髓穿刺镜检见利什曼原虫无鞭毛体, 确诊为内脏利什曼病。捕获的12只白蛉, 经剖检鉴定为亚历山大白蛉（Phlebotomus alexandri）, 其中1只雌蛉利什曼原虫前鞭毛体阳性。 结论 哈满沟矿区为内脏利什曼病流行区。     

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。     

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^...     

新疆维吾尔自治区伽师县卧里托格拉克乡居民内脏利什曼病流行现况调查

目的 调查新疆维吾尔自治区伽师县内脏利什曼病流行现况.方法 采用回顾性调查和现况调查相结合,对伽师县卧里托格拉克乡20村1组所有居民进行入户调查.调查内容包括既往有无疑似内脏利什曼病病史,部分居民进行利什曼素皮内试验,对3岁以下婴幼儿皮内试验阴性者进行查体(触诊肝、脾)和rk39免疫层析试条检测.在居民住宅内、外捕集白蛉,解剖鉴定并检查前鞭毛体有无自然感染.结果 接受利什曼素皮内试验149人,阳性率为68.46%(102/149);3岁以下年龄组阳性率为11.76%(2/17),4～75岁年龄组阳性率为75.76%(100/132).3岁以下年龄组皮内试验阴性者有15人接受rk39免疫层析试条检测,1例阳性者(女,1岁),确认为临床诊断病例.2001-2008年7月有既往病史者52例,其中治愈50例,死亡2例.捕获的374只雌蛉,经解剖鉴定为吴氏白蛉(Phlebotomus wui),其中3只雌蛉体内查见自然感染的利什曼原虫前鞭毛体.结论 伽师县仍有内脏利什曼病流行.     

新疆民丰县黑热病血清流行病学调查

目的调查塔里木盆地南部和田地区民丰县黑热病流行状况。方法以r K39免疫层析法和r K39-ELISA夹心法检测人群黑热病抗体,阳性标本复测反应滴度,B超仪检测黑热病抗体阳性者脾脏。结果 5个乡镇检测7~9岁学生1 060人和成年村民49人,县城和地势较高的叶亦克乡未检出黑热病抗体阳性者,地处荒漠腹地的亚瓦通古孜、安迪尔和萨勒吾则克3个乡,学生的阳性率分别为8.00%(2/25)、1.83%(3/164)和0.76%(2/266),亚瓦通古孜乡成年村民的阳性率为8.16%(4/49);B超检查11份黑热病抗体阳性者(女5份和男6份)均未见脾脏肿大。结论调查塔里木盆地南部和田地区民丰县,新发现地处荒漠腹地的安迪尔乡和萨勒吾则克乡也有黑热病流行。     

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。     

Comparison of an rK39 dipstick rapid test with direct agglutination test and splenic aspiration for the diagnosis of kala-azar in Sudan

We compared an rK39 dipstick rapid test (Amrad ICT, Australia) with a direct agglutination test (DAT) and splenic aspirate for the diagnosis of kala-azar in 77 patients. The study was carried out under field conditions in an endemic area of north-east Sudan. The sensitivity of the rK39 test compared with splenic aspiration was 92% (46/50), the specificity 59% (16/27), and the positive predictive value 81% (46/57). Compared with the diagnostic protocol used by Médecins sans Frontières, the sensitivity of the rK39 test was 93% (50/54), the specificity 70% (16/23), and the positive predictive value 88% (50/57). Compared with splenic aspirates, the sensitivity of a DAT with a titre > or =1:400 was 100% (50/50), but its specificity only 55% (15/27) and the positive predictive value was 80% (50/62). Using a DAT titre > or =1:6400, the sensitivity was 84% (42/50), the specificity 85% (23/27) and the positive predictive value 91% (42/46). All four patients with DAT titre > or =1:6400 but negative splenic aspirate were also rK39 positive; we consider these are probably 'true' cases of kala-azar, i.e. false negative aspirates, rather than false DAT and rK39 seropositives. There were no false negative DATs (DAT titre < or =1:400 and aspirate positive), but there were four false negative rK39 tests (rK39 negative and aspirate positive). The rK39 dipstick is a good screening test for kala-azar; but further development is required before it can replace the DAT as a diagnostic test in endemic areas of the Sudan.     

Prospective evaluation and comparison of the direct agglutination test and an rK39-antigen-based dipstick test for the diagnosis of suspected kala-azar in Nepal

The diagnosis of visceral leishmaniasis (kala-azar) remains difficult in rural endemic areas and practical and reliable tests are badly needed. Two serological tests, the Direct Agglutination Test (DAT) and an rK39-antigen-based dipstick test, were compared to parasitological diagnosis in a group of 184 patients presenting at a tertiary care centre in south-eastern Nepal with a history of fever > or = 14 days and splenomegaly; 139 patients had a parasitologically proven kala-azar and 45 patients had a negative parasitological work-up. The rK39 dipstick showed a sensitivity of 97% and a specificity of 71%. The DAT was up to 99% sensitive with a low cut-off titre (1:400) but its specificity did not exceed 82% even with a high cut-off titre (1:51 200). Both tests could be used for screening suspect patients in endemic areas. However, their use as confirmatory tests should be restricted to situations where the proportion of kala-azar among clinical suspect patients is high. The rK39 dipstick is cheaper and easier to use than the DAT and could be used widely provided that both its performance and production remain stable.     

Field validity, reproducibility and feasibility of diagnostic tests for visceral leishmaniasis in rural Nepal

OBJECTIVES: To assess the field accuracy, reproducibility and feasibility of the formol gel test (FGT), the urine latex agglutination test (KAtex) and a rK39 antigen-based dipstick for the diagnosis of visceral leishmaniasis (VL) in rural Nepal. METHOD: Patients with clinical suspicion of VL were recruited at Rangeli District Hospital (DH), a 15-bed government hospital located in south-eastern Nepal. FGT, KAtex and rK39 dipstick tests were performed on site and later repeated at a reference kala-azar diagnostic laboratory to assess reproducibility. Diagnosis of VL was confirmed by either a positive bone marrow aspirate examination or a positive direct agglutination test (DAT titre > or = 1:3200) in patients who later responded to anti-leishmanial therapy. RESULTS: Of 155 patients initially recruited, 142 (85 with VL and 57 with another diagnosis) were included in the study. The sensitivity of the rK39 dipstick [89%; 95% confidence interval (CI): 81-94] was significantly higher than that of the KAtex (57%; 95% CI: 46-67) and the FGT (52%; 95% CI: 41-62). All three tests had a specificity of at least 90%. Agreement was higher for the rK39 dipstick (kappa = 0.87) than for the FGT (0.68) and the KAtex (0.43). All tests required < or = 20 min of actual work and < or = 40 min to obtain the results. CONCLUSION: The rK39 dipstick was easy to do, more accurate and reproducible than other rapid diagnostic tests for VL in a DH of rural Nepal. It should be integrated into the field diagnostic algorithm of VL in this region and mechanisms to secure its availability should be found.     

Use of rK39 for diagnosis of post kala-azar dermal leishmaniasis in Nepal

A recently developed nitrocellulose-based dipstick test, rK39, has been widely used for the diagnosis of kala-azar. In this study, we evaluated its use for the diagnosis of post kala-azar dermal leishmaniasis (PKDL). We also investigated the time taken by patients to develop PKDL after apparent cure of kala-azar (visceral leishmaniasis, VL) and the time taken by patients to come to the hospital after the appearance of symptoms of PKDL. A majority of patients developed the disease within three years after the apparent cure of kala-azar (KA). A majority of patients sought treatment within five years after the onset of PKDL. The amastigotes of Leishmania donovani bodies (LDBs) were demonstrated in 70, 20, and 20% of slit-skin smears (SSS) prepared, respectively, from nodular, papular, and macular forms. The presence of highest density (6+) LDBs in the SSS of 20% of nodular PKDL patients indicated that they may have acted as reservoir in the community. Other reservoirs are not known in Nepal. Only 8% cases were detected by aldehyde test. Although this test is obsolete it is still used in rural parts of Nepal. The dipstick (rK39) was 96% sensitive and 100% specific to diagnose PKDL. Its positive predictive value, negative predictive value, and diagnostic efficacy were 100, 91, and 97% respectively. Due to the advantage of cost compared with the direct agglutination test (DAT), and being easy to use and store in field conditions, rK39 is a good tool to diagnose PKDL in rural situations. All the PKDL patients were cured of the disease after treatment by SAG.     

用重组 rK39抗原试纸条快速诊断内脏利什曼病

[目的 ]评价以恰氏利氏曼原虫类 kinesin基因中编码 39个氨基酸的基因片段 (r K39)为重组抗原 ,用于血清学诊断内脏利什曼病的价值。 [方法 ]在新疆喀什地区对 13例经脾检和骨髓穿刺阳性的内脏利什曼病患者 ,取一滴病人全血或血清滴在 r K39抗原试纸条底部的吸收垫上 ,血清中蛋白随缓冲液向试纸条上部移动 ,其中相应特异抗体可与 r K39抗原带结合 ,而产生阳性条带。同时 ,本文亦用相同阳性血清作了关于 r K 39抗原的 Western印迹分析对照。 [结果 ]EL ISA分析显示病人血清抗体滴度在 10 - 2～ 10 - 4 ,与所见到的 r K39试纸条上的反应强度符合。Western印迹分析亦显示阳性血清可识别 r K39蛋白条带。[结论 ]与传统诊断内脏利什曼病方法相比较 ,r K39试纸条更快速 ,特异 ,灵敏和低损伤性 ,可用于低发病率流行区的内脏利什曼病的诊断和筛选     

Evaluation of a new recombinant K39 rapid diagnostic test for Sudanese visceral leishmaniasis

A new rK39 rapid diagnostic dipstick test (DiaMed-IT-Leish) was compared with aspiration and a direct agglutination test (DAT) for diagnosis of visceral leishmaniasis (VL) in 201 parasitologically confirmed cases, 133 endemic controls, and in 356 clinical suspects in disease-endemic and -epidemic areas in Sudan. The sensitivity of the rK39 test in parasitologically confirmed VL cases was 90%, whereas the specificity in disease-endemic controls was 99%. The sensitivity of the DAT was 98%. In clinically suspected cases, the sensitivity of the rK39 test was 81% and the specificity was 97%. When compared with the diagnostic protocol based on the DAT and aspiration used by Médecins sans Frontières in epidemic situations, the positive predictive value was 98%, and the negative predictive value was 71%. This rK39 rapid diagnostic test is suitable for screening as well as diagnosis of VL. Further diagnostic work-up of dipstick-negative patients with clinically suspected VL is important. The ease and convenience of the dipstick test will allow decentralization and improved access to care in disease-endemic areas in Sudan.     

Short report: production of recombinant kinesin-related protein of Leishmania donovani and its application in the serodiagnosis of visceral leishmaniasis

To detect IgG antibody in the serodiagnosis of visceral leishmaniasis (VL), a recombinant antigen rK39, which is part of a Leishmania chagasi kinesin-related protein, has been used successfully and showed high sensitivity and specificity. We report production of a recombinant protein rKRP42, which is part of an L. donovani kinesin-related protein and a homolog of rK39, and its application in an enzyme-linked immunosorbent assay (ELISA) for the diagnosis of VL. When rKRP42 and rK39 were compared, amino acid sequence analysis showed 89.3% identity and 98.7% homology, with rKRP42 having 39 more amino acids than rK39. The ELISA using rKRP42 showed a sensitivity of 94.6% (70 positive samples among 74 from VL patients) and a specificity of 99.3% (148 negative samples among 149 samples from Japanese controls), whereas the sensitivity of the commercial rK39 dipstick test was 93.2% (69 positive samples among 74 from patients with VL). The rKRP42 is a promising new antigen in developing immunodiagnostic methods for VL.     

Use of the recombinant K39 dipstick test and the direct agglutination test in a setting endemic for visceral leishmaniasis in Nepal

We evaluated the field use of two serologic tests for visceral leishmaniasis (VL), the direct agglutination test (DAT) and rK39 dipstick test, in the context of a case-control study. Most VL cases in Nepal are currently diagnosed on clinical grounds and with relatively non-specific tests such as the formol-gel test. Among 14 newly diagnosed VL patients with bone-marrow slides confirmed positive in two independent laboratories, the sensitivity of both tests was 100%. Among 113 controls with no personal or household history of VL, the specificity of the rK39 was 100% while that of the DAT was 93%. The rK39 was less expensive than DAT, and has the advantages of ease of use and obtaining results within minutes. The wider use of the rK39 dipstick test could improve the specificity of VL diagnosis in Nepal.     

Immunologic tests in patients after clinical cure of visceral leishmaniasis

The results of five serologic tests (ELISA using promastigote antigen [ELISAp] and recombinant K39 [ELISArK39] and K26 [ELISArK26] antigens, indirect immunofluorescence test using promastigote antigen [IIFT], and immunochromatographic tests using the rK39 antigen [TRALd]) and of the Montenegro skin test (MST) were analyzed in 41 individuals treated for kala-azar and living in Porteirinha, Minas Gerais, Brazil. The tests were carried out 1 week to 12 years after specific treatment. All MSTs during the 8 months after treatment were negative, whereas after 1 year, 28 (84.8%) were positive. Negativity in all serologic tests was observed for 11 (26.8%) of the 41 individuals, whereas positivity in at least one test was observed for 70.3% of subjects evaluated > or = 2 years after treatment. With respect to each exam, positivity was 38.0% for TRALd, 61.9% for ELISA rK39, 47.6% for ELISA rK26, 38.0% for ELISAp, and 40.5% for IIFT. None of the individuals presented recurrence of the disease during the 4 years of follow-up. The tests were repeated in 24 of the 41 individuals, after some time, and the results were the same in 33.3% of the cases. We conclude that serological tests for kala-azar might continue to be positive after treatment of the disease, although this does not indicate a poor prognosis or a poor therapeutic response.