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Performance of KL914-ELISA and KL914-RDT to detect super-spreaders
Highly Mildly Never Sensitivity Specificity False positive False negative Degree of agreement1 Kappa RDT 14/23 (60%) 6/30 (20%) 2/56 (3.6%) 60.1% 96.4% 3.6% 39% 86.1% z=5.76 p<0.001 0.63 ELISA 23/23 (100%) 27/30 (90%) 7/56 (12.5%) 100% 87.5% 12.5% 0% 91.1% z=7.23 p<0.001 0.80
118 Finally, while comparing the results of ELISA and RDT, the degree of agreement was 61.16% (Table 6.3) which suggests improvement is needed, and a kappa coefficient of 0.26 (z=3.87, p<0.0001). Individuals results were also compared in the Table 6.3 for each group of infectious (super-spreaders, mildly and never-infectious)
Table 6.3 – Comparison of the detection between KL914-RDT and KL914-ELISA with
degree of agreement measured using Cohen’s method and the kappa coefficient.
Comparison of the individual detection between RDT and ELISA and degree of agreement using Cohen’s method
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6.4 Discussion
The development of the first prototype RDT for detecting super-spreaders was achieved. Due to the limited number of tests available for the experiment, the results presented here are considered to be preliminary; however, the project generated a proof- of-concept that the RDT can be developed. Further development to improve its sensitivity is needed (discussed below), before it can be evaluated under field conditions.
In the example of longitudinal follow-up of a super-spreader dog, the rapid diagnostic tests based on KL914 were run with all samples of the sera collection. The RDT used as screening method matched at all times points with the xenodiagnosis and the ELISA results, giving a 100 percent of agreement between these three methods. Hence, the RDT worked perfectly for this dog to identify sera sample of dogs with high infectiousness potential. However, due to individual variability, not all dogs offered a perfect longitudinal detection. After analysing all the dogs tested in the assays, the overall performance of KL914-RDT showed a strong degree of agreement (86.1%) with the xenodiagnoses used to determine the infectiousness of the dog (k=0.63), suggesting a high diagnostic potential for super-spreaders. However, the degree of agreement was higher for the KL914-ELISA and xenodiagnosis (91.1%) without being significant difference compared to the RDT and the correlation between ELISA and RDT was also lower by Pearson’s’ correlation. While comparing the sensitivity and specificity of the KL914-rapid test to its equivalent in immune-assays, the results highlight that ELISA is the more sensitive method, but that the RDT has a higher specificity in detecting super-spreaders. The specificity of RDT to identify super-spreaders increased compared to the immune-assays, to 96.4% and 87.5 respectively. The RDT had a low detection of mildly infectious dogs (20%); this is not considered a major issue as mildly infectious dogs are believed not to contribute to most transmission events. Moreover, only 3.6% of the samples were detected as false positives by the RDT which is an advantage to improve dog-owner compliance.
The reduced sensitivity of the RDT is likely the result of the conversion of the antigen into a rapid diagnostic tool, as observed in other studies (Pattabhi et al., 2010; de Silva Solcà et al., 2014). Similar discrepancies were observed between other antigens and
120 their respective RDTs for the detection infection in canine population. For example, the crude Leishmania antigen (CLA) was tested in ELISA and RDT, offering sensitivities of 78,3 % and 72,5%, and specificities of 90% and 84%, respectively, in detecting
Leishmania infection (de Silva Solcà et al., 2014). This shows a decrease in detection
when the antigen is converted to RDT. Moreover, the adaptation of rK28 into a dipstick was only measured for human VL infection: the RDT showed a lower sensitivity compared to the ELISA format, without being significantly different, in the detection of human VL infection (Pattabhi et al., 2010). No publications comparing rK28 in ELISA and in RDT could be found on canine trials. Very limited studies involve the detection of canine infectiousness; indeed, only one could be found in the literature comparing the performance of tests towards infectiousness. The performance of rK39 was compared in ELISA and in the format of Kalazar Detect™ Rapid Test, showing that the detection of highly infectious dogs dropped from 97% to 79% (Courtenay et al., 2014). The reason why antigens perform weaker in rapid diagnostic tools than in ELISA formats remains unclear. Several reasons could be suggested to explain the lack of performance such as the composition of the migration buffer or the adhesion of the protein to the nitrocellulose membrane. Moreover, during the conception of the RDT in IDRI/InBios, the sera conservation conditions were not optimal; indeed, some of the serum seems to be dried out in the wells, even if the reactivity of the samples was validated and confirmed by another ELISA during the secondment at IDRI. Therefore, further work is required to evaluate the reasons for this discrepancy.
In conclusion, the first prototype rapid diagnostic test for the detection of super- spreaders has been developed based on a new protein, for its adaption into a field- friendly tool. Further improvements could be made to the sensitivity of the RDT. On full development of the RDT, the impact of such a tool on transmission, when applied to the mixed canine population, will require quantification by mathematical modelling dynamic scenarios. This is the subject of the next chapter, with the aim of modelling the most efficient screening method in the field, and generating recommendations to improve the Visceral Leishmaniasis Control and Surveillance Programme (VLCSP).
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