Dinoop K P, Parija Subhash Chandra, Mandal Jharna, Swaminathan R P, Narayanan P
Department of Microbiology, Jawaharlal Institute of Postgraduate Medical Education & Research, Puducherry, India.
Indian J Med Res. 2016 Jan;143(1):49-56. doi: 10.4103/0971-5916.178592.
BACKGROUND & OBJECTIVES: Amoebiasis is a common parasitic infection caused by Entamoeba histolytica and amoebic liver abscess (ALA) is the most common extraintestinal manifestation of amoebiasis. The aim of this study was to standardise real-time PCR assays (Taqman and SYBR Green) to detect E. histolytica from liver abscess pus and stool samples and compare its results with nested-multiplex PCR.
Liver abscess pus specimens were subjected to DNA extraction. The extracted DNA samples were subjected to amplification by nested-multiplex PCR, Taqman (18S rRNA) and SYBR Green real-time PCR (16S-like rRNA assays to detect E. histolytica/E. dispar/E. moshkovskii). The amplification products were further confirmed by DNA sequence analysis. Receiver operator characteristic (ROC) curve analysis was done for nested-multiplex and SYBR Green real-time PCR and the area under the curve was calculated for evaluating the accuracy of the tests to dignose ALA.
In all, 17, 19 and 25 liver abscess samples were positive for E. histolytica by nested-multiplex PCR, SYBR Green and Taqman real-time PCR assays, respectively. Significant differences in detection of E. histolytica were noted in the real-time PCR assays evaluated ( P<0.0001). The nested-multiplex PCR, SYBR Green real-time PCR and Taqman real-time PCR evaluated showed a positivity rate of 34, 38 and 50 per cent, respectively. Based on ROC curve analysis (considering Taqman real-time PCR as the gold standard), it was observed that SYBR Green real-time PCR was better than conventional nested-multiplex PCR for the diagnosis of ALA.
INTERPRETATION & CONCLUSIONS: Taqman real-time PCR targeting the 18S rRNA had the highest positivity rate evaluated in this study. Both nested multiplex and SYBR Green real-time PCR assays utilized were evaluated to give accurate results. Real-time PCR assays can be used as the gold standard in rapid and reliable diagnosis, and appropriate management of amoebiasis, replacing the conventional molecular methods.
阿米巴病是由溶组织内阿米巴引起的常见寄生虫感染，阿米巴肝脓肿（ALA）是阿米巴病最常见的肠外表现。本研究的目的是标准化实时荧光定量PCR检测方法（Taqman和SYBR Green），以从肝脓肿脓液和粪便样本中检测溶组织内阿米巴，并将其结果与巢式多重PCR进行比较。
对肝脓肿脓液标本进行DNA提取。提取的DNA样本分别用巢式多重PCR、Taqman（18S rRNA）和SYBR Green实时荧光定量PCR（16S类rRNA检测法检测溶组织内阿米巴/迪斯帕内阿米巴/莫斯科维奇内阿米巴）进行扩增。扩增产物通过DNA序列分析进一步确认。对巢式多重PCR和SYBR Green实时荧光定量PCR进行受试者操作特征（ROC）曲线分析，并计算曲线下面积以评估诊断ALA的检测准确性。
巢式多重PCR、SYBR Green和Taqman实时荧光定量PCR检测分别显示，共有17、19和25份肝脓肿样本溶组织内阿米巴呈阳性。在所评估的实时荧光定量PCR检测中，溶组织内阿米巴的检测存在显著差异（P<0.0001）。所评估的巢式多重PCR、SYBR Green实时荧光定量PCR和Taqman实时荧光定量PCR的阳性率分别为34%、38%和50%。基于ROC曲线分析（以Taqman实时荧光定量PCR为金标准），观察到SYBR Green实时荧光定量PCR在诊断ALA方面优于传统的巢式多重PCR。
本研究中，靶向18S rRNA的Taqman实时荧光定量PCR阳性率最高。所使用的巢式多重PCR和SYBR Green实时荧光定量PCR检测均经评估可得出准确结果。实时荧光定量PCR检测可作为快速、可靠诊断及适当管理阿米巴病的金标准，取代传统分子方法。
