Design and optimization of an improved qPCR assay for the detection of Histoplasma capsulatum.

European journal of microbiology & immunology Pub Date : 2023-09-21 DOI:10.1556/1886.2023.00026

Bianca Kelly Neves Izidro da Silva, Ana Cláudia Alves Cortez, Luciana Aires Oliveira, Juan Diego Ribeiro de Almeida, Érica Simplício de Souza, Katia Santana Cruz, Ani Beatriz Jackisch Matsuura, Mauricio Morishi Ogusku, Karolina Jeaneth Solórzano Chavarría, Djane Baía-da-Silva, Quique Bassat, Marcus Vinícius Guimarães Lacerda, Hagen Frickmann, João Vicente Braga de Souza

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Abstract

Background: This study aimed at improving a real-time polymerase-chain-reaction (qPCR) assay for the detection of Histoplasma capsulatum, a fungal pathogen that can cause severe respiratory infections in humans, in clinical and soil samples.

Methods: Primer and probes were in-silico designed, in-silico and in-vitro evaluated including clinical biopsy materials and finally subjected to a real-world application with collected soil samples.

Results: Applying the qPCR assay with liver and lung biopsies from 71 patients each, including 59 patients infected with human immunodeficiency virus (HIV), as well as with Sabouraud (SAB) agar culture as the diagnostic reference standard, diagnostic accuracy of the qPCR assay of 100% (5/5) sensitivity and 96% (63/66) specificity for liver samples and 100% (4/4) sensitivity and 94% (63/67) specificity for the lung samples was recorded. When applying the assay with soil samples from caves near of Presidente Figueiredo city, Amazonas, Brazil, one sample from the Maroaga cave was confirmed as positive.

Conclusions: The improved qPCR assessed in this study was successful in detecting H. capsulatum with high efficiency and accuracy in in-vitro evaluation, including the identification of the target pathogen in both clinical and environmental samples.

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用于检测荚膜组织浆的改良qPCR检测方法的设计和优化。

背景：本研究旨在改进实时聚合酶链式反应（qPCR）检测方法，用于在临床和土壤样本中检测荚膜组织浆，这是一种可导致人类严重呼吸道感染的真菌病原体。方法：对引物和探针进行了计算机设计、计算机和体外评估，包括临床活检材料，并最终对收集的土壤样本进行了实际应用。结果：应用qPCR方法分别对71例患者（包括59例人类免疫缺陷病毒（HIV）感染者）的肝和肺活检进行检测，并以沙氏琼脂培养基为诊断参考标准，记录了qPCR检测对肝脏样本的诊断准确性为100%（5/5）敏感性和96%（63/66）特异性，对肺部样本的诊断准确率为100%（4/4）敏感性和94%（63/67）特异性。当对巴西亚马逊总统菲格雷多市附近洞穴的土壤样本进行分析时，Maroaga洞穴的一个样本被确认为阳性。结论：本研究中评估的改进qPCR在体外评估中成功地检测了荚膜H.capsultum，具有较高的效率和准确性，包括在临床和环境样本中鉴定目标病原体。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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European journal of microbiology & immunology

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