Enhancing the detection sensitivity of mNGS in Bronchoalveolar Lavage Fluid through cell counting: An empirical study

IF 3.2 3区医学 Q2 MEDICAL LABORATORY TECHNOLOGY

Clinica Chimica Acta Pub Date : 2025-04-26 DOI:10.1016/j.cca.2025.120311

Zhe Liu , Shangdong Yang , Shumei Xie , Depan Cao , Wen Xi , Yang Xiao , Xin Xu , Zhonglin Wang , Lifeng Li , Jian Hu , Xiaoqin Wang

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引用次数: 0

Abstract

Objectives

Lower respiratory tract infections pose significant clinical challenges due to their high morbidity and mortality rates. While metagenomic next-generation sequencing (mNGS) has emerged as a promising diagnostic tool, its sensitivity is often compromised by host DNA contamination that overwhelms microbial signals. Selective host DNA depletion through cell lysis effectively reduces host DNA; however, it has an impact on microorganisms with relatively thin cell walls, and samples with low host content may introduce more environment or reagent-derived microbial contamination, interfering the detection results. Methods for determining host DNA depletion based on sample type, sample characteristics or using spike-in controls to monitor sensitivity do not fully consider the potential limitations of host depletion technology on microbial detection, nor do they evaluate the possible significant impact on detection efficiency. This study aimed to develop a pre-analytical method for accurate host DNA content assessment.

Methods

We established a cell-counting-based method for precise cellular content measurement in clinical bronchoalveolar lavage fluid (BALF) samples. The protocol involved: (1) evaluating the linearity and robustness of cell-counting dyes in BALF samples with varying characteristics, (2) assessing the correlation between cell counts and extracted nucleic acid mass, (3) investigating cellular counting thresholds for host depletion in clinical BALF analysis, and (4) implementing the optimized cell-counting method in clinical mNGS testing to guide selective-lysis treatment.

Results

Acridine orange/propidium iodide (AO/PI) staining demonstrated superior performance compared to trypan blue and 4',6-diamidino-2-phenylindole (DAPI), particularly in turbid and bloody BALF samples. Implementing a host depletion threshold at 1 × 10⁶ cell counts significantly improves pathogen detection rates in high host background samples, while effectively preserving the detection sensitivity for pathogens in moderate and low host background samples.

Conclusions

Our findings demonstrate that cell counting serves as a reliable pre-analytical tool for determining optimal selective-lysis treatment in BALF mNGS testing, enhancing diagnostic accuracy while preserving pathogen integrity.

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细胞计数提高支气管肺泡灌洗液中mNGS检测灵敏度的实证研究

目的下呼吸道感染因其高发病率和死亡率而成为临床的一大挑战。虽然新一代宏基因组测序（mNGS）已成为一种很有前途的诊断工具，但其敏感性往往受到宿主DNA污染的影响，而宿主DNA污染会压倒微生物信号。通过细胞裂解选择性消耗宿主DNA，有效减少宿主DNA；然而，它对细胞壁相对较薄的微生物有影响，并且宿主含量低的样品可能引入更多的环境或试剂来源的微生物污染，干扰检测结果。基于样品类型、样品特征确定宿主DNA缺失的方法或使用峰值控制来监测灵敏度的方法没有充分考虑宿主缺失技术对微生物检测的潜在局限性，也没有评估对检测效率可能产生的重大影响。本研究旨在建立一种准确评估宿主DNA含量的分析前方法。方法建立一种基于细胞计数的临床支气管肺泡灌洗液（BALF）细胞含量精确测定方法。该方案涉及：(1)评估不同特征的BALF样品中细胞计数染料的线性和鲁棒性；(2)评估细胞计数与提取的核酸质量之间的相关性；(3)研究临床BALF分析中宿主耗损的细胞计数阈值；(4)在临床mNGS检测中实施优化的细胞计数方法，以指导选择性溶解治疗。结果吖啶橙/碘化丙啶（AO/PI）染色优于台盼蓝和4′，6-二氨基-2-苯基吲哚（DAPI）染色，特别是在浑浊和带血的BALF样品中。采用1 × 106细胞计数的宿主耗尽阈值可显著提高高宿主背景样品中的病原体检出率，同时有效地保持对中等和低宿主背景样品中的病原体的检测灵敏度。结论研究结果表明，细胞计数可作为确定BALF mNGS检测中最佳选择性裂解处理的可靠分析前工具，在保持病原体完整性的同时提高诊断准确性。

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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来源期刊

Clinica Chimica Acta 医学-医学实验技术

CiteScore

10.10

自引率

2.00%

发文量

1268

审稿时长

23 days

期刊介绍： The Official Journal of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) Clinica Chimica Acta is a high-quality journal which publishes original Research Communications in the field of clinical chemistry and laboratory medicine, defined as the diagnostic application of chemistry, biochemistry, immunochemistry, biochemical aspects of hematology, toxicology, and molecular biology to the study of human disease in body fluids and cells. The objective of the journal is to publish novel information leading to a better understanding of biological mechanisms of human diseases, their prevention, diagnosis, and patient management. Reports of an applied clinical character are also welcome. Papers concerned with normal metabolic processes or with constituents of normal cells or body fluids, such as reports of experimental or clinical studies in animals, are only considered when they are clearly and directly relevant to human disease. Evaluation of commercial products have a low priority for publication, unless they are novel or represent a technological breakthrough. Studies dealing with effects of drugs and natural products and studies dealing with the redox status in various diseases are not within the journal''s scope. Development and evaluation of novel analytical methodologies where applicable to diagnostic clinical chemistry and laboratory medicine, including point-of-care testing, and topics on laboratory management and informatics will also be considered. Studies focused on emerging diagnostic technologies and (big) data analysis procedures including digitalization, mobile Health, and artificial Intelligence applied to Laboratory Medicine are also of interest.