Le Govic Y, Guyot K, Certad G, Deschildre A, Novo R, Mary C, Sendid B, Viscogliosi E, Favennec L, Dei-Cas E, Fréalle E, Dutoit E
Laboratoire de Parasitologie-Mycologie, CHRU de Lille & Faculté de Médecine de Lille, Université de Lille, Villeneuve-d'Ascq, France.
Laboratoire de Parasitologie-Mycologie, Centre Hospitalier Universitaire d'Angers, France; Groupe d'Etude des Interactions Hôte-Pathogène, UPRES-EA 3142, UNAM Université, Université d'Angers, Angers, France.
Eur J Clin Microbiol Infect Dis. 2016 Jan;35(1):137-48. doi: 10.1007/s10096-015-2519-2. Epub 2015 Nov 26.
Cryptosporidiosis is an important though underreported public health concern. Molecular tools might be helpful in improving its diagnosis. In this study, ZR Fecal DNA MiniPrep™ Kit (ZR) and NucliSens® easyMAG® (EM) were compared using four Cryptosporidium-seeded feces and 29 Cryptosporidium-positive stools. Thereafter, ZR was selected for prospective evaluation of Cryptosporidium detection by 18S rDNA and LAXER quantitative PCR (qPCR) in 69 stools from 56 patients after Cryptosporidium detection by glycerin, modified Ziehl-Neelsen (ZN) and auramine-phenol (AP) stainings. The combination of any of the two extraction methods with 18S qPCR yielded adequate detection of Cryptosporidium in seeded stools, but the ZR kit showed the best performance. All 29 Cryptosporidium-positive samples were positive with 18S qPCR, after both ZR and EM extraction. However, false-negative results were found with LAXER qPCR or nested PCR. Cryptosporidiosis was diagnosed in 7/56 patients. All the microscopic methods enabled the initial diagnosis, but Cryptosporidium was detected in 12, 13, and 14 samples from these seven patients after glycerin, ZN, and AP staining respectively. Among these samples, 14 and 12 were positive with 18S and LAXER qPCR respectively. In two patients, Cryptosporidium DNA loads were found to be correlated with clinical evolution. Although little known, glycerin is a sensitive method for the initial detection of Cryptosporidium. When combined with 18S qPCR, ZR extraction, which had not been evaluated so far for Cryptosporidium, was an accurate tool for detecting Cryptosporidium and estimating the oocyst shedding in the course of infection.
隐孢子虫病是一个重要的公共卫生问题，尽管报告不足。分子工具可能有助于改善其诊断。在本研究中，使用四份接种隐孢子虫的粪便和29份隐孢子虫阳性粪便样本对ZR粪便DNA微量制备试剂盒（ZR）和NucliSens® easyMAG®（EM）进行了比较。此后，在通过甘油、改良齐-尼（ZN）染色和金胺酚（AP）染色检测到隐孢子虫后，选择ZR对56例患者的69份粪便样本进行18S rDNA和LAXER定量PCR（qPCR）检测隐孢子虫的前瞻性评估。两种提取方法中的任何一种与18S qPCR相结合，都能在接种粪便样本中充分检测到隐孢子虫，但ZR试剂盒表现最佳。在ZR和EM提取后，所有29份隐孢子虫阳性样本经18S qPCR检测均为阳性。然而，LAXER qPCR或巢式PCR检测出现了假阴性结果。56例患者中有7例被诊断为隐孢子虫病。所有显微镜检查方法均能进行初步诊断，但在这7例患者的样本中，甘油、ZN和AP染色后分别在12、13和14份样本中检测到隐孢子虫。在这些样本中，14份和12份样本分别经18S和LAXER qPCR检测为阳性。在两名患者中，发现隐孢子虫DNA载量与临床病程相关。甘油虽然鲜为人知，但却是隐孢子虫初始检测的敏感方法。当与18S qPCR结合使用时，此前未针对隐孢子虫进行评估的ZR提取法是检测隐孢子虫和估计感染过程中卵囊排出量的准确工具。
