Katiyar Manish, Gulati Reena, Pagal Sudhakar, Rajkumari Nonika, Singh Rakesh
Department of Microbiology, Jawaharlal Institute of Postgraduate Medical Education and Research , Puducherry, 605 006, India.
Department of Paediatrics, Jawaharlal Institute of Postgraduate Medical Education and Research , Puducherry, 605 006, India.
Indian J Gastroenterol. 2021 Oct;40(5):512-518. doi: 10.1007/s12664-021-01170-y. Epub 2021 Sep 27.
Cystoisospora belli (C. belli) is the only pathogenic species of the Cystoisospora genus responsible for severe diarrhea in immunocompromised patients. Most common microscopic method of diagnosis is less sensitive due to intermittent shedding of oocysts. We developed a new single-run polymerase chain reaction (PCR)-based diagnostic assay for C. belli.
A new single-run PCR-based diagnostic assay was standardized for the detection of C. belli. Diagnostic reproducibility and repeatability of the PCR assay were evaluated. A cross-sectional analytical study was done on a total of 354 stool samples collected from 331 immunocompromised patients with diarrhea. All the stool samples were tested for the presence of oocysts of C. belli and were also tested by our new PCR assay for C. belli. Three of the representative PCR products were confirmed by sequencing. Fisher's exact test was used to compare the two proportions.
Microscopy detected C. belli in 11/354 (3.1%) of stool samples, and the new PCR-based assay detected C. belli in 16/354 (4.5%). The new single-run PCR-based assay detected C. belli in all the stool samples which were tested positive by microscopy and additionally detected C. belli in five stool samples. The developed PCR assay detected statistically significant proportion of C. belli (p < 0.001) as compared to microscopy. The 795 base pair PCR product from one microscopy positive stool sample and two microscopy negative stool samples were confirmed by sequencing.
Our newly developed single-run PCR-based detection assay for C. belli is robust and reproducible. It may be used for molecular diagnosis of cystoisosporiasis especially in transplant, pediatrics, and human immunodeficiency virus (HIV) positive patients.
贝氏等孢球虫（C. belli）是等孢球虫属中唯一的致病物种，可导致免疫功能低下患者出现严重腹泻。由于卵囊间歇性排出，最常用的显微镜诊断方法敏感性较低。我们开发了一种基于单轮聚合酶链反应（PCR）的新型贝氏等孢球虫诊断检测方法。
对基于单轮PCR的新型贝氏等孢球虫诊断检测方法进行标准化。评估了该PCR检测方法的诊断重现性和重复性。对从331例腹泻的免疫功能低下患者收集的354份粪便样本进行了横断面分析研究。所有粪便样本均检测贝氏等孢球虫卵囊的存在情况，并通过我们新的贝氏等孢球虫PCR检测方法进行检测。对三个代表性的PCR产物进行测序确认。采用Fisher精确检验比较两个比例。
显微镜检查在354份粪便样本中的11份（3.1%）中检测到贝氏等孢球虫，而新的基于PCR的检测方法在354份粪便样本中的16份（4.5%）中检测到贝氏等孢球虫。新的基于单轮PCR的检测方法在所有显微镜检查呈阳性的粪便样本中均检测到贝氏等孢球虫，另外在5份粪便样本中也检测到贝氏等孢球虫。与显微镜检查相比，所开发的PCR检测方法检测到的贝氏等孢球虫比例具有统计学意义（p < 0.001）。对一份显微镜检查阳性粪便样本和两份显微镜检查阴性粪便样本的795碱基对PCR产物进行了测序确认。
我们新开发的基于单轮PCR的贝氏等孢球虫检测方法可靠且可重复。它可用于等孢球虫病的分子诊断，特别是在移植患者、儿科患者和人类免疫缺陷病毒（HIV）阳性患者中。
