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

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.

Design, Development, and Validation of a Culture-Independent Nucleic Acid Diagnostics Method for the Rapid Detection and Quantification of the Burkholderia cepacia Complex in Water with an Equivalence to ISO/TS 12869:2019.

In the wake of a series of outbreaks of finished pharmaceutical product-related Burkholderia cepacia complex (Bcc) human infections worldwide, the United States Food and Drug Administration (FDA) in 2017, and subsequently in 2021, issued advisory notifications to the pharmaceutical industry for stringent Bcc testing requirements for pharmaceutical manufacturing processes and for finished pharmaceutical products prior to release to the marketplace. The advisory notifications highlight non-sterile aqueous finished pharmaceutical products as being a major culprit associated with many of these human infection events. As such, there has been a significant number of Bcc-contaminated finished product recalls resulting in company revenue losses, delayed finished product release, finished product shortages for patients, and manufacturing plant shutdowns coupled with company reputational damage. With many of the finished product recall events, pharmaceutical grade water and/or manufacturing facility water distribution systems were identified as the primary origin source of Bcc contamination. Testing and monitoring regimes currently employed to identify Bcc contamination of water associated with pharmaceutical manufacturing are often limited by costly, laborious, lengthy, and nonspecific traditional microbial culture-based methodologies. Presently FDA approved, European Conformity (CE) marked, and International Organization for Standardization (ISO) standard microbial culture-independent rapid, quantitative, specific, and sensitive nucleic acid diagnostics (NAD) methodologies are now gaining greater widespread acceptance in their routine usage in testing laboratories. Here we present the development of a rapid (<4 hours from sample in to result out) single test culture-independent Bcc NAD method, incorporating a quantitative real-time polymerase chain reaction (qPCR) assay. This method can be used for the detection and simultaneous identification of all 24 Bcc species currently assigned, directly from water samples. This culture-independent Bcc NAD method is validated to the testing method equivalent of the ISO/TS 12869:2019 standard, which is a widely used rapid culture-independent NAD method for detecting Gram-negative Legionella species in water.