Accelerating the optimization of vertical flow assay performance guided by a rational systematic model-based approach.

Rapid diagnostic tests (RDTs) have shown to be instrumental in healthcare and disease control. However, they have been plagued by many inefficiencies in the laborious empirical development and optimization process for the attainment of clinically relevant sensitivity. While various studies have sought to model paper-based RDTs, most have relied on continuum-based models that are not necessarily applicable to all operation regimes, and have solely focused on predicting the specific interactions between the antigen and binders. It is also unclear how the model predictions may be utilized for optimizing assay performance. Here, we propose a streamlined and simplified model-based framework, only relying on calibration with a minimal experimental dataset, for the acceleration of assay optimization. We show that our models are capable of recapitulating experimental data across different formats and antigen-binder-matrix combinations. By predicting signals due to both specific and background interactions, our facile approach enables the estimation of several pertinent assay performance metrics such as limit-of-detection, sensitivity, signal-to-noise ratio and difference. We believe that our proposed workflow would be a valuable addition to the toolset of any assay developer, regardless of the amount of resources they have in their arsenal, and aid assay optimization at any stage in their assay development process.

Identification of colonies of cultured shellfish-associated Arcobacter species by Elastic Light Scatter Analysis.

An increasing number of Arcobacter species (including several regarded as emerging human foodborne pathogens) have been isolated from shellfish, an important food commodity. A method to distinguish these species and render viable isolates for further analysis would benefit epidemiological and ecological studies. We describe a method based on Elastic Light Scatter analysis (ELSA) for the detection and discrimination of eleven shellfish-associated Arcobacter species. Although substantive differences in the growth rates of some taxa were seen, ELSA was able to differentiate all the species studied, apart from some strains of A. butzleri and A. cryaerophilus, which were nonetheless distinguished from all other species examined. ELSA appears to be a promising new approach for the detection and identification of Arcobacter species in shellfish and may also be applicable for studies in other foods and matrices.

Developing a SARS-CoV-2 Antigen Test Using Engineered Affinity Proteins.

The ongoing COVID-19 pandemic has clearly established how vital rapid, widely accessible diagnostic tests are in controlling infectious diseases and how difficult and slow it is to scale existing technologies. Here, we demonstrate the use of the rapid affinity pair identification via directed selection (RAPIDS) method to discover multiple affinity pairs for SARS-CoV-2 nucleocapsid protein (N-protein), a biomarker of COVID-19, from in vitro libraries in 10 weeks. The pair with the highest biomarker sensitivity was then integrated into a 10 min, vertical-flow cellulose paper test. Notably, the as-identified affinity proteins were compatible with a roll-to-roll printing process for large-scale manufacturing of tests. The test achieved 40 and 80 pM limits of detection in 1× phosphate-buffered saline (mock swab) and saliva matrices spiked with cell-culture-generated SARS-CoV-2 viruses and is also capable of detection of N-protein from characterized clinical swab samples. Hence, this work paves the way toward the mass production of cellulose paper-based assays which can address the shortages faced due to dependence on nitrocellulose and current manufacturing techniques. Further, the results reported herein indicate the promise of RAPIDS and engineered binder proteins for the timely and flexible development of clinically relevant diagnostic tests in response to emerging infectious diseases.

Developing a SARS-CoV-2 Antigen Test Using Engineered Affinity Proteins.

The ongoing COVID-19 pandemic has clearly established how vital rapid, widely accessible diagnostic tests are in controlling infectious diseases and how difficult and slow it is to scale existing technologies. Here, we demonstrate the use of the rapid affinity pair identification via directed selection (RAPIDS) method to discover multiple affinity pairs for SARS-CoV-2 nucleocapsid protein (N-protein), a biomarker of COVID-19, from in vitro libraries in 10 weeks. The pair with the highest biomarker sensitivity was then integrated into a 10-minute, vertical-flow cellulose paper test. Notably, the as-identified affinity proteins were compatible with a roll-to-roll printing process for large-scale manufacturing of tests. The test achieved 40 pM and 80 pM limits of detection in 1×PBS (mock swab) and saliva matrices spiked with cell-culture generated SARS-CoV-2 viruses and is also capable of detection of N-protein from characterized clinical swab samples. Hence, this work paves the way towards the mass production of cellulose paper-based assays which can address the shortages faced due to dependence on nitrocellulose and current manufacturing techniques. Further, the results reported herein indicate the promise of RAPIDS and engineered binder proteins for the timely and flexible development of clinically relevant diagnostic tests in response to emerging infectious diseases.

Vertical Flow Cellulose-Based Assays for SARS-CoV-2 Antibody Detection in Human Serum.

Rapid and inexpensive serological tests for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) antibodies are essential to conduct large-scale seroprevalence surveys and can potentially complement nucleic acid or antigen tests at the point of care. During the COVID-19 pandemic, extreme demand for traditional lateral flow tests has stressed manufacturing capacity and supply chains. Motivated by this limitation, we developed a SARS-CoV-2 antibody test using cellulose, an alternative membrane material, and a double-antigen sandwich format. Functionalized SARS-CoV-2 antigens were used as both capture and reporter binders, replacing the anti-human antibodies currently used in lateral flow tests. The test could provide enhanced sensitivity because it labels only antibodies against SARS-CoV-2 and the signal intensity is not diminished due to other human antibodies in serum. Three-dimensional channels in the assay were designed to have consistent flow rates and be easily manufactured by folding wax-printed paper. We demonstrated that this simple, vertical flow, cellulose-based assay could detect SARS-CoV-2 antibodies in clinical samples within 15 min, and the results were consistent with those from a laboratory, bead-based chemiluminescence immunoassay that was granted emergency use approval by the US FDA.

Search for Campylobacter spp. Reveals High Prevalence and Pronounced Genetic Diversity of Arcobacter butzleri in Floodwater Samples Associated with Hurricane Florence in North Carolina, USA.

In September 2018, Hurricane Florence caused extreme flooding in eastern North Carolina, USA, a region highly dense in concentrated animal production, especially swine and poultry. In this study, floodwater samples (n = 96) were collected as promptly post-hurricane as possible and for up to approximately 30 days and selectively enriched for Campylobacter using Bolton broth enrichment and isolation on modified charcoal cefoperazone deoxycholate agar (mCCDA) microaerobically at 42°C. Only one sample yielded Campylobacter, which was found to be Campylobacter jejuni with the novel sequence type 2866 (ST-2866). However, the methods employed to isolate Campylobacter readily yielded Arcobacter from 73.5% of the floodwater samples. The Arcobacter isolates failed to grow on Mueller-Hinton agar at 25, 30, 37, or 42°C microaerobically or aerobically but could be readily subcultured on mCCDA at 42°C microaerobically. Multilocus sequence typing of 112 isolates indicated that all were Arcobacter butzleri The majority (85.7%) of the isolates exhibited novel sequence types (STs), with 66 novel STs identified. Several STs, including certain novel ones, were detected in diverse waterbody types (channel, isolated ephemeral pools, floodplain) and from multiple watersheds, suggesting the potential for regionally dominant strains. The genotypes were clearly partitioned into two major clades, one with high representation of human and ruminant isolates and another with an abundance of swine and poultry isolates. Surveillance of environmental waters and food animal production systems in this animal agriculture-dense region is needed to assess potential regional prevalence and temporal stability of the observed A. butzleri strains as well as their potential association with specific types of food animal production.IMPORTANCE Climate change and associated extreme weather events can have massive impacts on the prevalence of microbial pathogens in floodwaters. However, limited data are available on foodborne zoonotic pathogens such as Campylobacter or Arcobacter in hurricane-associated floodwaters in rural regions with intensive animal production. With a high density of intensive animal production as well as pronounced vulnerability to hurricanes, eastern North Carolina presents unique opportunities in this regard. Our findings revealed widespread incidence of the emerging zoonotic pathogen Arcobacter butzleri in floodwaters from Hurricane Florence. We encountered high and largely unexplored diversity while also noting the potential for regionally abundant and persistent clones. We noted pronounced partitioning of the floodwater genotypes into two source-associated clades. The data will contribute to elucidating the poorly understood ecology of this emerging pathogen and highlight the importance of surveillance of floodwaters associated with hurricanes and other extreme weather events for Arcobacter and other zoonotic pathogens.

Complete Genome Sequencing of Four Arcobacter Species Reveals a Diverse Suite of Mobile Elements.

Arcobacter species are recovered from a wide variety of sources, including animals, food, and both fresh and marine waters. Several Arcobacter species have also been recovered from human clinical samples and are thus associated tentatively with food- and water-borne human illnesses. Genome sequencing of the poultry isolate Arcobacter cibarius H743 and the Arcobacter acticola, Arcobacter pacificus, and Arcobacter porcinus type strains identified a large number and variety of insertion sequences. This study presents an analysis of these A. acticola, A. cibarius, A. pacificus, and A. porcinus IS elements. The four genomes sequenced here contain 276 complete and degenerate IS elements, representing 13 of the current 29 prokaryotic IS element families. Expansion of the analysis to include 15 other previously sequenced Arcobacter spp. added 73 complete and degenerate IS elements. Several of these IS elements were identified in two or more Arcobacter species, suggesting movement by horizontal gene transfer between the arcobacters. These IS elements are putatively associated with intragenomic deletions and inversions, and tentative movement of antimicrobial resistance genes. The A. cibarius strain H743 megaplasmid contains multiple IS elements common to the chromosome and, unusually, a complete ribosomal RNA locus, indicating that larger scale genomic rearrangements, potentially resulting from IS element-mediated megaplasmid cointegration and resolution may be occurring within A. cibarius and possibly other arcobacters. The presence of such a large and varied suite of mobile elements could have profound effects on Arcobacter biology and evolution.

Polymerization-Based Amplification for Target-Specific Colorimetric Detection of Amplified Mycobacterium tuberculosis DNA on Cellulose.

Loop-mediated isothermal amplification (LAMP) is an appealing method for low-cost, point-of-care nucleic acid diagnostic assays due to high sensitivity, minimal equipment requirements, and compatibility with user-friendly colorimetric detection methods. The enhanced sensitivity LAMP offers comes with vulnerability to cross-contamination, where negative samples are exposed to minute amounts of nucleic acids from positive samples. These amounts are insignificant in less sensitive amplification methods, but visible when LAMP is paired with common colorimetric methods. Here, we examined the use of eosin photopolymerization, a tunable reaction, for colorimetric detection of LAMP products to reduce this false positive risk. Using eosin and biotin end-labeled primers, we successfully amplified target regions of the Mycobacterium tuberculosis (MTB) genome using PCR and LAMP, captured amplicons on streptavidin-coated cellulose, and detected DNA targets via eosin photopolymerization, producing a bright pink color only if MTB DNA was present in the sample. Consistent with previous reports, the LAMP-based method exhibited high background signal, but tuning the illumination time for the photopolymerization reaction allowed readouts from samples with no added MTB DNA to remain blank and visually distinct from pink positives. This method yielded limits of detection of 30 and 300 copies/µL for LAMP and PCR amplification, respectively. When confronted with boiled MTB culture samples, this method gave clear positive readouts, compared to negligible signal from other Mycobacterium boiled culture samples. This new method of LAMP colorimetric detection has the potential to increase the utility of LAMP as a nucleic acid assay technique by mitigating sensitivity to cross-contamination.

Complete Genome Sequences of the Campylobacter fetus subsp. venerealis, Campylobacter lari subsp. concheus, Campylobacter sputorum bv. sputorum, and Campylobacter volucris Type Strains.

Campylobacter spp. are recovered from a wide variety of sources, including birds, livestock, shellfish, and human clinical samples. We present here the complete genomic data for the type strains of Campylobacter fetus subsp. venerealis, Campylobacter lari subsp. concheus, Campylobacter sputorum bv. sputorum, and Campylobacter volucris.

Complete Genome Sequence of the Arcobacter canalis Type Strain LMG 29148.

Arcobacter canalis was originally recovered from shellfish and from a sewage-contaminated canal. Arcobacter canalis is closely related to the marine bacterium Arcobacter marinus This study describes the complete whole-genome sequence of the A. canalis type strain LMG 29148 (=F138-33T; =CECT 8984T), which was recovered from oysters.

Complete Genome Sequence of the Arcobacter mytili Type Strain LMG 24559.

Multiple Arcobacter species have been recovered from fresh and contaminated waters, marine environments, and shellfish. Arcobacter mytili was recovered in 2006 from mussels collected from the Ebro River delta in Catalonia, Spain. This study describes the complete whole-genome sequence of the A. mytili type strain LMG 24559 (=F2075T =CECT 7386T).

Complete Genome Sequence of the Arcobacter bivalviorum Type Strain LMG 26154.

Arcobacters are routinely recovered from marine environments, and multiple Arcobacter species have been isolated from shellfish. Arcobacter bivalviorum was recovered from mussels collected in the Ebro Delta in northeastern Spain. This report describes the complete whole-genome sequence of the A. bivalviorum type strain LMG 26154 (= F4T = CECT 7835T).

Complete Genome Sequence of the Arcobacter trophiarum Type Strain LMG 25534.

Arcobacter species have been recovered from food and/or food animals, and several of these species are potential human pathogens. Arcobacter trophiarum was recovered from fecal samples taken from pigs on two Belgian farms. This study describes the whole-genome sequence of the A. trophiarum type strain LMG 25534 (=64T =CCUG 59229T).

Complete Genome Sequence of the Arcobacter halophilus Type Strain CCUG 53805.

Many Arcobacter spp. are free living and are routinely recovered from marine environments. Arcobacter halophilus was isolated from hypersaline lagoon water in the Hawaiian islands, and it was demonstrated to be an obligate halophile. This study describes the complete whole-genome sequence of the A. halophilus type strain, CCUG 53805 (= LA31BT = ATCC BAA-1022T).

Complete Genome Sequence of the Arcobacter marinus Type Strain JCM 15502.

Arcobacter species are often recovered from marine environments and are isolated from both seawater and shellfish. Arcobacter marinus was recovered from the homogenate of a sample containing surface seawater, seaweed, and a starfish. This study describes the whole-genome sequence of the A. marinus type strain JCM 15502 (= CL-S1T = KCCM 90072T).

Complete Genome Sequence of the Arcobacter molluscorum Type Strain LMG 25693.

As components of freshwater and marine microflora, Arcobacter spp. are often recovered from shellfish, such as mussels, clams, and oysters. Arcobacter molluscorum was isolated from mussels from the Ebro Delta in Catalonia, Spain. This article describes the whole-genome sequence of the A. molluscorum strain LMG 25693T (= F98-3T = CECT 7696T).

Complete Genome Sequence of the Arcobacter ellisii Type Strain LMG 26155.

Arcobacter spp. are highly prevalent in contaminated environmental waters and have been recovered from both freshwater and seawater, with several species isolated from shellfish. Arcobacter ellisii was recovered from mussels collected in Catalonia, Spain. This study describes the whole-genome sequence of the A. ellisii type strain LMG 26155 (=F79-6T =CECT 7837T).

Complete Genome Sequence of the Arcobacter skirrowii Type Strain LMG 6621.

Arcobacter skirrowii is a species of veterinary importance, originally recovered from the feces, aborted fetuses, and preputial fluids of livestock. We present here the whole-genome sequence of the A. skirrowii type strain LMG 6621 (= 449/80T = CCUG 10374T), isolated in the United Kingdom from a lamb diarrheal fecal sample.

Complete Genome Sequence of the Arcobacter suis Type Strain LMG 26152.

Arcobacter species are prevalent in pigs, and strains have been isolated from pig feces and pork meat; some Arcobacter strains may be porcine abortifacients. Arcobacter suis was recovered from pork meat in Spain. This study describes the whole-genome sequence of the A. suis type strain LMG 26152 (=F41T =CECT 7833T).

Complete Genome Sequences of the Arcobacter cryaerophilus Strains ATCC 43158T and ATCC 49615.

Arcobacter cryaerophilus was originally recovered from aborted bovine and porcine fetuses, but it has been subsequently isolated from meat, water, and human clinical samples. This study describes the complete whole-genome sequences of two A. cryaerophilus strains, ATCC 43158T (=A 169/BT =LMG 24291T) and ATCC 49615 (=CDC D2610 =LMG 10829).