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。     

rK39抗原试条法检测家犬内脏利什曼病

在中国西部 6省 /自治区 (包括 :新疆 ,甘肃 ,四川 ,陕西 ,山西和内蒙古 )的一些山区和荒漠 ,目前黑热病(内脏利什曼病 )呈散发状态[1 ] ,其病原体为婴儿利什曼原虫 (Ｌｅｉｓｈｍａｎｉａｉｎｆａｎｔｕｍ) [2 ] 。患内脏利什曼病的家犬为山丘地带黑热病的主要动物宿主 ,而在新疆和内蒙古的荒漠地带 ,在黑热病的疫区内却未查见病犬 ,动物宿主不明[3 ] 。近期来的研究表明 ,在各种能引起黑热病的利什曼原虫的无鞭毛体中 ,均存在编码 39氨基酸的基因片段 (Ｋ39) ,以该基因片段或其重组抗原 (ｒＫ39)制成的ｄｉｐｓｔｉｃｋ试条 ,检测针对利什…     

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     

Dipstick法诊断黑热病患者28例

诊断黑热病的传统方法 ,是以骨髓穿刺物涂片镜检或培养 ,但骨髓穿刺不仅给患者造成痛苦 ,而且易漏诊。 70年代以来 ,黑热病的血清学检查在国内外发展很快 ,如ＩＦＡ[1 ] 、ＩＨＡ[2 ] 、ＥＬＩＳＡ[3 ] 和ＭｃＡｂ ＡＳＴ[4] 等方法。Ｂｕｒｎｓ等[5 ] 获得了利什曼原虫类Ｋｉｎｅｓｉｎ基因中编码 39个氨基酸重组片段的表达产物ｒＫ39,用于美洲内脏利什曼病的诊断 ,阳性率 10 0 %。Ｄｉｐｓｔｉｃｋ是近年发展起来的一种快速诊断方法 ,它将免疫分子亲和原理和免疫印迹法、经典的薄层层析技术相结合。由于其敏感性高、操作简便和报告结果快…     

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^...     

rK39免疫层析试纸条用于诊断甘肃省内脏利什曼病人和病犬

1997年 6月 ,美国芝加哥大学张光朴教授和中国预防医学科学院寄生虫病研究所瞿靖琦研究员来本省武都县考察内脏利什曼病 (黑热病 ,VL ) ,并用诊断VL的 r K3 9免疫层析试纸条 (dipstick)检测了该县的一些 VL 病例 ,证实该法具有快速、正确诊断 VL 的效果。之后 ,我们又用张光朴教授提供的 dipstick于 1997～ 1999年在更大范围内对 VL患者和疫区内家犬进行了检测 ,结果如下。1　病例选择被测试者来自本省 VL流行区的武都、迭部、舟曲和静宁 4县共 6 0例患者。凡患者具有下列三项之一者 ,即为 VL病例。(1)骨髓穿刺涂片 (BMP)镜检查见利…     

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.     

聚合酶链反应用于黑热病诊断的实验研究

用ＰＣＲ方法对２５名确诊的黑热病病人和６０例对照进行黑热病诊断的实验研究，结果显示：其灵敏度９６％（２４／２５）特异度１００％（６０／６０），假阳性率０％，假阴性率４％，符合率９９％，约登指数为０．９６，ＰＣＲ方法能在外周血清仅为１μｌ情况下测出其中原虫的ＤＮＡ。     

rK39 antigen for the diagnosis of visceral leishmaniasis by using human saliva

The rK39 rapid immunochromatographic test (ICT) is now being widely used in the diagnosis of visceral leishmaniasis (VL) using serum. We evaluated the presence of anti-rK-39 antibody in human saliva being noninvasive to replace the invasive procedures of diagnosis. Enzyme-linked immunosorbent assay (ELISA) and ICT assays were performed in 300 subjects: 114-confirmed VL patients, 95 and 47 healthy controls from endemic and nonendemic regions, respectively, and 44 subjects with different diseases. Sensitivity in saliva was 83.3% by ELISA and 82.5% by ICT, compared with 100% for both ICT and ELISA in serum. Specificity in saliva was 100%, 90.5%, and 88.6% with ELISA, and 91.48%, 91.57%, and 84.06% using ICT, in nonendemic, endemic, and different diseases, respectively. In serum, specificity was 97%, 88.5%, and 89% by ELISA and 100%, 94.7%, and 95.5% by ICT in nonendemic, endemic, and different diseases, respectively. Saliva is not suitable for diagnosis of VL because of low sensitivity.     

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.     

Evaluation of two rK39 dipstick tests, direct agglutination test, and indirect fluorescent antibody test for diagnosis of visceral leishmaniasis in a new epidemic site in highland Ethiopia

We assessed the performance characteristics of two rK39 immunochromatographic tests, a direct agglutination test (DAT), and an indirect immunofluorescent antibody test (IFAT) in the site of a new epidemic of visceral leishmaniasis (VL) in northwestern Ethiopia. The study population was composed of 179 patients with suspected VL and 67 controls. The sensitivities of Kalazar Detect^®, DiaMed-IT Leish^®, DAT, and IFAT in 35 polymerase chain reaction–confirmed VL cases were 94.3%, 91.4%, 91.4%, and 100%, respectively, and the specificities were 98.5%, 94%, 98.5%, and 98.5%, respectively. In a Bayesian latent class analysis of all 246 specimens, the estimated sensitivities were 90.5%, 89%, 88.8%, and 96% for Kalazar Detect^®, DiaMed-IT Leish^®, DAT, and IFAT, respectively; DAT showed the highest estimated specificity (97.4%). Both rK39 immunochromatographic tests perform as well as DAT, and are suitable for VL diagnosis in first-level health centers in this area of Ethiopia.     

Case series of misdiagnosis with rK39 strip test in Indian leishmaniasis

This report presents three cases where the rK39 strip test failed to diagnose two cases of post-kala-azar dermal leishmaniasis and one case of visceral leishmaniasis. However, a strong clinical suspicion prompted further evaluation by polymerase chain reaction (PCR), which established the etiology. The present case series highlights the usefulness of PCR in the diagnosis of leishmaniasis.     

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.     

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.     

Recombinant Leishmania (Leishmania) infantum Ecto-Nucleoside Triphosphate Diphosphohydrolase NTPDase-2 as a new antigen in canine visceral leishmaniasis diagnosis

Canine visceral leishmaniasis is an important public health concern. In the epidemiological context of human visceral leishmaniasis, dogs are considered the main reservoir of Leishmania parasites; therefore, dogs must be epidemiologically monitored constantly in endemic areas. Furthermore, dog to human transmission has been correlated with emerging urbanization and increasing rates of leishmaniasis infection worldwide. Leishmania (Leishmania) infantum (L. chagasi) is the etiologic agent of visceral leishmaniasis in the New World. In this work, a new L. (L.) infantum (L. chagasi) recombinant antigen, named ATP diphosphohydrolase (rLic-NTPDase-2), intended for use in the immunodiagnosis of CVL was produced and validated. The extracellular domain of ATP diphosphohydrolase was cloned and expressed in the pET21b-Escherichia coli expression system. Indirect ELISA assays were used to detect the purified rLic-NTPDase-2 antigen using a standard canine sera library. This library contained CVL-positive samples, leishmaniasis-negative samples and samples from Trypanosoma cruzi-infected dogs. The results show a high sensitivity of 100% (95% CI = 92.60–100.0%) and a high specificity of 100% (95% CI = 86.77–100.0%), with a high degree of confidence (k = 1). These findings demonstrate the potential use of this recombinant protein in immune diagnosis of canine leishmaniasis and open the possibility of its application to other diagnostic approaches, such as immunochromatography fast lateral flow assays and human leishmaniasis diagnosis.