Background: Melioidosis, an infectious disease caused by Burkholderia pseudomallei, is endemic in many tropical developing countries and has a high mortality. Here we evaluated combinations of a lateral flow immunoassay (LFI) detecting B. pseudomallei capsular polysaccharide (CPS) and enzyme-linked immunosorbent assays (ELISA) detecting antibodies against hemolysin co-regulated protein (Hcp1) or O-polysaccharide (OPS) for diagnosing melioidosis. Methodology/Principal findings: We conducted a cohort-based case-control study. Both cases and controls were derived from a prospective observational study of patients presenting with community-acquired infections and sepsis in northeast Thailand (Ubon-sepsis). Cases included 192 patients with a clinical specimen culture positive for B. pseudomallei. Controls included 502 patients who were blood culture positive for Staphylococcus aureus, Escherichia coli or Klebsiella pneumoniae or were polymerase chain reaction assay positive for malaria or dengue. Serum samples collected within 24 hours of admission were stored and tested using a CPS-LFI, Hcp1-ELISA and OPS-ELISA. When assessing diagnostic tests in combination, results were considered positive if either test was positive. We selected ELISA cut-offs corresponding to a specificity of 95%. Using a positive cut-off OD of 2.912 for Hcp1-ELISA, the combination of the CPS-LFI and Hcp1-ELISA had a sensitivity of 67.7% (130/192 case patients) and a specificity of 95.0% (477/502 control patients). The sensitivity of the combination (67.7%) was higher than that of the CPS-LFI alone (31.3%, p<0.001) and that of Hcp1-ELISA alone (53.6%, p<0.001). A similar phenomenon was also observed for the combination of CPS-LFI and OPS-ELISA. In case patients, positivity of the CPS-LFI was associated with a short duration of symptoms, high modified Sequential (sepsis-related) Organ Failure Assessment (SOFA) score, bacteraemia and mortality outcome, while positivity of Hcp1-ELISA was associated with a longer duration of symptoms, low modified SOFA score, non-bacteraemia and survival outcome. Conclusions/Significance: A combination of antigen-antibody diagnostic tests increased the sensitivity of melioidosis diagnosis over individual tests while preserving high specificity. Point-of-care tests for melioidosis based on the use of combination assays should be further developed and evaluated.

Patients with signs of COVID-19 were tested through diagnostic RT-PCR for SARS-CoV-2 using RNA extracted from the nasopharyngeal/nasal swabs. To determine the variants of SARS-CoV-2 circulating in the state of Nevada, specimens from 200 COVID-19 patients were sequenced through our robust sequencing platform, which enabled sequencing of SARS-CoV-2 from specimens with even very low viral loads, without the need of culture-based amplification. High genome coverage allowed the identification of single and multi-nucleotide variants in SARS-CoV-2 in the community and their phylogenetic relationships with other variants present during the same period of the outbreak. We report the occurrence of a novel mutation at 323aa (314aa of orf1b) of nsp12 (RNA dependent RNA Polymerase) changed to Phenylalanine (F) from Proline (P), in the first reported isolate of SARS-CoV-2, Wuhan-Hu-1. This 323F variant was present at a very high frequency in Northern Nevada. Structural modeling determined this mutation in the interface domain, which is important for the association of accessory proteins required for the polymerase. In conclusion, we report the introduction of specific SARS-CoV-2 variants at very high frequency in distinct geographic locations, which is important for understanding the evolution and circulation of SARS-CoV-2 variants of public health importance, while it circulates in humans.

Francisella tularensis is the causative agent of tularemia, a zoonotic bacterial infection that is often fatal if not diagnosed and treated promptly. Natural infection in humans is relatively rare, yet persistence in animal reservoirs, arthropod vectors, and water sources combined with a low level of clinical recognition make tularemia a serious potential threat to public health in endemic areas. F. tularensis has also garnered attention as a potential bioterror threat, as widespread dissemination could have devastating consequences on a population. A low infectious dose combined with a wide range of symptoms and a short incubation period makes timely diagnosis of tularemia difficult. Current diagnostic techniques include bacterial culture of patient samples, PCR and serological assays; however, these techniques are time consuming and require technical expertise that may not be available at the point of care. In the event of an outbreak or exposure a more efficient diagnostic platform is needed. The lipopolysaccharide (LPS) component of the bacterial outer leaflet has been identified previously by our group as a potential diagnostic target. For this study, a library of ten monoclonal antibodies specific to F. tularensis LPS were produced and confirmed to be reactive with LPS from type A and type B strains. Antibody pairs were tested in an antigen-capture enzyme-linked immunosorbent assay (ELISA) and lateral flow immunoassay format to select the most sensitive pairings. The antigen-capture ELISA was then used to detect and quantify LPS in serum samples from tularemia patients for the first time to determine the viability of this molecule as a diagnostic target. In parallel, prototype lateral flow immunoassays were developed, and reactivity was assessed, demonstrating the potential utility of this assay as a rapid point-of-care test for diagnosis of tularemia.

Background: Yersinia pestis is the causative agent of plague, a zoonosis associated with small mammals. Plague is a severe disease, especially in the pneumonic and septicemic forms, where fatality rates approach 100% if left untreated. The bacterium is primarily transmitted via flea bite or through direct contact with an infected host. The 2017 plague outbreak in Madagascar resulted in more than 2,400 cases and was highlighted by an increased number of pneumonic infections. Standard diagnostics for plague include laboratory-based assays such as bacterial culture and serology, which are inadequate for administering immediate patient care for pneumonic and septicemic plague. Principal findings: The goal of this study was to develop a sensitive rapid plague prototype that can detect all virulent strains of Y. pestis. Monoclonal antibodies (mAbs) were produced against two Y. pestis antigens, low-calcium response V (LcrV) and capsular fraction-1 (F1), and prototype lateral flow immunoassays (LFI) and enzyme-linked immunosorbent assays (ELISA) were constructed. The LFIs developed for the detection of LcrV and F1 had limits of detection (LOD) of roughly 1–2 ng/mL in surrogate clinical samples (antigens spiked into normal human sera). The optimized antigen-capture ELISAs produced LODs of 74 pg/mL for LcrV and 61 pg/mL for F1 when these antigens were spiked into buffer. A dual antigen LFI prototype comprised of two test lines was evaluated for the detection of both antigens in Y. pestis lysates. The dual format was also evaluated for specificity using a small panel of clinical near-neighbors and other Tier 1 bacterial Select Agents. Conclusions: LcrV is expressed by all virulent Y. pestis strains, but homologs produced by other Yersinia species can confound assay specificity. F1 is specific to Y. pestis but is not expressed by all virulent strains. Utilizing highly reactive mAbs, a dual-antigen detection (multiplexed) LFI was developed to capitalize on the diagnostic strengths of each target.

Importance: Variants of SARS-CoV-2 have sequence variations in the viral genome that may alter the accuracy of rapid diagnostic tests.Objective: To assess the analytical and clinical accuracy of 2 rapid diagnostic tests for detecting SARS-CoV-2 during 3 phases of variants.Design, Setting, and Participants: This diagnostic study included participants aged 18 years or older who reported onset of COVID-19–like symptoms within the prior 5 days and were tested at multiple COVID-19 testing locations in King County, Washington, from February 17, 2021, to January 11, 2022, during 3 distinct phases of SARS-CoV-2 infection (pre-Delta, Delta, and Omicron).Interventions: Two anterior nasal swab specimens were collected from each participant—1 for onsite testing by the SCoV-2 Ag Detect Rapid Self-Test and 1 for reverse transcriptase–polymerase chain reaction (RT-PCR) testing.Main Outcomes and Measures: The analytical limit of detection of the 2 rapid diagnostic tests (SCoV-2 Ag Detect Rapid Self-Test and BinaxNOW COVID-19 Ag Card) was assessed using Omicron (B.1.1.529/BA.1), Delta (B.1.617.2), and a wild-type (USA-WA1/2020) variant. Diagnostic sensitivity and specificity of clinical testing for the rapid antigen tests were compared with that of RT-PCR testing.Results: A total of 802 participants were enrolled (mean [SD] age, 37.3 [13.3] years; 467 [58.2%] female), 424 (52.9%) of whom had not received COVID-19 vaccination and presented a median of 2 days (IQR, 1-3 days) from symptom onset. Overall, no significant differences were found in the analytical limit of detection or clinical diagnostic accuracy of rapid antigen testing across SARS-CoV-2 variants. The estimated limit of detection for both rapid nucleocapsid antigen tests was at or below a 50% tissue culture infectious dose of 62.5, and the positive percent agreement of the SCoV-2 Ag Detect Rapid Self-Test ranged from 81.2% (95% CI, 69.5%-89.9%) to 90.7% (95% CI, 77.9%-97.4%) across the 3 phases of variants. The diagnostic sensitivity increased for nasal swabs with a lower cycle threshold by RT-PCR, which correlates with a higher viral load.Conclusions and Relevance: In this diagnostic study, analytical and clinical performance data demonstrated accuracy of 2 rapid antigen tests among adults with COVID-19 symptoms across 3 phases of SARS-CoV-2 variants. The findings suggest that home-based rapid antigen testing programs may be an important intervention to reduce global SARS-CoV-2 transmission.

The 2013–2016 Ebola virus (EBOV) outbreak in West Africa and the ongoing cases in the Democratic Republic of the Congo have spurred development of a number of medical countermeasures, including rapid Ebola diagnostic tests. The likelihood of transmission increases as the disease progresses due to increasing viral load and potential for contact with others. Early diagnosis of EBOV is essential for halting spread of the disease. Polymerase chain reaction assays are the gold standard for diagnosing Ebola virus disease (EVD), however, they rely on infrastructure and trained personnel that are not available in most resource-limited settings. Rapid diagnostic tests that are capable of detecting virus with reliable sensitivity need to be made available for use in austere environments where laboratory testing is not feasible. The goal of this study was to produce candidate lateral flow immunoassay (LFI) prototypes specific to the EBOV glycoprotein and viral matrix protein, both targets known to be present during EVD. The LFI platform utilizes antibody-based technology to capture and detect targets and is well suited to the needs of EVD diagnosis as it can be performed at the point-of-care, requires no cold chain, provides results in less than twenty minutes and is low cost. Monoclonal antibodies were isolated, characterized and evaluated in the LFI platform. Top performing LFI prototypes were selected, further optimized and confirmed for sensitivity with cultured live EBOV and clinical samples from infected non-human primates. Comparison with a commercially available EBOV rapid diagnostic test that received emergency use approval demonstrates that the glycoprotein-specific LFI developed as a part of this study has improved sensitivity. The outcome of this work presents a diagnostic prototype with the potential to enable earlier diagnosis of EVD in clinical settings and provide healthcare workers with a vital tool for reducing the spread of disease during an outbreak. Ebola virus (EBOV) causes a severe hemorrhagic fever and has an extremely high fatality rate that ranges from 60%-90%. There is no approved treatment or vaccine for this infectious disease and halting spread of the virus relies on identifying and isolating infected patients quickly. The current gold standard, polymerase chain reaction assay, requires patient samples be transported to regional reference laboratories where it often takes days to get results. A handful of Ebola rapid diagnostic tests have been developed, but lack the sensitivity required to detect the virus in earlier stages of the disease. There is great need for more sensitive rapid diagnostic tests that can identify the EBOV infected patients when they first become symptomatic. This study focused on production of high affinity mAbs to two target EBOV proteins for development of a more sensitivity rapid diagnostic test. Efforts have resulted in production of prototype detecting the EBOV glycoprotein that shows a notable improvement in sensitivity and offers the potential for earlier diagnosis of infection.