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一种用于检测和定量水性成品药物制剂中洋葱伯克霍尔德菌复合污染的非培养核酸诊断方法。

A culture-independent nucleic acid diagnostics method for use in the detection and quantification of Burkholderia cepacia complex contamination in aqueous finished pharmaceutical products.

机构信息

Nucleic Acid Diagnostics Research Laboratory (NADRL), School of Biological and Chemical Sciences, University of Galway, Galway, Ireland.

Microbial Diagnostics Research Laboratory, School of Biological and Chemical Sciences, University of Galway, Galway, Ireland.

出版信息

PLoS One. 2024 May 16;19(5):e0303773. doi: 10.1371/journal.pone.0303773. eCollection 2024.

Abstract

The Burkholderia cepacia complex (Bcc) is the number one bacterial complex associated with contaminated Finished Pharmaceutical Products (FPPs). This has resulted in multiple healthcare related infection morbidity and mortality events in conjunction with significant FPP recalls globally. Current microbiological quality control of FPPs before release for distribution depends on lengthy, laborious, non-specific, traditional culture-dependent methods which lack sensitivity. Here, we present the development of a culture-independent Bcc Nucleic Acid Diagnostic (NAD) method for detecting Bcc contaminants associated with Over-The-Counter aqueous FPPs. The culture-independent Bcc NAD method was validated to be specific for detecting Bcc at different contamination levels from spiked aqueous FPPs. The accuracy in Bcc quantitative measurements was achieved by the high degree of Bcc recovery from aqueous FPPs. The low variation observed between several repeated Bcc quantitative measurements further demonstrated the precision of Bcc quantification in FPPs. The robustness of the culture-independent Bcc NAD method was determined when its accuracy and precision were not significantly affected during testing of numerous aqueous FPP types with different ingredient matrices, antimicrobial preservative components and routes of administration. The culture-independent Bcc NAD method showed an ability to detect Bcc in spiked aqueous FPPs at a concentration of 20 Bcc CFU/mL. The rapid (≤ 4 hours from sample in to result out), robust, culture-independent Bcc NAD method presented provides rigorous test specificity, accuracy, precision, and sensitivity. This method, validated with equivalence to ISO standard ISO/TS 12869:2019, can be a valuable diagnostic tool in supporting microbiological quality control procedures to aid the pharmaceutical industry in preventing Bcc contamination of aqueous FPPs for consumer safety.

摘要

伯克霍尔德氏菌复合群(Bcc)是与污染的已完成药品(FPP)相关的头号细菌复合群。这导致了与全球范围内多次与医疗保健相关的感染发病率和死亡率事件以及大量 FPP 召回。目前,在放行用于分发之前,对 FPP 的微生物质量控制取决于冗长、费力、非特异性、传统的基于培养的方法,这些方法缺乏敏感性。在这里,我们提出了一种用于检测与非处方水性 FPP 相关的 Bcc 污染物的非培养 Bcc 核酸诊断(NAD)方法的开发。该非培养 Bcc NAD 方法经过验证,可特异性检测不同污染水平的水性 FPP 中的 Bcc。通过从水性 FPP 中高度回收 Bcc 来实现 Bcc 定量测量的准确性。在几个重复的 Bcc 定量测量之间观察到的低变化进一步证明了 FPP 中 Bcc 定量的精度。当该非培养 Bcc NAD 方法在测试具有不同成分基质、抗菌防腐剂成分和给药途径的多种水性 FPP 类型时,其准确性和精密度没有受到显著影响时,确定了该非培养 Bcc NAD 方法的稳健性。该非培养 Bcc NAD 方法能够以 20 Bcc CFU/mL 的浓度检测到水性 FPP 中的 Bcc。该快速(从样品到结果的时间≤4 小时)、稳健、非培养的 Bcc NAD 方法提供了严格的测试特异性、准确性、精密度和灵敏度。该方法通过与 ISO 标准 ISO/TS 12869:2019 的等效性进行验证,可成为支持微生物质量控制程序的有价值的诊断工具,以帮助制药行业防止 Bcc 污染水性 FPP,确保消费者安全。

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