Kostidis Sarantos, Kokova Daria, Dementeva Natalia, Saltykova Irina V, Kim Hye Kyong, Choi Young Hae, Mayboroda Oleg A
Center for Proteomics and Metabolomics, Leiden University Medical Centre, Leiden, The Netherlands.
Department of Parasitology, Leiden University Medical Center, Leiden, The Netherlands.
BMC Infect Dis. 2017 Apr 17;17(1):275. doi: 10.1186/s12879-017-2351-7.
Analysis of the stool samples is an essential part of routine diagnostics of the helminthes infections. However, the standard methods such Kato and Kato-Katz utilize only a fraction of the information available. Here we present a method based on the nuclear magnetic resonance spectroscopy (NMR) which could be auxiliary to the standard procedures by evaluating the complex metabolic profiles (or phenotypes) of the samples.
The samples were collected over the period of June-July 2015, frozen at -20 °C at the site of collection and transferred within four hours for the permanent storage at -80 °C. Fecal metabolites were extracted by mixing aliquots of about 100 mg thawed stool material with 0.5 mL phosphate buffer saline, followed by the homogenization and centrifugations steps. All NMR data were recorded using a Bruker 600 MHz AVANCE II spectrometer equipped with a 5 mm triple resonance inverse cryoprobe and a z-gradient system.
Here we report an optimized method for NMR based metabolic profiling/phenotyping of the stools samples. Overall, 62 metabolites were annotated in the pool sample using the 2D NMR spectra and the Bruker Biorefcode database. The compounds cover a wide range of the metabolome including amino acids and their derivatives, short chain fatty acids (SCFAs), carboxylic acids and their derivatives, amines, carbohydrates, purines, alcohols and others. An exploratory analysis of the metabolic profiles reveals no strong trends associated with the infection status of the patients. However, using the penalized regression as a variable selection method we succeeded in finding a subset of eleven variables which enables to discriminate the patients on basis of their infections status.
A simple method for metabolic profiling/phenotyping of the stools samples is reported and tested on a pilot opisthorchiasis cohort. To our knowledge this is the first report of a NMR-based feces analysis in the context of the helminthic infections.
粪便样本分析是蠕虫感染常规诊断的重要组成部分。然而，诸如加藤法和加藤-凯茨法等标准方法仅利用了可用信息的一小部分。在此，我们提出一种基于核磁共振波谱（NMR）的方法，该方法可通过评估样本的复杂代谢谱（或表型）辅助标准程序。
样本于2015年6月至7月期间采集，在采集地点于-20°C冷冻，并在四小时内转移至-80°C进行长期储存。通过将约100mg解冻的粪便材料等分试样与0.5mL磷酸盐缓冲盐水混合，随后进行匀浆和离心步骤来提取粪便代谢物。所有NMR数据均使用配备5mm三共振逆低温探头和z梯度系统的布鲁克600MHz AVANCE II光谱仪记录。
在此，我们报告了一种用于粪便样本基于NMR的代谢谱分析/表型分析的优化方法。总体而言，使用二维NMR光谱和布鲁克生物参考代码数据库在混合样本中注释了62种代谢物。这些化合物涵盖了广泛的代谢组，包括氨基酸及其衍生物、短链脂肪酸（SCFAs）、羧酸及其衍生物、胺、碳水化合物、嘌呤、醇类等。对代谢谱的探索性分析显示，未发现与患者感染状态相关的强烈趋势。然而，使用惩罚回归作为变量选择方法，我们成功找到了一个由11个变量组成的子集，该子集能够根据患者的感染状态对其进行区分。
报告了一种用于粪便样本代谢谱分析/表型分析的简单方法，并在一个华支睾吸虫病试点队列中进行了测试。据我们所知，这是在蠕虫感染背景下基于NMR的粪便分析的首次报告。
