Laboratório de Interação Parasito-Hospedeiro e Epidemiologia, Instituto Gonçalo Moniz-FIOCRUZ, Salvador, Bahia, Brazil.
Centro de Integração de Dados e Conhecimentos para Saúde, Instituto Gonçalo Moniz-FIOCRUZ, Salvador, Bahia, Brazil.
PLoS Negl Trop Dis. 2018 Oct 26;12(10):e0006871. doi: 10.1371/journal.pntd.0006871. eCollection 2018 Oct.
Canine Visceral leishmaniasis (CVL) is a serious public health problem, thus for its control, the Ministry of Health in Brazil recommends the rapid diagnosis and euthanasia of seropositive dogs in endemic areas. Therefore, our group had previously selected six recombinant proteins (rLci1, rLci2, rLci4, rLci5, rLci8, and rLci12) due to their high potential for CVL diagnostic testing. The present study aims to produce an immunodiagnostic test using the aforementioned antigens, to improve the performance of the diagnosis of CVL recommended by Brazilian Ministry of Health.
METHODOLOGY/PRINCIPAL FINDINGS: To evaluate the recombinant proteins in the serological assays, positive and negative samples were selected based on parasitological test (culture) and molecular test (qPCR) of splenic aspirate. Initially, we selected 135 dog serum samples, 73 positives (symptomatic and asymptomatic) and 62 negatives to screen recombinant proteins on ELISA platform. Then, for rLci5 ELISA validation, 361 serum samples collected in a cross-sectional study were selected, being 183 positives (symptomatic and asymptomatic) and 178 negatives. In the screening of the recombinant proteins, rLci5 was the only protein to present a performance statistically higher than the performance presented by EIE-LVC test, presenting 96% (IC 95%; 85-99%) vs. 83% (IC 95%; 69-92%) of sensitivity for symptomatic dogs, 71% (IC 95%; 49-97%) vs. 54% (IC 95%; 33-74%) for asymptomatic dogs and 94% (IC 95%; 83-99%) vs, 88% (IC 95%; 76-95% of specificity. Thus, the rLci5 protein was selected to compose a final ELISA test. Validation of rLci5 ELISA showed 87% (IC 81-91%) of sensitivity, 94% (IC 95%; 90-97%) of specificity and 90% accuracy. Testing the EIE-LVC with the same validation panel, we observed a lower performance when compared to ELISA rLci5 (sensitivity of 67% (IC 95%; 59-74%), specificity of 87% (IC 95%; 81-92%), and accuracy of 77%). Finally, the performance of current CVL diagnostic protocol recommended by Brazilian Ministry of Health, using DPP-LVC as screening test and EIE-LVC as confirmatory test, was compared with a modified protocol, replacing EIE-LVC by rLci5 ELISA. The current protocol presented a sensitivity of 59% (IC 95%; 52-66%), specificity of 98% (IC 95%; 95-99%) and accuracy of 80% (IC 95%; 76-84%), while the modified protocol presented a sensitivity of 71% (IC 95%; 63-77%), specificity of 99% (IC 95%; 97-100%) and accuracy of 86% (IC 95%; 83-89%).
Thus, we concluded that rLci5 ELISA is a promising test to replace EIE-LVC test and increase the diagnostic performance of CVL in Brazil.
犬内脏利什曼病（CVL）是一个严重的公共卫生问题，因此巴西卫生部建议在流行地区快速诊断和对血清阳性犬实施安乐死。因此，我们小组之前选择了 6 种重组蛋白（rLci1、rLci2、rLci4、rLci5、rLci8 和 rLci12），因为它们在 CVL 诊断测试方面具有很高的潜力。本研究旨在使用上述抗原生产免疫诊断测试，以提高巴西卫生部推荐的 CVL 诊断性能。
方法/主要发现：为了评估重组蛋白在血清学检测中的性能，根据脾脏抽吸的寄生虫学检测（培养）和分子检测（qPCR）选择了阳性和阴性样本。最初，我们筛选了 135 份狗血清样本，其中 73 份为阳性（有症状和无症状），62 份为阴性，用于 ELISA 平台上的重组蛋白筛选。然后，为了验证 rLci5 ELISA，我们选择了 361 份在横断面研究中收集的血清样本，其中 183 份为阳性（有症状和无症状），178 份为阴性。在重组蛋白的筛选中，rLci5 是唯一一种表现优于 EIE-LVC 检测的蛋白，对有症状犬的敏感性为 96%（95%CI；85-99%），比 EIE-LVC 检测的 83%（95%CI；69-92%）高，对无症状犬的敏感性为 71%（95%CI；49-97%），比 EIE-LVC 检测的 54%（95%CI；33-74%）高，特异性为 94%（95%CI；83-99%），比 EIE-LVC 检测的 88%（95%CI；76-95%）高。因此，rLci5 蛋白被选中用于组成最终的 ELISA 检测。rLci5 ELISA 的验证显示敏感性为 87%（81-91%），特异性为 94%（95%CI；90-97%），准确性为 90%。用相同的验证面板检测 EIE-LVC，我们发现其性能低于 rLci5 ELISA（敏感性为 67%（95%CI；59-74%），特异性为 87%（95%CI；81-92%），准确性为 77%）。最后，比较了巴西卫生部目前推荐的 CVL 诊断方案，使用 DPP-LVC 作为筛查试验，EIE-LVC 作为确认试验，与修改后的方案进行比较，用 rLci5 ELISA 代替 EIE-LVC。现行方案的敏感性为 59%（95%CI；52-66%），特异性为 98%（95%CI；95-99%），准确性为 80%（95%CI；76-84%），而修改后的方案的敏感性为 71%（95%CI；63-77%），特异性为 99%（95%CI；97-100%），准确性为 86%（95%CI；83-89%）。
因此，我们得出结论，rLci5 ELISA 是一种很有前途的替代 EIE-LVC 检测的测试方法，可以提高巴西 CVL 的诊断性能。
