Leathwick Dave, Green Peter, Bouchet Charlotte, Chambers Alex, Waghorn Tania, Sauermann Christian
AgResearch, Grasslands Research Centre, Private Bag 11008, Palmerston North, 4442, New Zealand.
AgResearch, Grasslands Research Centre, Private Bag 11008, Palmerston North, 4442, New Zealand.
Int J Parasitol Drugs Drug Resist. 2025 Apr;27:100589. doi: 10.1016/j.ijpddr.2025.100589. Epub 2025 Mar 26.
In the faecal egg count reduction test, visual identification of larvae cultured from faeces enables the egg counts to be apportioned to species/genera, resulting in a more accurate test. However, morphology cannot reliably differentiate some species meaning that, in some cases, efficacy can only be estimated at the genus or species-complex level. We investigated the benefits of identifying larvae to species using DNA to determine how often this would alter the diagnosis of resistance and whether increasing the number of larvae identified would alter the repeatability of an efficacy estimate. Data on faecal nematode egg counts and the corresponding larval species mixes were acquired from tests conducted on commercial sheep farms. The proportion of each species present in faecal culture was determined using DNA. Efficacy was then compared for individual species and for those genera/species complexes which cannot reliably be differentiated visually. The proportion of each species present was subsequently resampled 10,000 times (repeated random sampling) and efficacy recalculated to produce the median efficacy, along with the 5 % and 95 % simulation percentiles. Subsequently, the number of larvae sampled to determine the species mix in each sample was varied from 50 to 6400 and the process repeated. Of 152 comparisons of efficacy, 25 % of cases where genus-level identification resulted in a finding of 'susceptible' for that category, species-level identification returned at least one diagnosis of 'resistant' i.e., genus-level identification resulted in a 25 % false negative diagnosis. When the number of larvae sampled for species identification was low (<400) variation in efficacy estimates was high, however, as sample size increased the confidence interval around the efficacy estimate decreased. The results indicate that identifying large numbers of larvae to species using DNA has the potential to increase the accuracy and confidence in efficacy estimates achieved using the faecal egg count reduction test.
在粪便虫卵计数减少试验中，通过目视识别从粪便中培养出的幼虫，可将虫卵计数按物种/属进行分配，从而使试验更加准确。然而，形态学无法可靠地区分某些物种，这意味着在某些情况下，疗效只能在属或物种复合体水平上进行估计。我们研究了使用DNA将幼虫鉴定到物种的益处，以确定这种方法会在多大程度上改变耐药性诊断，以及增加鉴定的幼虫数量是否会改变疗效估计的可重复性。粪便线虫虫卵计数数据以及相应的幼虫物种混合数据来自在商业养羊场进行的试验。使用DNA确定粪便培养物中每个物种的比例。然后比较各个物种以及那些无法通过目视可靠区分的属/物种复合体的疗效。随后，对每个物种的比例进行10000次重新采样（重复随机抽样），并重新计算疗效，以得出中位疗效以及5%和95%的模拟百分位数。随后，将每个样本中用于确定物种混合的幼虫采样数量从50只变化到6400只，并重复该过程。在152次疗效比较中，有25%的情况在属水平鉴定时该类别结果为“敏感”，而在物种水平鉴定时至少有一次诊断为“耐药”，即属水平鉴定导致了25%的假阴性诊断。当用于物种鉴定的幼虫采样数量较低（<400只）时，疗效估计的变异性较高，然而，随着样本量的增加，疗效估计周围的置信区间会减小。结果表明，使用DNA将大量幼虫鉴定到物种有可能提高使用粪便虫卵计数减少试验获得的疗效估计的准确性和可信度。
