Saliva as a testing specimen with or without pooling for SARS-CoV-2 detection by multiplex RT-PCR test

Background Sensitive and high throughput molecular detection assays are essential during the coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The vast majority of the SARS-CoV-2 molecular assays use nasopharyngeal swab (NPS) or oropharyngeal swab (OPS) specimens collected from suspected individuals. However, using NPS or OPS as specimens has apparent drawbacks, e.g. the collection procedures for NPS or OPS specimens can be uncomfortable to some people and may cause sneezing and coughing which in turn generate droplets and/or aerosol particles that are of risk to healthcare workers, requiring heavy use of personal protective equipment. There have been recent studies indicating that self-collected saliva specimens can be used for molecular detection of SARS-CoV-2 and provides more comfort and ease of use for the patients. Here we report the performance of QuantiVirus™ SARS-CoV-2 test using saliva as the testing specimens with or without pooling. Methods Development and validation studies were conducted following FDA-EUA and molecular assay validation guidelines. Using SeraCare Accuplex SARS-CoV-2 reference panel, the limit of detection (LOD) and clinical performance studies were performed with the QuantiVirus™ SARS-CoV-2 test. For clinical evaluation, 85 known positive and 90 known negative clinical NPS samples were tested. Additionally, twenty paired NPS and saliva samples collected from recovering COVID-19 patients were tested and the results were further compared to that of the Abbott m2000 SARS-CoV-2 PCR assay. Results of community collected 389 saliva samples for COVID-19 screening by QuantiVirus™ SARS-CoV-2 test were also obtained and analyzed. Additionally, testing of pooled saliva samples was evaluated. Results The LOD for the QuantiVirus™ SARS-CoV-2 test was confirmed to be 100–200 copies/mL. The clinical performance studies using contrived saliva samples indicated that the positive percentage agreement (PPA) of the QuantiVirus™ SARS-CoV-2 test is 100% at 1xLOD, 1.5xLOD and 2.5xLOD. No cross-reactivity was observed for the QuantiVirus™ SARS-CoV-2 test with common respiratory pathogens. Testing of clinical samples showed a positive percentage agreement (PPA) of 100% (95% CI: 94.6% to 100%) and a negative percentage agreement (NPA) of 98.9% (95% CI: 93.1% to 99.9%). QuantiVirus™ SARS CoV-2 test had 80% concordance rate and no significant difference (p = 0.13) between paired saliva and NPS specimens by Wilcoxon matched pairs signed rank test. Positive test rate was 1.79% for 389 saliva specimens collected from local communities for COVID-19 screening. Preliminary data showed that saliva sample pooling up to 6 samples (1:6 pooling) for SARS-CoV-2 detection is feasible (sensitivity 94.8% and specificity 100%). Conclusion The studies demonstrated that the QuantiVirus™ SARS-CoV-2 test has a LOD of 200 copies/mL in contrived saliva samples. The clinical performance of saliva-based testing is comparable to that of NPS-based testing. Pooling of saliva specimens for SARS-CoV-2 detection is feasible. Saliva based and high-throughput QuantiVirus™ SARS-CoV-2 test offers a highly desirable testing platform during the ongoing COVID-19 pandemic.

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Saliva as a testing specimen with or without pooling for SARS-CoV-2 detection by multiplex RT-PCR test

10.1101/2020.10.27.20219196 ◽

2020 ◽

Author(s):

Qing Sun ◽

Jonathan Li ◽

Hui Ren ◽

Larry Pastor ◽

Yulia Loginova ◽

...

Keyword(s):

Clinical Evaluation ◽

High Throughput ◽

Molecular Detection ◽

Limit Of Detection ◽

Saliva Sample ◽

Clinical Samples ◽

Rank Test ◽

Percentage Agreement ◽

Testing Specimen ◽

Significant Difference

AbstractBackgroundSensitive and high throughput molecular detection assays are essential during the coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The vast majority of the SARS-CoV-2 molecular assays use nasopharyngeal swab (NPS) or oropharyngeal swab (OPS) specimens collected from suspected individuals. However, using NPS or OPS as specimens has apparent drawbacks, e.g. the collection procedures for NPS or OPS specimens can be uncomfortable to some people and may cause sneezing and coughing which in turn generate droplets and/or aerosol particles that are of risk to healthcare workers, requiring heavy use of personal protective equipment. There have been recent studies indicating that self-collected saliva specimens can be used for molecular detection of SARS-CoV-2 and provides more comfort and ease of use for the patient. Here we report the performance of QuantiVirus™ SARS-CoV-2 multiplex test using saliva as the testing specimens with or without pooling.MethodsDevelopment and validation studies were conducted following FDA-EUA and molecular assay validation guidelines. Using SeraCare Accuplex SARS-CoV-2 reference panel, the limit of detection (LOD) and clinical evaluation studies were performed with the QuantiVirus™ SARS-CoV-2 multiplex test. For clinical evaluation, 85 known positive and 90 known negative clinical NPS samples were tested. Additionally, twenty paired NPS and saliva samples collected from recovering COVID-19 patients were tested and the results were further compared to that of the Abbott m2000 SARS-CoV-2 PCR assay. Results of community collected 389 saliva samples for COVID-19 screening by QuantiVirus™ SARS-CoV-2 multiplex test were also obtained and analyzed. Moreover, saliva pooling with 6 and 12 samples together were also evaluated.ResultsThe LOD for the QuantiVirus™ SARS-CoV-2 multiplex test was confirmed to be 100-200 copies/mL. The clinical evaluation using contrived saliva samples indicated that the positive percentage agreement (PPA) of the QuantiVirus™ SARS-CoV-2 multiplex test is 100% at 1xLOD, 1.5xLOD and 2.5xLOD. No cross-reactivity was observed for the QuantiVirus™ SARS-CoV-2 multiplex test with common respiratory pathogens. Testing of clinical samples showed a positive percentage agreement (PPA) of 100% (95% CI: 94.6% to 100%) and a negative percentage agreement (NPA) of 98.9% (95% CI: 93.1% to 99.9%). QuantiVirus ™SARS CoV-2 multiplex test had 80% concordance rate and no significant difference (p=0.13) in paired saliva and NPS specimens by Wilcoxon matched pairs signed rank test. Positive test rate was 1.79% for 389 saliva specimens collected from the communities for COVID-19 screening. Preliminary data showed that saliva sample pooling up to 6 samples for SARS-CoV-2 detection is feasible (sensitivity 94.8% and specificity 100%).ConclusionThe studies demonstrated that the QuantiVirus™ SARS-CoV-2 multiplex test has a LOD of 200 copies/mL in contrived saliva samples. The clinical performance of saliva-based testing is comparable to that of NPS-based testing. Pooling of saliva specimens for SARS-CoV-2 detection is feasible. Saliva based and high-throughput QuantiVirus™SARS-CoV-2 multiplex test offers a highly desirable test during the ongoing COVID-19 pandemic.

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Direct diagnostic testing of SARS-CoV-2 without the need for prior RNA extraction

Scientific Reports ◽

10.1038/s41598-021-81487-y ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Shan Wei ◽

Esther Kohl ◽

Alexandre Djandji ◽

Stephanie Morgan ◽

Susan Whittier ◽

...

Keyword(s):

Limit Of Detection ◽

Point Of Care ◽

Diagnostic Testing ◽

Rna Extraction ◽

Cost Effective ◽

Clinical Samples ◽

Nasopharyngeal Swab ◽

Percentage Agreement ◽

Testing Method ◽

Viral Transport

AbstractThe COVID-19 pandemic has resulted in an urgent need for a rapid, point of care diagnostic testing that could be rapidly scaled on a worldwide level. We developed and tested a highly sensitive and robust assay based on reverse transcription loop mediated isothermal amplification (RT-LAMP) that uses readily available reagents and a simple heat block using contrived spike-in and actual clinical samples. RT-LAMP testing on RNA-spiked samples showed a limit of detection (LoD) of 2.5 copies/μl of viral transport media. RT-LAMP testing directly on clinical nasopharyngeal swab samples in viral transport media had an 85% positive percentage agreement (PPA) (17/20), and 100% negative percentage agreement (NPV) and delivered results in 30 min. Our optimized RT-LAMP based testing method is a scalable system that is sufficiently sensitive and robust to test for SARS-CoV-2 directly on clinical nasopharyngeal swab samples in viral transport media in 30 min at the point of care without the need for specialized or proprietary equipment or reagents. This cost-effective and efficient one-step testing method can be readily available for COVID-19 testing world-wide, especially in resource poor settings.

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Validation of a real-time PCR assay for high-throughput detection of Avibacterium paragallinarum in chicken respiratory sites

Journal of Veterinary Diagnostic Investigation ◽

10.1177/1040638719866484 ◽

2019 ◽

Vol 31 (5) ◽

pp. 714-718 ◽

Cited By ~ 2

Author(s):

Kristin A. Clothier ◽

Simone Stoute ◽

Andrea Torain ◽

Beate Crossley

Keyword(s):

Real Time ◽

High Throughput ◽

Real Time Pcr ◽

Limit Of Detection ◽

Bacterial Flora ◽

Clinical Samples ◽

Single Tube ◽

Avibacterium Paragallinarum ◽

Normal Bacterial Flora ◽

Close Relatives

Avibacterium paragallinarum is the causative agent of infectious coryza, a highly contagious respiratory disease in chickens. Given its fastidious nature, this bacterium is difficult to recover and identify, particularly from locations colonized by normal bacterial flora. Standard PCR methods have been utilized for detection but are labor-intensive and not feasible for high-throughput testing. We evaluated a real-time PCR (rtPCR) method targeting the HPG-2 region of A. paragallinarum, and validated a high-throughput extraction for this assay. Using single-tube extraction, the rtPCR detected 4 A. paragallinarum (ATCC 29545T and 3 clinical) isolates with a limit of detection (LOD) of 10 cfu/mL and a PCR efficiency of 89–111%. Cross-reaction was not detected with 33 non– A. paragallinarum, all close relatives from the family Pasteurellaceae. Real-time PCR testing on extracts of 66 clinical samples (choana, sinus, or trachea) yielded 98.2% (35 of 36 on positives, 30 of 30 on negatives) agreement with conventional PCR. Duplicate samples tested in a 96-well format extraction in parallel with the single-tube method produced equivalent LOD on all A. paragallinarum isolates, and 96.8% agreement on 93 additional clinical samples extracted with both procedures. This A. paragallinarum rtPCR can be utilized for outbreak investigations and routine monitoring of susceptible flocks.

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High throughput Next-Generation Sequencing Respiratory Viral Panel: A Diagnostic and Epidemiologic Tool for SARS-CoV-2 and Other Viruses.

10.1101/2021.07.24.21261046 ◽

2021 ◽

Author(s):

Nikhil Shri Sahajpal ◽

Ashis K Mondal ◽

Allan Njau ◽

Zachary Petty ◽

Jiani Chen ◽

...

Keyword(s):

Next Generation Sequencing ◽

High Throughput ◽

Performance Metrics ◽

Limit Of Detection ◽

Virus Transmission ◽

Respiratory Viruses ◽

Clinical Samples ◽

Current Phase ◽

Next Generation ◽

Generation Sequencing

Background: In the current phase of the COVID-19 pandemic, we are facing two serious public health challenges that include deficits in SARS-CoV-2 variant monitoring and neglection of other co-circulation respiratory viruses. Additionally, accurate assessment of the evolution, extent, and dynamics of the outbreak are required to understand the transmission of the virus amongst seemingly unrelated cases and provide critical epidemiological information. To address these challenges, we evaluated a new high-throughput next-generation sequencing (NGS) panel that includes 40 viral pathogens to analyze viral subtypes, mutational variants of SARS-CoV-2, model to understand the spread of the virus in the state of Georgia, USA, and to assess other circulating viruses in the same population. Methods This study evaluated a total of 522 samples that included 483 patient samples and 42 synthetic positive control materials. The performance metrics were calculated for both clinical and reference control samples by comparing detection results with the RT-PCR assay. The limit of detection (LoD) studies were conducted as per the FDA guidelines. Inference and visualization of the phylogeny of the SARS-CoV-2 sequences were performed through the Nextstrain Command-Line Interface (CLI) tool, utilizing the associated augur and auspice toolkits. Result The performance metric was calculated using both the clinical samples and the reference control with a PPA, NPA, and accuracy of 95.98%, 85.96%, and 94.4%, respectively. The LoD was determined to be 10 copies/ml with all 25 replicates detected across two different runs. The clade for pangolin lineage B contains certain distant variants, including P4715L in ORF1ab, Q57H in ORF 3a and, S84L in ORF8 covarying with the D614G spike protein mutation were found to be prevalent in the early pandemic in Georgia, USA. Isolates from the same county formed paraphyletic groups in our analysis, which indicated virus transmission between counties. Conclusion The study demonstrates the clinical utility of the NGS panel to identify novel variants that can provide actionable information to prevent or mitigate emerging viral threats, models that provide insights into viral transmission patterns and predict transmission/ resurgence of regional outbreaks and provide critical information on co-circulating respiratory viruses that might be independent factors contributing to the global disease burden.

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Evaluation of RT-qPCR and Loop-Mediated Isothermal Amplification (LAMP) Assays for the Detection of SARS-CoV-2 in Argentina

Genes ◽

10.3390/genes12050659 ◽

2021 ◽

Vol 12 (5) ◽

pp. 659

Author(s):

María Dolores Fellner ◽

Romina Bonaventura ◽

Jorge Basiletti ◽

Martín Avaro ◽

Estefanía Benedetti ◽

...

Keyword(s):

World Health Organization ◽

Reverse Transcription ◽

Diagnostic Performance ◽

Limit Of Detection ◽

Clinical Performance ◽

Isothermal Amplification ◽

World Health ◽

Clinical Samples ◽

Loop Mediated Isothermal Amplification ◽

Health Organization

Our aim was to evaluate the analytical and clinical performance of the SARS-CoV-2 molecular detection kits used in Argentina. Nine real-time reverse-transcription polymerase chain reaction (RT-qPCR) and three reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assays were evaluated using the World Health Organization (WHO) recommended test as reference method. A secondary standard calibrated for the E, N and RdRp genes against the Pan American Health Organization—World Health Organization—International Standard was used to calculate the limit of detection (LoD). A panel of artificial clinical samples, 32 positive and 30 negative for SARS-CoV-2, were analyzed to estimate the kappa concordance (κ) and the diagnostic performance. Differences among the LoD values for the target genes amplified by each kit were >1 log copies/reaction. The κ for the RT-qPCR kits was greater than 0.9, whereas that for the RT-LAMP assays ranged from 0.75 to 0.93. The clinical performance of RT-qPCR kits showed 100% specificity and high sensitivity, although with variations according to the gene analyzed. The E and N genes provided greater clinical sensitivity, whereas the RdRp gene increased the clinical specificity. The RT-LAMP assays revealed a variable diagnostic performance. The information provided can be useful to choose the most appropriate diagnostic test and may contribute to the establishment of a consensus in the diagnosis of SARS-CoV-2 in Argentina and the region.

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Saliva Is a Valid Alternative to Nasopharyngeal Swab in Chemiluminescence-Based Assay for Detection of SARS-CoV-2 Antigen

Journal of Clinical Medicine ◽

10.3390/jcm10071471 ◽

2021 ◽

Vol 10 (7) ◽

pp. 1471

Author(s):

Alessandra Amendola ◽

Giuseppe Sberna ◽

Eleonora Lalle ◽

Francesca Colavita ◽

Concetta Castilletti ◽

...

Keyword(s):

Limit Of Detection ◽

Clinical Performance ◽

Diagnostic Methods ◽

Clinical Samples ◽

Nasopharyngeal Swab ◽

Promising Alternative ◽

Viral Loads ◽

Healthy Donors ◽

Antigen Assay ◽

Highly Correlated

Diagnostic methods based on SARS-CoV-2 antigens detection are a promising alternative to SARS-CoV-2 RNA amplification. We evaluated the automated chemiluminescence-based Lumipulse® G SARS-CoV-2 Ag assay on saliva samples, using Simplexa™ COVID-19 Direct assay as a reference test. Analytical performance was established on a pool of healthy donors’ saliva samples spiked with the 2019-nCoV/Italy-INMI1 isolate, whereas clinical performance was assessed on fresh saliva specimens collected from hospitalized patients with suspect or confirmed COVID-19 diagnosis. The limit of detection (LOD) was 0.65 Log TCID50/mL, corresponding to 18,197 copies/mL of SARS-CoV-2 RNA. Antigen concentrations and SARS-CoV-2 RNA were highly correlated (r = 0.99; p < 0.0001). Substantial agreement (80.3%) and significant correlation (r = −0.675; p = 0.0006) were observed between Lumipulse® G assay results and Ct values on clinical samples, with 52.4% sensitivity and specificity 94.1%. Sensitivity exceeded 90.0% when calculated on samples with Ct < 25, and specificity was 100% when excluding samples from recovered patients with previous COVID-19 diagnosis. Overall, chemiluminescence-based antigen assay may be reliably applied to saliva samples to identify individuals with high viral loads, more likely to transmit the virus. However, the low positive predictive value in a context of low SARS-CoV-2 prevalence underscores the need for confirmatory testing in SARS-CoV-2 antigen-positive cases.

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An isothermal method for sensitive detection of Mycobacterium tuberculosis complexes using CRISPR/Cas12a cis- and trans-cleavage

10.1101/2020.02.03.933101 ◽

2020 ◽

Author(s):

Haipo Xu ◽

Xiaolong Zhang ◽

Zhixiong Cai ◽

Xiuqing Dong ◽

Geng Chen ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Limit Of Detection ◽

Clinical Performance ◽

High Specificity ◽

Culture Method ◽

Sensitive Detection ◽

Clinical Samples ◽

Target Sequence ◽

Guide Rna ◽

Specificity And Sensitivity

AbstractTuberculosis is still one of the most serious infectious diseases resulting in lethal death worldwide. The traditional method is still not enough to meet the clinical requirements of rapid diagnosis, high specificity and sensitivity. Fast, sensitive and accurate detection of mycobacterium tuberculosis (MTB) is an urgent need for the treatment and control of tuberculosis disease. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated proteins (Cas12a) exhibits strongly nonspecific degradation ability of exogenous single-strand nucleic acid (trans-cleavage) after specific recognition of target sequence. We purified Cas12a protein and selected a proper guide RNA (gRNA) based on conserved sequences of MTB from gRNA library we designed. Then, we proposed a novel method based on recombinase polymerase amplification (RPA) and CRISPR/Cas12a nuclease system for specific and sensitive detection of MTB DNA. The assay based on fluorescence detection pattern showed 4.48 fM of limit of detection (LOD) and good linear correlation of concentration and fluorescence value (R2=0.9775). Also, it showed good performance in distinguishing other bacteria. Furthermore, its clinical performance was evaluated by 193 samples and showed sensitivity of 99.29% (139/140) and specificity of 100% (53/53) at 99% confidence interval, respectively, compared with culture method. The CRISPR/Cas12a system showed good specificity, excellent sensitivity and accuracy for MTB detection, and it meets requirements of MTB detection in clinical samples and has great potential for clinical translation.

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Rapid and sensitive point-of-care detection of Leptospira by RPA-CRISPR/Cas12a targeting lipL32

PLoS Neglected Tropical Diseases ◽

10.1371/journal.pntd.0010112 ◽

2022 ◽

Vol 16 (1) ◽

pp. e0010112

Author(s):

Sirawit Jirawannaporn ◽

Umaporn Limothai ◽

Sasipha Tachaboon ◽

Janejira Dinhuzen ◽

Patcharakorn Kiatamornrak ◽

...

Keyword(s):

Limit Of Detection ◽

Clinical Performance ◽

Point Of Care ◽

Limited Resource ◽

Cross Reactivity ◽

Clinical Samples ◽

Detection Assay ◽

Short Palindromic Repeat ◽

Flow Detection ◽

Point Of Care Detection

Background One of the key barriers preventing rapid diagnosis of leptospirosis is the lack of available sensitive point-of-care testing. This study aimed to develop and validate a clustered regularly-interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 12a (CRISPR/Cas12a) platform combined with isothermal amplification to detect leptospires from extracted patient DNA samples. Methodology/Principal findings A Recombinase Polymerase Amplification (RPA)-CRISPR/Cas12a-fluorescence assay was designed to detect the lipL32 gene of pathogenic Leptospira spp. The assays demonstrated a limit of detection (LOD) of 100 cells/mL, with no cross-reactivity against several other acute febrile illnesses. The clinical performance of the assay was validated with DNA extracted from 110 clinical specimens and then compared to results from qPCR detection of Leptospira spp. The RPA-CRISPR/Cas12a assay showed 85.2% sensitivity, 100% specificity, and 92.7% accuracy. The sensitivity increased on days 4–6 after the fever onset and decreased after day 7. The specificity was consistent for several days after the onset of fever. The overall performance of the RPA-CRISPR/Cas12a platform was better than the commercial rapid diagnostic test (RDT). We also developed a lateral flow detection assay (LFDA) combined with RPA-CRISPR/Cas12a to make the test more accessible and easier to interpret. The combined LFDA showed a similar LOD of 100 cells/mL and could correctly distinguish between known positive and negative clinical samples in a pilot study. Conclusions/Significance The RPA-CRISPR/Cas12 targeting the lipL32 gene demonstrated acceptable sensitivity and excellent specificity for detection of leptospires. This assay might be an appropriate test for acute leptospirosis screening in limited-resource settings.

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Comparison of Two High-Throughput Reverse Transcription-PCR Systems for the Detection of Severe Acute Respiratory Syndrome Coronavirus 2

Journal of Clinical Microbiology ◽

10.1128/jcm.00890-20 ◽

2020 ◽

Vol 58 (8) ◽

Cited By ~ 20

Author(s):

Arryn R. Craney ◽

Priya D. Velu ◽

Michael J. Satlin ◽

Kathy A. Fauntleroy ◽

Katrina Callan ◽

...

Keyword(s):

Severe Acute Respiratory Syndrome ◽

High Throughput ◽

Reverse Transcription ◽

Clinical Performance ◽

Threshold Values ◽

Limited Data ◽

Rt Pcr ◽

Reverse Transcription Pcr ◽

Significant Difference ◽

Pcr Assays

ABSTRACT Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has emerged as the cause of a worldwide pandemic. Many commercial SARS-CoV-2 reverse transcription-PCR (RT-PCR) assays have received Emergency Use Authorization from the U.S. Food and Drug Administration. However, there are limited data describing their performance, in particular the performance of high-throughput SARS-CoV-2 RT-PCR systems. We analyzed the diagnostic performance of two high-throughput systems: cobas 6800 and Panther Fusion, and their associated RT-PCR assays, with a collection of 389 nasopharyngeal specimens. The overall agreement between the platforms was 96.4% (375/389). Cohen’s kappa analysis rated the strength of agreement between the two platforms as “almost perfect” (κ = 0.922; standard error, 0.051). Furthermore, there was no significant difference between corresponding cycle threshold values generated on the two systems (P value = 0.88; Student’s t test). Taken together, these data imply that the two platforms can be considered comparable in terms of their clinical performance. We believe that this information will be useful for those who have already adopted these platforms or are seeking to implement high-throughput RT-PCR testing to stem the SARS-CoV-2 pandemic.

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High-throughput nanofluidic real-time PCR to discriminate Pneumococcal Conjugate Vaccine (PCV)-associated serogroups 6, 18, and 22 to serotypes using modified oligonucleotides.

10.21203/rs.3.rs-798778/v1 ◽

2021 ◽

Author(s):

Sarah Leah Downs ◽

Shabir Ahmed Madhi ◽

Lara van Der Merwe ◽

Marta Coelho Nunes ◽

Courtney Paige Olwagen

Keyword(s):

Real Time ◽

High Throughput ◽

Real Time Pcr ◽

Limit Of Detection ◽

Locked Nucleic Acid ◽

Clinical Samples ◽

Taqman Assay ◽

Vaccine Serotypes ◽

Serotype Replacement ◽

The Impact

Abstract Current real-time Polymerase Chain Reaction (qPCR) methods are unable to distinguish serotypes 6A from 6B, 18C from 18A/B and 22F from 22A. We established a nanofluidic real-time PCR (Fluidigm) for serotyping that included Dual-Priming-Oligonucleotides (DPO), a Locked-Nucleic-Acid (LNA) probe and TaqMan assay-sets for high-throughput serotyping. The designed assay-sets target capsular gene wciP in serogroup 6, wciX and wxcM in serogroup 18, and wcwA in serogroup 22. An algorithm combining results from published assay-sets (6A/B/C/D; 6C/D; 18A/B/C; 22A/F) and designed assay-sets for 6A/C; 18B/C/F; 18C/F, 18F and 22F was validated through blind analysis of 1973 archived clinical samples collected from South African children ≤ 5-years-old (2009-11), previously serotyped with the culture-based Quellung method. All assay-sets were efficient (92–101%), had low variation between replicates (R2 > 0.98), and were able to detect targets at a limit of detection (LOD) of < 100 Colony-Forming-Units (CFU)/ml of sample. There was high concordance (Kappa = 0.73–0.92); sensitivity (85–100%) and specificity (96–100%) for Fluidigm compared with Quellung for serotyping 6A; 6B; 6C; 18C and 22F. Fluidigm distinguishes vaccine-serotypes 6A, 6B, 18C, next-generation PCV-serotype 22F and non-vaccine-serotypes 6C, 6D, 18A, 18B, 18F and 22A. Discriminating single serotypes is important for assessing serotype replacement and the impact of PCVs on vaccine- and non-vaccine serotypes.

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