Department of Food and Environmental Virology, National Veterinary Research Institute, Al. Partyzantów 57, 24-100 Puławy, Poland.
Department of Hygiene of Animal Feedingstuffs, National Veterinary Research Institute, Al. Partyzantów 57, 24-100 Puławy, Poland.
Int J Food Microbiol. 2019 May 2;296:14-20. doi: 10.1016/j.ijfoodmicro.2019.02.011. Epub 2019 Feb 19.
Growing demand for fresh, unprocessed food favours the emergence of Cryptosporidium infections in humans. Mainly it is food of plant origin or unpasteurized milk which have been involved in food-borne outbreaks of cryptosporidiosis. So far consumption of shellfish contaminated with Cryptosporidium were not associated with human infections although such as possibility exists. In this study an attempt was undertaken to evaluate the analytical performance of three commonly used methods for recovery of Cryptosporidium oocysts from shellfish: i) pepsin digestion of shellfish in conjunction with immunomagnetic separation (IMS) of oocysts (method A), ii) pepsin-HCl treatment of shellfish homogenate without IMS (method B), and iii) a strainer method with direct oocyst extraction and separation from shellfish tissue using IMS (method C). Each method's performance was assessed according to the ISO standard requirements by testing shellfish homogenates seeded with different numbers of C. parvum oocysts. Two groups of parameters were compared, encompassing precision (coefficient of variation (CV)) and accuracy of measurements. These were described by linear regression models allowing calculation of the methods' limits of detection (LOD) and quantification (LOQ). In addition, oocyst recovery efficiencies from shellfish were calculated for each method. All three compared methods allowed for at least 66% recovery of Cryptosporidium oocysts from the tested samples. The best recovery (83.3-100%) in the whole range of tested suspensions was obtained for method C. The accuracy of method B was better (linearity of r = 0.9996 in the full measurement range) than that of method A (r = 0.968). Method C showed the best accuracy (r = 1) and precision (CV 0.2-14.1). Compared to other methods it was also characterised by the best LOD and LOQ, attaining ≅4 and ≅12 oocysts per 3 g of tested shellfish sample respectively. Despite a lack of the ability of method A to give the proportional results in oocysts recovery (non-linearity of the method) compared to the reference values, it achieved the highest LOD and LOQ values among the tested methods. As demonstrated here, the most efficient method for extraction of Cryptosporidium oocysts from shellfish tissues was method C employing sample homogenisation and separation of oocysts from tissue debris using IMS. Used alone this method does not in fact allow for identification of Cryptosporidium species but delivers quantitative results concerning the level of food contamination by parasites.
对新鲜、未加工食品的需求不断增长，有利于人类中隐孢子虫感染的出现。主要是植物来源的食品或未经巴氏消毒的牛奶，这些食品曾涉及隐孢子虫病的食源性暴发。迄今为止，虽然存在这种可能性，但食用受隐孢子虫污染的贝类与人类感染无关。在这项研究中，我们试图评估三种常用于从贝类中回收隐孢子虫卵囊的常用方法的分析性能：i）贝类胃蛋白酶消化结合卵囊免疫磁分离（IMS）（方法 A），ii）无 IMS 的贝类匀浆胃蛋白酶 -HCl 处理（方法 B），以及 iii）一种使用 IMS 从贝类组织中直接提取和分离卵囊的筛网方法（方法 C）。根据 ISO 标准要求，通过测试用不同数量的 C. parvum 卵囊接种的贝类匀浆，评估每种方法的性能。比较了两组参数，包括测量的精密度（变异系数 (CV)）和准确性。这些通过线性回归模型来描述，允许计算方法的检测限 (LOD) 和定量限 (LOQ)。此外，还计算了每种方法从贝类中回收卵囊的效率。所有三种比较方法都允许从测试样品中至少回收 66%的隐孢子虫卵囊。方法 C 在整个测试悬浮液范围内获得了最佳的回收（83.3-100%）。方法 B 的准确性更好（在整个测量范围内线性 r=0.9996），而方法 A 的准确性较差（r=0.968）。方法 C 显示出最佳的准确性（r=1）和精密度（CV 0.2-14.1）。与其他方法相比，它还具有最佳的 LOD 和 LOQ，分别达到 ≅4 和 ≅12 个卵囊/3g 测试贝类样本。尽管方法 A 缺乏在卵囊回收方面给出比例结果的能力（方法的非线性）与参考值相比，它在测试方法中实现了最低的 LOD 和 LOQ 值。如这里所示，从贝类组织中提取隐孢子虫卵囊最有效的方法是方法 C，该方法采用样品匀浆和 IMS 从组织碎片中分离卵囊。单独使用该方法实际上无法鉴定隐孢子虫种类，但可提供有关寄生虫污染食物程度的定量结果。
