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^...     

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。     

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。     

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.     

ABC-ELISA检测喜马拉雅旱獭鼠疫FI抗体的研究

用ABC-ELISA和常规ELISA检测了19份血凝阳性喜马拉雅旱獭(Marmota himalayana)血清和48份血凝阴性喜马拉雅旱獭血清及247份近年采自疫区的该旱獭血清,比较了两法的敏感性与特异性。两法的阳性、阴性符合率均为100％,但检测阳性血清的OD均值ABC-ELISA法明显地比常规ELISA法高(P<0.01)。ABC-ELISA与常规ELISA法对疫区血清标本的阳性检出率分别为9.7％与8.1%,后者出现1.6％的漏检率,滴度也显著低于前者(P<0.01)。表明ABC-ELISA用于检测旱獭鼠疫FI抗体可明显提高ELISA法的敏感性,且能保持良好的特异性。     

ELISA与IHA测定弓形虫抗体评价及正确运用

<正> 用ELISA与IHA同时测定2252份人血清,IHA阳性率4.00％,ELISA阳性率4.57％,P>0.05。其中共同阳性89份,共同阴性2148份,ELISA阳性而IHA阴性14份,ELISA阴性而IHA阳性1份,阳性符合率85.58％,总符合率99.33％。对38份不同血凝滴度的标本,用ELISA稀释定量,1:16~+5例中,ELISA滴度均<1:200;1:16~(+++)5例中,有2例ELISA滴度1:400;1:64~(++)16例中,有1例ELISA为1:200,>1:64的12例中,ELISA滴度均>1:400,最高1:12800。IHA与ELISA的阳性误差往往出自结果在临界值附近的标本。抗人IgG-ELISA不适于动物与禽类血清抗体检测,使用IHA,能显示其优越性。本文测定结果见(附表)。     

四川省黑水县利什曼原虫家犬感染率的检测

目的 了解四川省黑水县利什曼病流行区家犬的利什曼原虫感染现状,并比较PCR和ELISA检测利什曼原虫无症状感染的效能.方法 2009年5月在犬源型利什曼病疫区四川省黑水县随机选取的无内脏利什曼病现症的家犬,采集静脉血,采用ELISA方法检测血清中利什曼原虫特异性抗体,用两组PCR引物RV1、RV2和K13A、K13B检测血样中利什曼原虫特异DNA,并比较两种检测方法的敏感性.结果 PCR法检测抗凝静脉血阳性检出率为30.47%(32/105),ELISA法检测血清阳性检出率为19.05%(20/105).结论 四川省黑水县存在大量的利什曼原虫无症状感染犬,PCR是检测无症状感染较敏感的方法.     

rK39免疫层析试条在新疆黑热病诊断和流行病学调查中的应用

目的观察rK39免疫层析试条对新疆不同类型黑热病的诊断及流行病学调查中的应用价值。方法用rK39试条检查不同地区确诊的黑热病患者;用rK39-ELISA和rK39免疫层析试条检查不同流行地区居民血清标本与利什曼素皮内反应结果进行比较分析。结果对来自新疆不同地区的黑热病患者共1204例,用rK39试条检测抗体阳性者1169例,阳性率97.09%。其中人源型黑热病患者1052例中1031例阳性,阳性率98.0%;荒漠型黑热病患者158例中阳性143例,阳性率90.5%。二者之间差异显著(P<0.001),荒漠型患者阳性率较低。人源型黑热病流行区居民中rK39抗体阳性率在既往有黑热病史者中为42.7%～59.3%,在无既往史的居民中仅为2.2%～5.8%。在随访的rK39阳性,皮内反应阴性的18人中有4人在4和6个月后出现症状,确诊为黑热病。在黑热病治愈后2年内94.6%的患者rK39抗体仍为阳性。9年后仍有82.5%保持阳性。结论rK39层析试条对新疆黑热病患者有很高的诊断价值。其保存、携带方便,操作简单适于在基层现场使用。由于其可在潜伏期中发现黑热病患者。故可用于在流行地区皮内反应阴性的居民中进行免疫学检测,早期发现病人。由于黑热病患者治愈后血清中rK39抗体长期存在,故不适用于预后判定和疗效评价。     

与人类关系密切的7种动物对戊型肝炎病毒易感性的初步研究

目的　用双抗原夹心法ELISA (DS -ELISA)和逆转录巢式PCR法 (RT -nPCR)研究与人类生活关系密切的猪、犬、山羊、鸡、鸭、鸽子和兔等 7种动物对戊型肝炎病毒 (HEV)的易感性。方法　用多基因型HEV重组蛋白建立检测HEV抗体的DS -ELISA ,并用于 5 91份猪、警犬、家犬、宠物犬、山羊、鸡、鸭、鸽子和兔等多种动物血清标本的HEV抗体检测 ;用HEV多基因型通用性引物RT -nPCR扩增 14 3份犬血清标本HEVRNA。结果　在猪、家犬和宠物犬血清标本中检测到HEV抗体 ,阳性率分别为 4 3 93%、15 19%和 4 6 5 % ,但在警犬、山羊、鸡、鸭、鸽子和兔血清标本中未检测到HEV抗体 ;14 3份犬血清标本用RT -nPCR未扩增出HEVRNA。结论　用多基因型HEV重组蛋白建立的双抗原夹心法ELISA适用于多种动物血清标本HEV抗体的检测 ;猪和犬对HEV易感 ,在HEV传播中可能起重要作用。     

碳粒免疫试验(CIA)检测实验家兔弓形体抗体的研究

碳粒免疫试验(Carbon immunoassay——简称CIA)是一种直接的血清学方法。本文首次报道在国内应用CIA法对实验家兔弓形体抗体进行检测。共检测25只实验弓形体感染家兔的血清(55份),24只实验弓形体感染家兔的血滴(53份),两者弓形体抗体阳性率均为100％,无假阴性。血清抗体滴度高者可达1:5120,血滴抗体滴度高者可达1:64;前者几何平均滴度为1527,后者几何平均滴度为11.27。用正常家兔作对照,共检测正常家兔血清39份,正常家兔滤纸干血滴16份,结果均为阴性,未发现假阳性。还应用CIA法检测了10只实验家兔弓形体感染1周的血清和血滴,其弓形体抗体滴度均已能检出,而用酶联金葡菌A蛋白酶联免疫吸附试验(PPA-ELISA法)检测,10只兔中有1只尚未能检测出,提示前法似较后法敏感。另用同一份阳性和阴性血清分别进行PPA-ELISA法检测,两种方法的阳性符合率为98～100％,阴性符合率为100％。本文认为,CIA法是一种比较简便、经济、快速、灵敏的弓形体抗体检测方法,有推广应用的价值。     

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.     

Recombinant K28 antigen in ELISA in the diagnosis of canine visceral leishmaniosis

Crude total antigen (CTA) from Leishmania infantum and recombinant antigen K39 (rK39) and recombinant antigen K28 (rK28) were compared using an ELISA for the diagnosis of canine visceral leishmaniosis (CVL). Forty‐two blood samples from healthy dogs from a nonendemic area and 80 blood samples from an endemic area for dogs with visceral leishmaniosis (VL), confirmed with positive parasitological tests for Leishmania spp., were used in an ELISA. The parasitological diagnosis was chosen as a gold standard. The ELISA with rK28 antigen showed sensitivity of 100% and specificity of 100%, high agreement with CTA and rK39, indicating that the rK28 antigen is useful for ELISA serological diagnosis of CVL.     

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     

Latent class analysis of diagnostic tests for visceral leishmaniasis in Brazil

Objective To estimate the sensitivities and specificities of different diagnostic tests for visceral leishmaniasis (VL) using latent class analysis (LCA). Methods This study was performed using data from a prospective study conducted in four Brazilian states from May 2004 to May 2007. Five diagnostic tests for VL were evaluated in 285 VL cases and 119 non‐cases: microscopy, indirect fluorescence antibody test (IFAT), enzyme‐linked immunosorbent assay using recombinant K39 antigen (rK39‐ELISA), direct agglutination test (DAT) and the rK39 rapid test. Results Microscopy showed sensitivity of 77.0% (CI: 71.5–81.5) and specificity of 99.0% (CI: 94.0–99.7). The IFAT and the DAT showed similar sensitivities, 88.3% (CI: 84.0–92.0) and 88.5% (CI: 84.1–92.0), respectively, but the DAT had a higher specificity (95.4%, CI: 89.2–98.1) than did the IFAT (83.0%, CI: 75.0–88.2). The rK39‐ELISA and the rK39 rapid test showed sensitivities of 99.0% (CI: 96.3–99.6) and 94.0% (CI: 90.1–96.3), and specificities of 82.5% (CI: 75.0–88.3) and 100% (CI: 97.0–100.0%), respectively. Conclusions Considering the lack of an adequate reference standard, LCA proved to be a useful tool in validating diagnostic methods for VL. The DAT and the rK39 rapid test showed better performance. Thus, clinically suspected cases of VL in a Brazilian endemic area could be treated based on the positivity of one of these tests.     

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.     

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.     

Canine visceral leishmaniasis: performance of a rapid diagnostic test (Kalazar Detect) in dogs with and without signs of the disease

Current visceral leishmaniasis (VL) control programs in Brazil include the infected dog elimination but, despite this strategy, the incidence of human VL is still increasing. One of the reasons is the long delay between sample collection, analysis, control implementation and the low sensitivity of diagnostic tests. Due to the high prevalence of asymptomatic dogs, the diagnosis of these animals is important considering their vector infection capacity. Hence, a rapid and accurate diagnosis of canine visceral leishmaniasis is essential for an efficient surveillance program. In this study we evaluated the performance of rK39 antigen in an immunochromatographic format to detect symptomatic and asymptomatic Leishmania chagasi infection in dogs and compared the results with those using a crude antigen ELISA. The sensitivity of rK39 dipstick and ELISA were 83% vs. 95%, respectively, while the specificity was both 100%. Our results also demonstrated that the dipstick test was able to detect infected dogs presenting different clinical forms.     

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.     

The sensitivity and specificity of Leishmania chagasi recombinant K39 antigen in the diagnosis of American visceral leishmaniasis and in differentiating active from subclinical infection

The sensitivity and specificity of a Leishmania chagasi recombinant K39 (rK39)-based enzyme-linked immunosorbent assay (ELISA) for visceral leishmaniasis (VL) was assessed in Natal, Brazil. Anti-rK39 antibodies were detected in 93.3% of patients with parasitologically confirmed VL (n = 120) and in 33 others with clinically diagnosed disease. Anti-rK39 antibodies decreased significantly following treatment. The presence of antibodies was inversely correlated with development of a positive leishmanin skin test result. Anti-rK39 antibodies were detected in only 2.9% of asymptomatic subjects with a positive skin test result (n = 168). They were not detected in healthy controls (n = 30) or in persons with Chagas' disease (n = 13) or active tuberculosis (n = 31). Antibodies were found in only one of 13 patients with cutaneous leishmaniasis. In contrast, an ELISA using total L. chagasi promastigote antigen was sensitive, but not specific. The results indicate that the rK39-based ELISA is a sensitive and specific diagnostic test for symptomatic VL and can differentiate progressive from self-resolving infection.     

Performance of recombinant K39 antigen in the diagnosis of Brazilian visceral leishmaniasis

This study evaluated the performance of recombinant K39 (rK39) antigen in a immunochromatographic format (strip test) and a crude antigen enzyme-linked immunosorbent assay in the diagnosis of Brazilian visceral leishmaniasis (VL) in 128 consecutive patients with parasitologically proven infections (by microscopy and/or culture). For each patient, a medical history was obtained and a complete physical examination was performed. Controls included 10 healthy volunteers and 50 patients with other diseases: malaria (10), leprosy (9), Chagas' disease (10), tuberculosis (10), and cutaneous leishmaniasis (11). The sensitivities of the rK39 antigen strip test and the ELISA were 90% and 89%, respectively, while the specificities were 100% and 98%, respectively. Our study confirms the accuracy of the rK39 antigen strip test in the diagnosis of VL in a high prevalence population.