Wednesday, August 17, 2022

A Fast, Accurate, Equipment-free Diagnostic Test for SARS-CoV-2 and its Variants

More than two years into the pandemic, the virus that causes COVID—SARS-CoV-2—continues to spread worldwide. Testing for the virus and its variants can help limit transmission and inform treatment decisions, and is therefore an important pillar of the public health response.

Now, a team of researchers from Princeton University and the Broad Institute of MIT and Harvard have created an easy-to-use diagnostic test for COVID infection that is more sensitive than the commonly used at-home antigen tests, and that also allows for the rapid and specific detection of SARS-CoV-2 variants in point-of-care settings. The study was published May 30, 2022 in the journal Nature Biomedical Engineering.

“Viral infections are often challenging to detect – as we all realized in the early days of the SARS-CoV-2 pandemic,” said Cameron Myhrvold, an assistant professor in the Department of Molecular Biology at Princeton.

The improved test detects virus using a different mechanism than the more familiar clinic-based PCR or at-home antigen tests. Instead, the test detects virus using a technology known as CRISPR, which has found widespread use in gene editing. CRISPR originated from a system used by bacteria to detect and defend against viral infections, and is uniquely suited to rapidly identifying specific genetic sequences.

SHINEv.2 is a CRISPR-based diagnostic test for SARS-CoV-2 and its variants that can be performed with only five simple steps. Depicted is a version of SHINE v2 that reads out its results via a method similar to other viral home tests.

Myhrvold previously worked with Broad Institute member and Harvard University professor Pardis Sabeti to develop a CRISPR-based test that could be tailored to diagnose specific viral infections. In the wake of the pandemic’s first wave in 2020, Myhrvold’s team of researchers modified their test, which they called SHINE, to detect SARS-CoV-2 with remarkable sensitivity.

“The original version of SHINE was more of a lab-based test,” said Myhrvold. “We wanted to make it possible to perform outside of a lab, with the ultimate goal of enabling people to self-test or test at home.”

Jon Arizti-Sanz, a graduate student in medical engineering in the joint Harvard-MIT Health Sciences and Technology department and whom Myhrvold is mentoring, spearheaded a collaborative effort between the two scientists’ research teams to improve SHINE. First, the group focused on eliminating the need for specialized equipment to prepare patient samples for testing. Next, they optimized the test so that its reagents don’t need to be kept in a freezer, which ensures that the test can be easily transported long distances.

They dubbed the improved test “SHINEv.2.” After confirming that the altered test worked well in their own laboratory, the group sent a test kit to a laboratory in Nigeria to see whether it could survive a long period in transit and still retain its accuracy and sensitivity. It did. But then a new challenge arose for the team to address.

“While we were working on SHINEv.2, new SARS-CoV-2 variants emerged, so we wanted to make tests for them,” said Myhrvold.

The team was able to quickly adapt SHINEv.2 to discern between infections mounted by the Alpha, Beta, Delta or Omicron SARS-CoV-2 variants in patient samples, and say the test can be rapidly modified to pick up on any other variants that may arise.

“The data indicating this approach can be used to distinguish SARS-CoV-2 variants of concern by differential detection of genomic mutations could enable us to better prepare for the appearance of the next mutation,” said Tony Hu, director of the Center for Intelligent Molecular Diagnostics and Weatherhead Presidential Chair at Tulane University. Hu reviewed the manuscript for Nature Biomedical Engineering.

Because it tests for the different variants all at once, this version of the test needs one special, but relatively inexpensive, piece of equipment to read out its results. Nonetheless, SHINEv.2 is much faster to perform, and requires much less equipment and expertise, than current approaches used to identify SARS-CoV-2 variants. The team envisions that it would mainly be used at doctors’ offices, even ones with limited resources in remote locations, to help physicians determine which viral variant is afflicting their patient, and tailor therapies appropriately.

The team also wanted to create a version of their test that would be usable by people at home, which meant optimizing it so that it can be performed without any special equipment.

“We created a fully equipment-free version that can be performed by incubating the reaction using body heat—holding it in the underarm—or at room temperature,” said Myhrvold. “Our goal was to minimize the need for heating as much as possible.”

“The approach reported by authors could be a significant contribution for pandemic control by providing a method that requires minimal sample processing or heating,” Hu said.

The home test doesn’t discern between the different viral variants, but the minimal sample processing involved in this version of SHINEv.2 would be a boon to the home user—especially with a test that can accurately detect an infection with high sensitivity, picking up on cases that might be missed by other types of home tests. Myhrvold is optimistic that SHINEv.2 will soon be joining the front lines in the battle against SARS-CoV-2, and any other new pathogen that arises in the future.

Citation: Jon Arizti-Sanz, A’Doriann Bradley, Yibin B. Zhang, Chloe K. Boehm, Catherine A. Freije, Michelle E. Grunberg, Tinna-Solveig F. Kosoko-Thoroddsen, Nicole L. Welch, Priya P. Pillai, Sreekar Mantena, Gaeun Kim, Jessica N. Uwanibe, Oluwagboadurami G. John, Philomena E. Eromon, Gregory Kocher, Robin Gross, Justin S. Lee, Lisa E. Hensley, Bronwyn L. MacInnis, Jeremy Johnson, Michael Springer, Christian T. Happi, Pardis C. Sabeti  and Cameron Myhrvold. Simplified Cas13-based assays for the fast identification of SARS-CoV-2 and its variants. Nature Biomedical Engineering. 2022. DOI: 10.1038/s41551-022-00889-z

Funding: Funding for the work described in this story was provided by DARPA D18AC00006; the Flu Lab; donors through TED’s Audacious Project (the ELMA Foundation, MacKenzie Scott, the Skoll Foundation and Open Philanthropy); the ‘la Caixa’ Foundation (ID 100010434, code LCF/BQ/AA18/11680098); start-up funds from Princeton University; Agreement No. HSHQDC-15-C-00064 awarded to Battelle National Biodefense Institute (BNBI)by the Department of Homeland Security (DHS) Science and Technology (S&T) Directorate for the management and operation of the National Biodefense Analysis and Countermeasures Center (NBACC), a Federally Funded Research and Development Center; the Howard Hughes Medical Institute; Merck KGaA Future Insight Prize; and the National Institutes of Health (RO1 GM120122- 01, U01AI151812 and U54HG007480).

Grant Numbers: D18AC00006, 100010434, code LCF/BQ/AA18/11680098, HSHQDC-15-C-00064, RO1 GM120122- 01, U01AI151812, U54HG007480

Funders: DARPA, Flu Lab, ELMA Foundation, MacKenzie Scott, TED’s Audacious Project, Skoll Foundation, Open Philanthropy, ‘la Caixa’ Foundation, Princeton University, Battelle National Biodefense Institute, Department of Homeland Security Science and Technology Directorate for the management and operation of the National Biodefense Analysis and Countermeasures Center, National Institutes of Health, Howard Hughes Medical Institute, Merck KGaA Future Insight Prize

Source: Department of Molecular Biology, Princeton University (https://molbio.princeton.edu/) 

Wednesday, August 10, 2022

New Approaches to BCC detection Using Loop-Mediated Isothermal Amplification

The pharmaceutical, personal care and medical device industries have experienced significant BCC contamination events in recent years,. As such, I will be presenting an overview of BCC investigation and remediation case studies at this year's PDA Global Conference on Microbiology.  

Numerous detection methods are currently in use, including those described in USP <60> and rapid PCR and real-time quantitative PCR (qPCR) assays. However, there is a continued desire to develop more rapid and sensitive BCC detection strategies.

To meet this need, a team of scientists from FDA, academia and industry recently published a paper titled, "Loop-Mediated Isothermal Amplification (LAMP) Assay for Detecting Burkholderia cepacia Complex in Non-Sterile Pharmaceutical Products."

Their abstract is reproduced below, and I encourage you to review the publication at your leisure.

Loop-Mediated Isothermal Amplification (LAMP) Assay for Detecting Burkholderia cepacia Complex in Non-Sterile Pharmaceutical Products. Soumana Daddy Gaoh, Ohgew Kweon, Yong-Jin Lee, John J. LiPuma, David Hussong, Bernard Marasa and Youngbeom Ahn. Pathogens 2021, 10(9), 1071. 

Abstract

Simple and rapid detection of Burkholderia cepacia complex (BCC) bacteria, a common cause of pharmaceutical product recalls, is essential for consumer safety. In this study, we developed and evaluated a ribB-based colorimetric loop-mediated isothermal amplification (LAMP) assay for the detection of BCC in (i) nuclease-free water after 361 days, (ii) 10 μg/mL chlorhexidine gluconate (CHX) solutions, and (iii) 50 μg/mL benzalkonium chloride (BZK) solutions after 184 days. The RibB 5 primer specifically detected 20 strains of BCC but not 36 non-BCC strains. The limit of detection of the LAMP assay was 1 pg/μL for Burkholderia cenocepacia strain J2315. Comparison of LAMP with a qPCR assay using 1440 test sets showed higher sensitivity: 60.6% in nuclease-free water and 42.4% in CHX solution with LAMP vs. 51.3% and 31.1%, respectively, with qPCR. These results demonstrate the potential of the ribB-based LAMP assay for the rapid and sensitive detection of BCC in pharmaceutical manufacturing.

Monday, August 8, 2022

EU Methods for the Detection and Characterisation of SARS-CoV-2 Variants Document Updated

The European Centre for Disease Prevention and Control has recently published its second edition of the "Methods for the detection and characterisation of SARS-CoV-2 variants" document

In the past year, several SARS-CoV-2 variants of concern (VOCs) have emerged and it is of key importance o monitor their circulation in all countries. Whole Genome Sequencing (WGS), or at least complete or partial spike (S)-gene sequencing, is the best method for characterising a specific variant. Alternative methods, such as diagnostic screening nucleic acid amplification technique (NAAT)-based assays, have been developed for early detection and pre-screening to allow prevalence calculation of VOCs, variants of interest (VOI) and variants under monitoring (VUM). Many of these methods can accurately identify the different variants, while others will require confirmation by sequencing of at least the complete or partial S-gene genomic region in a subset of samples.

Genomic monitoring should be integrated into the overall respiratory virus surveillance strategies. Specific objectives for the detection and identification of variants include assessment of the circulation of different SARS-CoV-2 variants in the community by selecting representative samples for sequencing, and genetic characterisation to monitor virus evolution and inform vaccine composition decisions or outbreak analyses. When NAAT-based assays are used, confirmatory sequencing of at least a subset of samples should be performed to use these assay results as indicators of community circulation of virus variants, particularly VOCs. Before introducing a new testing method or a new assay, a validation and verification exercise should be carried out to ensure that the laboratory testing system is reliably detecting the circulating viruses. Variant typing results should be reported to The European Surveillance System (TESSy) and SARS-CoV-2 consensus sequences should be deposited in the Global Initiative on Sharing All Influenza Data (GISAID) database, or other public databases. If available, related sequencing raw data should be deposited in the European Nucleotide Archive (ENA) and raw data, if available, in the European Nucleotide Archive (ENA). This should be done in a timely manner (ideally on a weekly basis).

This document was developed by technical experts from ECDC and the World Health Organization (WHO) European Region and previous versions have been reviewed by experts at WHO’s referral laboratories and in the SARS-CoV-2 Characterisation Working Group.

New in this Update

- Information related to detection assays specific for Omicron variants BA.4 and BA.5 has been included.
- The chapter on rapid antigen detection tests (RADT) has been updated and includes available information on their performance for Omicron variants.
- The chapter on neutralisation assays has been updated with information on the isolation of SARS-CoV-2 BA.4/5 variants.

The document may be downloaded here: 

https://www.ecdc.europa.eu/sites/default/files/documents/Methods-for-the-detection-char-SARS-CoV-2-variants_2nd%20update_final.pdf

Wednesday, August 3, 2022

Identifying Bacteria Using Optical Properties of Nanometer-Scaled Metal-Organic Hybrids

A recent study published in Analytical Chemistry proposes a strategy for optical detection of multiple bacterial species based on the optical properties of nanohybrid structures of polymer-coated metal nanoparticles. A review of this exciting research was discussed in AZO Optics

Rapid detection of bacteria is essential due to the rise in antibiotic-resistant microbes, the global food trade, and their application in pharmaceutical, bioremediation, and food production. Optical detection techniques have piqued the curiosity of researchers due to their potential for fast, high-throughput, non-destructive, amplification-free identification.

Developments in Bacterial Detection Techniques

Several bacterial species are useful for enhancing safety and quality of life in medication, food and energy production; yet, some bacteria are dangerous.

Bacterial identification tests performed in the food, environmental and medical field must satisfy selectivity, sensitivity, cost, and speed standards. Recent years have seen extensive research into the development of bacterial testing, ranging from absorption, luminescence, or current response-based detections to the integration of spectroscopy or microscopy and deep learning.

While these advancements have numerous advantages, they require adequate development time to replace conventional approaches.

Challenges of Conventional Bacterial Detection Techniques

Although conventional bacterial tests offer advantages, they also present various obstacles.

Culturing

Culturing is a popular method for identifying bacterial species based on their biological activity. However, results take at least one day because of the culture time.

Gram staining

Gram staining can be done more quickly than culture. It distinguishes between gram-negative and gram-positive bacteria under a microscope but cannot distinguish bacterial species.

Fluorescent labeling

Fluorescent labeling detects dye-conjugated antibody-labeled bacteria using flow cytometry or a microscope. It has issues related to intensity adjustment and limited fluorescence lifespan.

Lateral flow test

The lateral flow test uses antibody-conjugated gold nanoparticles (AuNP) to label target bacteria for naked-eye detection. The label does not fade, based on the AuNP's localized surface plasmon resonance. Stable inspection is easier with the lateral flow test than fluorescent labeling. However, due to the label's low optical intensity, the considerable antigen must be cultured to see the color.

Using Nanometer-Scaled Metal−Organic Hybrids to Detect Bacteria

Researchers used the optical properties of nanometer-scaled metal-organic hybrids to identify various bacteria in their study.

Metal nanoparticles (NPs) are valuable for optical detection and have strong affinities to biological components. Darkfield microscopy (DFM) was utilized to investigate scattering light induced by target substances due to its ability to observe metal nanoparticles smaller than the theoretical resolution limit.

A reaction system that autonomously controls nanostructure production was developed using aniline and metal ions to produce organic metal NHs.

E. coli O157, E. coli O26, and S. aureus were added to the mixture to generate an assessment solution with 13% bacteria density of the total cells. The capacity of NHs to mark individual cells was investigated using sample suspensions isolated from rotting chicken mince.

A dark field microscope and a field emission scanning electron microscope helped examine the mixture of the antibody-conjugated NH dispersion and bacterial solution. Images from a dark field microscope were captured using an optical microscope equipped with a halogen lamp, darkfield condenser, and a charge-coupled device camera.

The light-scattering spectra were recorded with a small grating spectrometer connected via an optical fiber to the dark field microscope. Focusing on the NH labels' light-scattering features helped identify the bacterial species.

Important Findings of the Study

Organic metal NHs are an excellent identification tool, facilitating quantitative and qualitative investigations of bacterial species in the same reaction area.

The optical properties of the nanohybrid structures (NHs) depend heavily on the individual metal elements of nanoparticles.

The rate of false negatives was estimated to be around 6%, while false positives were not confirmed.

Integrating antibodies into NHs leads to the binding of antigens to the cells, allowing bacteria to be identified by light scattering. Multiple bacterial species deposited on a slide were recognized within one field of view of a dark field microscope using scattered light colors.

Future Developments

There are currently no rapid techniques for detecting several bacterial species in a small number of samples. However, the proposed approach will allow for the simultaneous identification of many bacterial species in a single reaction area, which is not currently attainable with current technology.

The advancement of bacterial testing methods will improve quality and safety in various sectors of our life, including food production, medicine, and energy harvesting.

Reference

Tanabe, S., Itagaki, S., Matsui, K., Nishii, S., Yamamoto, Y., Sadanaga, Y., & Shiigi, H. (2022). Simultaneous Optical Detection of Multiple Bacterial Species Using Nanometer-Scaled Metal–Organic Hybrids. Analytical Chemistry. https://pubs.acs.org/doi/10.1021/acs.analchem.2c01188

Source: AZO Optics.