Data for: Reliability of techniques used in the diagnosis of canine visceral leishmaniasis: A survey in an area of recent transmission in southeastern Brazil

One of the key components of the Brazilian Program for the Control of Visceral Leishmaniasis (PCLV) is the euthanasia of Leishmania-infected canine reservoirs, the detection of which depends on a screening procedure involving a Dual Path Platform® (DPP) immunoassay and a confirmatory enzyme-linked immunosorbent assay (ELISA). The aims of the present study were to evaluate the reliability of these techniques in a region of recent transmission of canine VL, to follow up the seroconversion 3 to 4 months after the initial diagnosis of DPP reactive but ELISA indeterminate or non-reactive dogs, and to identify the species of Leishmania in circulation in the area. Each animal was submitted to DPP under field conditions, performed by municipal health workers using peripheral blood (DPP-field), to DPP under laboratory conditions using serum (DPP-lab) and to ELISA using serum. The agreements between the tests were determined using McNemar's χ2 test, Cohen’s kappa coefficient (k) at the 95% confidence interval and prevalence-adjusted bias-adjusted kappa (PABAK). Of the 1130 dogs examined, 74.2% were non-reactive in all three tests applied. Based on the PCLV positive-infection criterion, seroprevalence was 8.9% (101/1130) with 83.2% (84/101) of infected animals showing reactivity in all three tests while 7.8% (8/101) were reactive in DPP-field and ELISA and 8.9% (9/101) in DPP-lab and ELISA. The proportions of disagreements were substantial in all comparisons. Inter-rater reliability between DPP-field and ELISA (k = 0.55; PABAK = 0.78) and DPP-lab and ELISA (k = 0.59; PABAK = 0.81) were considered moderate, while that between DPP-field and DPP-lab (k = 0.61; PABAK = 0.79) was classified as marginally good. The proportion of seroconversions in DPP reactive animals that were initially ELISA indeterminate was significantly higher than in those that were DPP reactive but initially ELISA non-reactive. Restriction fragment length polymorphism analysis revealed the presence of Leishmania infantum, the etiologic agent of VL, in bone marrow samples from VL-infected animals. Our data showed that the techniques and protocols currently employed in the PCLV screening approach are not entirely reliable. Further consideration should be given to monitoring dogs with undetermined results in ELISA and a better training should be provided for health workers responsible for performing DPP tests applied under field conditions.

巴西内脏利什曼病控制计划（Brazilian Program for the Control of Visceral Leishmaniasis, PCLV）的核心举措之一，是对感染利什曼原虫的犬类储存宿主实施安乐死；该类宿主的检测需依托联合筛查流程，即采用双路径平台®（Dual Path Platform®, DPP）免疫分析法，辅以验证用酶联免疫吸附试验（enzyme-linked immunosorbent assay, ELISA）。本研究的研究目的包括：评估上述两种检测技术在近期暴发犬内脏利什曼病（canine Visceral Leishmaniasis, VL）的区域内的可靠性；对初始DPP反应呈阳性但ELISA结果不确定或呈阴性的犬只，在确诊后3至4个月开展血清转换情况的追踪随访；明确该区域流行的利什曼原虫菌种。所有受试犬只均接受三类检测：由市政卫生工作人员使用外周血在野外条件下开展的DPP检测（简称DPP-野外组）、使用血清在实验室条件下开展的DPP检测（简称DPP-实验室组），以及血清ELISA检测。检测间的一致性采用麦克尼马尔χ²检验、95%置信区间下的科恩kappa系数（k）以及患病率校正偏倚校正kappa值（PABAK）进行评估。本次研究共纳入1130只受试犬，其中74.2%的犬只在三项检测中均呈阴性反应。依据PCLV的阳性感染判定标准，本次研究的血清阳性率为8.9%（101/1130）；其中83.2%（84/101）的感染犬只在三项检测中均呈阳性反应，7.8%（8/101）的感染犬只仅DPP-野外组与ELISA呈阳性，8.9%（9/101）的感染犬只仅DPP-实验室组与ELISA呈阳性。所有检测对比组的不一致比例均较高。DPP-野外组与ELISA（k=0.55；PABAK=0.78）、DPP-实验室组与ELISA（k=0.59；PABAK=0.81）的组间信度被评定为中等水平；而DPP-野外组与DPP-实验室组的组间信度（k=0.61；PABAK=0.79）则被归类为轻度良好。初始ELISA结果不确定的DPP反应阳性犬只，其血清转换率显著高于初始ELISA结果呈阴性的DPP反应阳性犬只。限制性片段长度多态性分析显示，内脏利什曼病的病原体——婴儿利什曼原虫（Leishmania infantum）存在于感染内脏利什曼病的犬只骨髓样本中。本研究数据表明，当前PCLV筛查流程所采用的技术与方案并非完全可靠。未来应进一步关注对ELISA结果不确定的犬只的监测工作，并为负责开展野外DPP检测的卫生工作人员提供更完善的培训。