Badr Mohamed S, El-Askary Hala M, Elmalawany Marwa, Kamal Reham, Alsamra Nashwa
J Egypt Soc Parasitol. 2016 Dec;46(3):549-556.
Low parasite density in chronic infection with S. stercoralis is a challenging diagnostic issue. Generally, molecular techniques don't depend on parasite viability while copro-culture or Baermann concentration method relies on the presence of living larvae in fecal samples. Therefore, evaluation of PCR-based methods is important to increase the detection rates in light or chronic infection. This study was designed to analyze the sensitivity of quantitative PCR (qPCR) and nested PCR (nPCR) regarding the minimum amount of S. stercoralis DNA template that can be reliably detected by each molecular technique. Strongyloides larvae were collected from cultured stool samples from suspected infected Egyptian children. After counting larvae present in a known volume under the microscope, DNA extraction was done and se- rial dilution of genomic materials was prepared. Then, qPCR and nPCR targeting the, small subunit of the rRNA gene were performed. Regarding qPCR, the limit of detection was 0.0005 S. stercoralis larvae/μl, with crossing threshold (Ct) Value ranged from 17.8 to 38.7 while, nPCR did not detect from (0.002 to 0.0012 S tercopilis larvae/μl) with minimum limit 0.004 S. stercoralis larvae/μl. Real-time quantitative PCR is very sensitive technique that can detect very low genomic load up to about 10 (9.765) genome copies/reaction compared to. nested PCR which started positivity from 78.125 genome copies/reaction. Therefore, qPCR is recommended to detect chronic strongyloidiasis especially in high-risk groups to prevent life- threatening spread of such infection.
粪类圆线虫慢性感染时寄生虫密度较低是一个具有挑战性的诊断问题。一般来说，分子技术不依赖于寄生虫的活力，而粪便培养或贝曼氏浓集法依赖于粪便样本中活幼虫的存在。因此，评估基于PCR的方法对于提高轻度或慢性感染的检出率很重要。本研究旨在分析定量PCR（qPCR）和巢式PCR（nPCR）对于每种分子技术能够可靠检测的最低粪类圆线虫DNA模板量的敏感性。从疑似感染的埃及儿童的培养粪便样本中收集粪类圆线虫幼虫。在显微镜下对已知体积内的幼虫进行计数后，进行DNA提取并制备基因组材料的系列稀释液。然后，针对rRNA基因的小亚基进行qPCR和nPCR。对于qPCR，检测限为0.0005条粪类圆线虫幼虫/μl，交叉阈值（Ct）值范围为17.8至38.7，而nPCR在（0.002至0.0012条粪类圆线虫幼虫/μl）未检测到，最低检测限为0.004条粪类圆线虫幼虫/μl。实时定量PCR是一种非常敏感的技术，与巢式PCR相比，它可以检测到非常低的基因组载量，高达约10（9.765）个基因组拷贝/反应，巢式PCR从78.125个基因组拷贝/反应开始出现阳性。因此，建议使用qPCR检测慢性粪类圆线虫病，尤其是在高危人群中，以防止这种感染的危及生命的传播。
