新型双链聚合酶链反应快速检测眼内型棘阿米巴角膜炎患者角膜标本中的棘阿米巴原虫。

Novel Duplex Polymerase Chain Reaction for the Rapid Detection of Pythium insidiosum Directly From Corneal Specimens of Patients With Ocular Pythiosis.

机构信息

L&T Microbiology Research Centre, Vision Research Foundation, Chennai, India; and.

Sankara Nethralaya Referral Laboratory, Medical Research Foundation, Chennai, India.

出版信息

Cornea. 2020 Jun;39(6):775-778. doi: 10.1097/ICO.0000000000002284.

DOI:10.1097/ICO.0000000000002284

PMID:32118672

Abstract

PURPOSE

To standardize a novel duplex polymerase chain reaction (PCR) targeting 18S rRNA gene and internal transcribed spacer region for the identification of Pythium insidiosum isolates and also to detect P. insidiosum genome directly from corneal specimens of patients with suspected ocular pythiosis.

METHODS

A total of 42 nonsporulating molds culturally and morphologically resembling suspected unidentified fungal isolates (corneal buttons 33 and corneal scrapings 9) and 14 clinical specimens (corneal buttons 7 and corneal scrapings 7) clinically suspected to be ocular pythiosis were included in the present study. Standardization of uniplex PCRs and duplex PCRs targeting 18S rRNA gene and internal transcribed spacer region and further application of the standardized PCRs on both clinical isolates and clinical specimens suspected to have fungal keratitis. The sensitivity and specificity of the standardized duplex PCR were calculated using Medcal.net software.

RESULTS

The standardized uniplex and duplex PCRs were found specific for the detection of only P. insidiosum DNA, and the analytical sensitivities of the primers were 1.36 Zg. Of the 14 clinical specimens analyzed, 13 were positive in both corneal specimens and their respective P. insidiosum isolates. The specificity of the novel duplex PCR was 100% when applied on corneal specimens and clinical isolates, but the sensitivity was 92.8% (13/14) and 100% (42/42), respectively, for the clinical specimens and fungal isolates from suspected ocular pythiosis patients included in the study.

CONCLUSIONS

The novel duplex PCR developed in this study will aid in rapid identification of P. insidiosum clinical isolates and clinical specimens from suspected ocular pythiosis specimens, which in turn will help the ophthalmologists to initiate appropriate treatment.

摘要

目的

标准化一种针对 18S rRNA 基因和内部转录间隔区的新型双重聚合酶链反应（PCR），用于鉴定皮炎霉属分离株，并直接从疑似眼内皮炎患者的角膜标本中检测皮炎霉属基因组。

方法

本研究共纳入 42 株非孢子形成的霉菌，其在文化和形态上与疑似未鉴定真菌分离株（角膜纽扣 33 个和角膜刮片 9 个）相似，以及 14 份临床标本（角膜纽扣 7 个和角膜刮片 7 个），临床疑似眼内皮炎。本研究对针对 18S rRNA 基因和内部转录间隔区的单重 PCR 和双重 PCR 进行了标准化，并将标准化 PCR 进一步应用于临床分离株和疑似真菌性角膜炎的临床标本。使用 Medcal.net 软件计算标准化双重 PCR 的灵敏度和特异性。

结果

标准化的单重和双重 PCR 仅发现对皮炎霉属 DNA 的检测具有特异性，引物的分析灵敏度为 1.36 Zg。在分析的 14 个临床标本中，13 个在角膜标本及其各自的皮炎霉属分离株中均为阳性。当应用于角膜标本和临床分离株时，新型双重 PCR 的特异性为 100%，但对研究中包括的疑似眼内皮炎患者的临床标本和真菌分离株的灵敏度分别为 92.8%（13/14）和 100%（42/42）。