Adequately designed and clinically relevant quality controls (QCs) are a cornerstone in ensuring the reliability of test results, which is particularly critical for Group B Streptococcus testing given the possible dramatic outcomes.
By David Gauthier, Chance Garcia, and Charles Rzadkowolski
The study presented in this article highlights the necessity for quality controls (QCs) to simulate patient specimens, considering both the clinical and compliance (CAP and ISO 15189:2022) aspects. Here, we show the impact of the matrix effect and the significance of monitoring a pre-analytical step, illustrated with the example of Group B Streptococcus (GBS).
GBS colonization in expectant mothers poses a risk of infection and transmission to newborns, potentially leading to severe illness or even death. In the U.S., screening for GBS in pregnant mothers is performed using culture or nucleic acid amplification tests (NAATs – e.g., real-time PCR) around week 37 of gestation, and after broth enrichment, typically in Lim broth.
Adequately designed QCs play a crucial role in ensuring accurate and reliable patient results. These controls should truly simulate patient specimens. To achieve this, they must be formulated in clinically-relevant matrices in addition to containing whole organisms. This allows the comprehensive monitoring of the entire NAAT and pre-analytical processes, thus exposing these controls to similar challenges as the patient specimens they simulate.
Unlike the testing of other pathogens, GBS screening involves the additional broth enrichment step and, in some instances, subsequent swab dipping, as is the case with the Cepheid GBS LB and Cepheid GBS LB XC assays.
Here, we evaluate the effects of matrix type and swab immersion timing on the detection of GBS.
Anatomy of Group B Streptococcus
Streptococcus agalactiae, also known as Group B Streptococcus (GBS), is a gram-positive bacterium that commonly colonizes healthy adults—including 20-30% of pregnant women—without causing noticeable symptoms. More importantly, 50%-70% of neonates become infected with GBS when born from colonized mothers1. This poses a severe risk of potentially harmful complications and life-threatening infections to these newborns if GBS goes undetected2. Therefore, it is vital for expecting mothers to be screened for GBS, which typically occurs at weeks 35-37 of gestation3.
Identifying Group B Streptococcus may involve laborious culture-based techniques with long turnaround times and lower sensitivity. These traditional approaches often lead to diagnostic delays and the risk of yielding false negative outcomes. As a result, the gold standard bacterial culture is being replaced more and more frequently with NAAT molecular testing (real-time PCR in particular), which more generally represents a significant advancement in the realm of infectious disease diagnostics4. Both the culture and NAAT approaches require prior broth enrichment of the specimens for 18-24 hours5, which is widely performed in Lim broth. However, although direct-from-specimen (without broth enrichment) NAAT is not recommended for screening, it finds utility in cases such as when expecting mothers with unknown GBS status and no prior antibiotic prophylaxis go into labor.
Even after extraction, it has been shown that the nature of the matrix in clinical specimens and calibration standards have an effect on the results generated by various BKV assays6. Therefore, the effect of the matrices was not abolished by the various extraction methods associated with the assays tested.
Adequately designed and clinically relevant quality controls (QCs) are a cornerstone in ensuring the reliability of test results, which is particularly critical for GBS testing given the possible dramatic outcomes. Whether for assay verification/validation or routine run controls, QCs should be clinically-relevant and truly simulate patient specimens.
Hence, to replicate clinical conditions, QCs should carry the following attributes:
- Be made of whole organisms
- Be formulated in a matrix identical to or derived from the same physiological matrix (plasma, urine, etc.), transport medium (UTM, PreservCyt, etc.) or enrichment broth (Lim broth, etc.) constituting the patient specimens tested.
Designed this way, QCs can successfully mirror the same challenges and workflow intrinsic to clinical specimens.
This is recognized by the International Organization for Standardization 15189 (ISO 15189) on Medical Laboratories – Requirements for Quality and Competence, whose fourth edition of December 2022 indicates that “when selecting Internal quality control (IQC) material, factors to be considered shall include: […] the matrix is as close as possible to that of patient samples; […] the IQC material reacts to the examination method in a manner as close as possible to patient samples; […] the IQC material provides a clinically relevant challenge to the examination method, has concentration levels at or near clinical decision limits.”7
The College of American Pathologists echoes these requirements by stating in the Molecular Microbiology section of its Microbiology checklist that “controls are samples that act as surrogates for patient/client specimens. They are processed like a patient/client sample to monitor the ongoing performance of the entire analytic process in every run.”8
Therefore, QCs formulated in a clinically-relevant matrix and truly simulating patient specimens enable the comprehensive monitoring of steps related to the pre-analytical and analytical processes in patient testing. GBS molecular testing, in particular, introduces an additional layer of complexity by involving broth enrichment and the subsequent swab dipping within this enriched broth, a characteristic seen in tests like the Cepheid GBS LB and GBS LB XC assays.
Hence, QCs should also serve in the evaluation of the pre-analytical swab dipping step (which is only possible when the QC is in a liquid format) because the extent of GBS bacteria adsorption from the broth onto the swab influences the results.
GBS QC Study Methodology
We performed a study to better understand the adequacy and robustness of Exact Diagnostics Group B Streptococcus QC materials and techniques using both the Cepheid GBS LB assay and the newer Cepheid GBS LB XC assay by thoroughly evaluating (i) three swab dipping conditions; and (ii) two different matrices9.
The first component of our study involved dipping a clean single-use disposable swab in Exact Diagnostics GBS QC in adapted Lim broth (consisting of Lim broth with a preservative). Both the swab type and Lim broth required in Cepheid’s instructions for use were utilized.
The Exact Diagnostics GBS QC materials used for this study were prepared using absolute quantification as determined by a validated in-house laboratory-developed test (LDT) on Bio-Rad’s droplet digital PCR (ddPCR) platform. Comparative analysis of Ct values was performed after rapidly (<1 second) dipping a swab versus introducing a deliberate 3-second delay and 30-second delay during that dipping step using both the Cepheid assays.
To gain deeper insights into the impact of the swabbing technique on QC performance, we tested various Group B Streptococcus concentrations (100, 1000, and 3000 copies/mL) on the Cepheid GBS LB XC assay. Additionally, we compared the performance on the Cepheid GBS LB assay of the Exact Diagnostics QC in Lim broth versus when formulated in a synthetic matrix (resembling universal transport medium or UTM).
Lastly, for a more complete perspective supplementing the analyzing of GBS adsorption onto the swab, we took the alternative approach of directly introducing 100 mL of GBS QC in Lim broth into Cepheid cartridges.
Deciphering the Data
Our data indicates that when a clean swab is rapidly (<1 second) dipped into Exact Diagnostics GBS QC in Lim broth, it introduces an average delay of 2.1 and 2.4 Ct values when compared to a longer (3 seconds) dipping duration at a working concentration of 1,000 copies/mL with the Cepheid GBS LB and Cepheid GBS LB XC assays, respectively. Additionally, we observed that this rapid dipping also has the potential to generate false negative QC results when compared to the longer (3 seconds) dipping duration.
It is noteworthy that preliminary data associated with a substantially longer (and not workflow-friendly) 30-second dipping duration was not significantly different from dipping for 3 seconds. Moreover, pipetting the Exact Diagnostics GBS QC in Lim broth directly into Cepheid cartridges also resulted in similar Ct values compared to those observed when the cartridges were loaded with swabs dipped into this control. Taken together, these observations emphasize the criticality for the QC to control the pre-analytical swab dipping step (if required by the assay, as in the case of the Cepheid GBS LB and GBS LB XC assays) in addition to the entire real-time PCR process.
Furthermore, the rapid (<1 second) swabbing technique was also performed using the same Exact Diagnostics GBS QC in Lim broth but formulated in a synthetic matrix this time, and before processing both versions of this QC on the Cepheid GBS LB assay. At a working concentration of 1000 copies/mL, the results showed an average 1.7-Ct delay in Lim broth compared to the synthetic matrix. One of the replicates was even not detected in the Lim broth version. This suggests that the efficiency of GBS adsorption onto the swab surface differs significantly between Lim broth and the synthetic matrix. Although both matrices contained full Group B Streptococcus bacteria, Lim broth proved to be a more challenging and realistic matrix compared to the synthetic matrix tested. This underscores the significance of the matrix effect and emphasizes the importance of selecting a QC in the appropriate matrix to ensure true simulation of patient specimens.
Improving Group B Streptococcus Testing Quality
In the U.S., routine Group B Streptococcus screening requires subjecting vaginal-rectal swabs collected from expecting mothers to broth enrichment, then dipping a clean swab into that broth before loading it into the testing platform of assays such as the Cepheid GBS LB and GBS LB XC used in this study. Our findings specifically emphasize the importance for GBS QCs made of whole bacteria to be formulated in the clinically-relevant Lim broth matrix, and that these QCs are in a liquid format to also provide the ability to monitor the pre-analytical swab dipping step, when integrated into the patient specimen workflow. Hence, the criticality of the matrix effect and controlling the adsorption step of GBS onto a swab (when applicable) are demonstrated here.
As recognized by both CAP and ISO 15189:2022, it is imperative for laboratories to rely on quality controls appropriately designed to truly simulate patient specimens so that they can undergo the same workflow and challenges as those clinical specimens, whether those QCs are used for assay verification/validation or as routine run controls. By incorporating these measures, laboratory staff can establish a solid foundation to help obtain reliable testing results.
Our findings highlight key factors that must be considered when selecting meaningful QCs to verify/validate and routinely monitor assays, ultimately contributing to improved outcomes for patients.
4 Critically Important Steps to Take When Testing for GBS
As demonstrated by the study data in this article, when testing for Group B Streptococcus (or any other pathogen), it is critical to:
- Make sure that the QCs are appropriately designed to truly simulate patient specimens
- Ensure that the QCs undergo the same workflow as the clinical specimens
- Use Group B Streptococcus QC formulated in the same enrichment matrix (Lim broth here) as the patient specimens to be processed
- Control the post-enrichment swab-dipping step, if integrated in the laboratory workflow
By incorporating these measures, laboratory users can benefit from using QCs to help ensure reliable results. These findings emphasize the necessity for careful attention to these factors during testing, ultimately contributing to improved outcomes for patients.
Featured Image: The Exact Diagnostics GBS QC materials used for this study were prepared using absolute quantification as determined by a validated in-house laboratory-developed test (LDT) on Bio-Rad’s droplet digital PCR (ddPCR) platform. Photo: Bio-Rad
ABOUT THE AUTHORS
David Gauthier, MA, is senior product manager at Bio-Rad Laboratories. He is responsible for the Exact Diagnostics branded portfolio of molecular controls and verification panels at Bio-Rad.
Chance Garcia, MS MB(ASCP)CM, serves as an R&D scientist for the Fort Worth Bio-Rad Laboratories, which develops and manufactures the Exact Diagnostics branded molecular controls and verification panels at Bio-Rad.
Charles Rzadkowolski, MS, serves as the R&D group leader for the Fort Worth Bio-Rad Laboratories which develops and manufactures the Exact Diagnostics branded molecular controls and verification panels at Bio-Rad.
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7 ISO 15189:2022. “Medical laboratories – Requirements for quality and competence”. International Organization for Standardization; Fourth edition; December 2022 (Geneva, Switzerland)
8 College of American Pathologists (CAP). “Laboratory accreditation checklist – Microbiology”; 2020 (Northfield, IL)
9 Garcia C, Gauthier D, Rzadkowolski C. “Optimizing Exact Diagnostics Group B Streptococcus Quality Control in Lim Broth Processed on the Cepheid GBS LB Assay: Importance of Swabbing and Matrix Influence”. Poster presented at: 2023 Clinical Virology Symposium; September 2023; (West Palm Beach, FL)