Shahiduzzaman M, Dyachenko V, Obwaller A, Unglaube S, Daugschies A
Institute of Parasitology, University of Leipzig, An den Tierkliniken 35, 04103 Leipzig, Germany.
Vet Parasitol. 2009 Jun 10;162(3-4):271-7. doi: 10.1016/j.vetpar.2009.03.009. Epub 2009 Mar 13.
Cryptosporidium parvum is a zoonotic pathogen causing self-limiting diarrhea in immunocompetent patients. An assay combining cell culture and real time quantitative PCR (qPCR) is reported here to verify drug efficacy against C. parvum in vitro. The monolayers of Human ileocecal adenocarcinoma cells (HCT-8) were infected by sporozoites excysted directly on the cells and were incubated with monensin, halofuginone bromide and hexadecylphosphocholine until 45h post infection (p.i.). The genomic DNA was extracted at 3, 27 and 45h p.i. and subjected to qPCR targeting the 70kDa heat shock protein gene to quantify the development of C. parvum. The reliability of the method was validated by testing of monensin and halofuginone bromide, which are well known to be effective in vitro. With the dose dependency monensin and halofuginone showed a maximum inhibition of 98.15% and 98.05% at 0.144 and 25microM, respectively, compared with non-treated controls at the endpoint incubation, confirming previous reports. The reduction of the parasite DNA reproduction over 27h p.i. compared with 3h p.i. was found to be as 97-99% in 0.144microM monensin and 99% in 25microM halofuginone treated cells. The new antileishmanial compound hexadecylphosphocholine (24.5microM, Miltefosine) showed 78-98% inhibition at 45h p.i., however, the reproduction of parasite DNA was reduced to 96-98% over 27h p.i. The method has the potential to easily and reliably assess anticryptosporidial compounds in adequately equipped routine laboratories.
微小隐孢子虫是一种人畜共患病原体，可导致免疫功能正常的患者出现自限性腹泻。本文报道了一种结合细胞培养和实时定量PCR（qPCR）的检测方法，用于在体外验证抗微小隐孢子虫药物的疗效。人回盲部腺癌细胞（HCT-8）单层细胞直接被接种在细胞上的子孢子感染，并与莫能菌素、溴化常山酮和十六烷基磷酸胆碱一起孵育，直至感染后45小时（p.i.）。在感染后3、27和45小时提取基因组DNA，并进行靶向70kDa热休克蛋白基因的qPCR，以量化微小隐孢子虫的发育情况。通过检测莫能菌素和溴化常山酮验证了该方法的可靠性，这两种药物在体外的有效性是众所周知的。与终点孵育时的未处理对照相比，莫能菌素和常山酮呈剂量依赖性，分别在0.144和25μM时显示出最大抑制率98.15%和98.05%，证实了先前的报道。在感染后27小时与3小时相比，0.144μM莫能菌素处理的细胞中寄生虫DNA繁殖减少了97-99%，25μM常山酮处理的细胞中减少了99%。新型抗利什曼原原化合物化合物十六烷基磷酸胆碱（24.5μM，米替福新）在感染后45小时显示出78-98%的抑制率，然而，在感染后27小时，寄生虫DNA的繁殖减少到96-98%。该方法有可能在设备齐全的常规实验室中轻松可靠地评估抗隐孢子虫化合物。
