Rapid culture independent nucleic acid diagnostics methodologies for the specific detection of the Burkholderia cepacia complex – addressing a microbiological problem associated with pharmaceutical product manufacturing
Duong, Thu Huong
Duong, Thu Huong
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Publication Date
2023-02-10
Type
Thesis
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Abstract
Burkholderia cepacia complex (Bcc) consists of multiple phylogenetically related Gram negative opportunistic pathogens with a high potential to cause severe infections in vulnerable populations with underlying illnesses, particularly in patients with cystic fibrosis (CF) and other immunocompromised individuals. Bcc species are considered especially problematic for the pharmaceutical manufacturing industries where they are the most prevalent microbial contaminants found in pharmaceutical products, being the main reason for product recalls and leading to a significant negative impact on the pharmaceutical industry from an economic and reputational perspective. In the context of serious Bcc contamination in pharmaceutical manufacturing and its potential health hazard to humans, pharmaceutical manufacturers are responsible for strict monitoring of Bcc at all stages of the manufacturing process. Since water systems of pharmaceutical companies have been identified as the primary source of Bcc contamination, extensive and comprehensive microbiological testing of water for Bcc is required. Manufactured products are also stringently necessary to be examined to establish the absence of Bcc to ensure the quality and safety of finished pharmaceutical products (FPPs) before being released into the market. Currently, routine testing and monitoring regimes employed in pharmaceutical microbiological control laboratories to identify microbial contamination rely on laborious, lengthy, low sensitivity and non-specific traditional culture-based methodologies, resulting in the frequently undetected Bcc species. Culture-independent nucleic acid diagnostics (NAD) technologies have grown in their use over the years and they can provide high sensitivity, accuracy, specificity and rapid turnaround time-to-result. These features are significant for the pharmaceutical manufacturing industry because the rapid and accurate detection of Bcc contamination will allow prompt identification of contamination sources and subsequently implementation of adequate infection control actions can be taken quickly. This PhD study focuses on the design, development, optimization and validation of rapid and robust Bcc qPCR NAD-based methodologies to industry standards that can support the Bcc contamination control strategy in the pharmaceutical industry sector to detect these problematic bacteria. This was achieved using a definitive diagnostics assay development strategy and in accordance with highly regulated standards, such as the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines and International Organization for Standardization (ISO). In the first study, an internally controlled Bcc qPCR NAD assay was developed and validated for the specific quantitative detection of all Bcc species with 100% specificity, high analytical sensitivity and a broad range of calibration function. Subsequently, a rapid culture-independent Bcc NAD method incorporating the Bcc qPCR NAD assay was developed to detect and simultaneously identify all 24 Bcc species directly from water samples. This culture independent Bcc NAD method was validated with an equivalence to the ISO/TS 12869:2019 standard, a widely used rapid culture-independent NAD method for detecting Gram-negative Legionella species in water. The culture-independent Bcc NAD method could detect Bcc in spiked water samples at different contamination levels with high recovery efficiency, high reproducibility and a total analysis time of <4 hours. Method robustness was not significantly impacted by water types or by the natural microbiota within water samples. The validated culture-independent Bcc NAD method could be a valuable diagnostic tool for the detection of Bcc in water systems used for manufacturing, supporting water risk management strategy in the pharmaceutical sector. In the second study, a rapid culture-independent Bcc NAD method incorporating the Bcc qPCR NAD was developed for the specific detection of all Bcc species in aqueous FPPs. This culture independent Bcc NAD method was demonstrated to be specific for detecting Bcc at different contamination levels from spiked Over The Counter (OTC) aqueous FPPs with high accuracy, high precision and a total analysis time of <4 hours. Method robustness was not significantly impacted by aqueous FPP types, product ingredient matrices and antimicrobial preservative (AP) components. The validated culture-independent Bcc NAD method could be a valuable diagnostic tool for the detection of Bcc contaminants associated with aqueous FPPs, supporting microbiological quality control procedures of FPPs before releasing to the market. In the third study, two novel individual internally controlled qPCR NAD assays were developed for the rapid and quantitative detection and specific identification of the two most clinically problematic Bcc species, B. cenocepacia and B. multivorans. These qPCR NAD assays were validated with all parameters in compliance with the ISO/TS 12869:2019 with 100% specificity for the target bacteria, high analytical sensitivity and a broad range of calibration function. The application of these two qPCR NAD assays in testing water systems or FPPs could be valuable diagnostic tools to rapidly and accurately investigate contamination events caused by B. cenocepacia and B. multivorans in pharmaceutical industries while also delivering critical epidemiological data about the specific contaminating Bcc species in a manufacturing setting. In summary, internally controlled qPCR NAD assays and culture-independent NAD methods described in this PhD study with the appropriate validation adhering to highly regulated standards offer rapid, accurate, robust NAD methodologies for the detection, identification and quantification of problematic Bcc contaminants associated with the pharmaceutical manufacturing industry sector. We propose that these methodologies could contribute toward microbiological contamination control strategies to assure the safety and quality of manufactured FPPs to be administered to customers.
Publisher
NUI Galway