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Development and Validation of a Sensitive and Robust Multiplex Antigen Capture Assay to Quantify Streptococcus
Gowrisankar Rajam1, Yuhua Zhang1, Joseph M Antonello1
1Merck & Co., Inc., Rahway, New Jersey, USA.
Insights
A new urine test accurately detects 15 types of Streptococcus pneumoniae, aiding pneumonia research. This assay helps understand pneumococcal disease burden and vaccine impact in non-invasive cases.
Area of Science:
- Microbiology and Immunology
- Vaccinology
- Clinical Diagnostics
Background:
- Streptococcus pneumoniae causes significant community-acquired pneumonia (CAP), especially in vulnerable populations.
- Pneumococcal vaccines reduce invasive disease, but their impact on overall CAP burden is unclear due to limited serotype testing.
- Current methods for pneumococcal serotyping in respiratory samples are unreliable and not routine, leaving the burden of non-invasive disease largely unknown.
Purpose of the Study:
- To develop, qualify, and validate a high-throughput, multiplex serotype-specific urine antigen detection (SSUAD) assay.
- To address the unmet need for reliable laboratory techniques in pneumococcal epidemiology, particularly for non-invasive CAP.
- To enable accurate quantification of 15 specific pneumococcal serotypes (STs) included in the V114 vaccine.
Main Methods:
- Developed a 15-plex SSUAD assay utilizing ST-specific monoclonal antibodies and a Luminex microfluidics system.
- Optimized and qualified the assay using healthy adult urine spiked with pneumococcal polysaccharides.
- Validated the assay with culture-positive clinical urine samples (n=34), assessing accuracy, precision, sensitivity, specificity, selectivity, and parallelism.
Main Results:
- The SSUAD assay demonstrated high sensitivity and specificity for the 15 targeted pneumococcal serotypes.
- The assay met all prespecified validation acceptance criteria, confirming its reliability.
- The assay is suitable for quantifying pneumococcal STs in urine for disease burden assessments.
Conclusions:
- The validated SSUAD assay provides a reliable method to estimate pneumococcal disease burden and serotype distribution in non-bacteremic conditions.
- This assay will be crucial for understanding the impact of pneumococcal vaccines on non-invasive pneumococcal diseases.
- Data from this assay will inform the selection of pneumococcal serotypes for future vaccine development.
Abstract:
Streptococcus pneumoniae is a major cause of community-acquired pneumonia (CAP) in young children, older adults, and those with immunocompromised status. Since the introduction of pneumococcal vaccines, the burden of invasive pneumococcal disease caused by vaccine serotypes (STs) has decreased; however, the effect on the burden of CAP is unclear, potentially due to the lack of testing for pneumococcal STs. We describe the development, qualification, and clinical validation of a high-throughput and multiplex ST-specific urine antigen detection (SSUAD) assay to address the unmet need in CAP pneumococcal epidemiology. The SSUAD assay is sensitive and specific to the 15 STs in the licensed pneumococcal conjugate vaccine V114 (STs 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F) and uses ST-specific monoclonal antibodies for rapid and simultaneous quantification of the 15 STs using a Luminex microfluidics system. The SSUAD assay was optimized and qualified using healthy adult urine spiked with pneumococcal polysaccharides and validated using culture-positive clinical urine samples (n = 34). Key parameters measured were accuracy, precision, sensitivity, specificity, selectivity, and parallelism. The SSUAD assay met all prespecified validation acceptance criteria and is suitable for assessments of disease burden associated with the 15 pneumococcal STs included in V114. IMPORTANCE Streptococcus pneumoniae has more than 90 serotypes capable of causing a range of disease manifestations, including otitis media, pneumonia, and invasive diseases, such as bacteremia or meningitis. Only a minority (<10%) of pneumococcal diseases are bacteremic with known serotype distribution. Culture and serotyping of respiratory specimens are neither routine nor reliable. Hence, the serotype-specific disease burden of the remaining (>90%) noninvasive conditions is largely unknown without reliable laboratory techniques. To address this need, a 15-plex urine antigen detection assay was developed and validated to quantify pneumococcal serotype-specific capsular polysaccharides in urine. This assay will support surveillance to estimate the pneumococcal disease burden and serotype distribution in nonbacteremic conditions. Data obtained from this assay will be critical for understanding the impact of pneumococcal vaccines on noninvasive pneumococcal diseases and to inform the choice of pneumococcal serotypes for next-generation vaccines.
