On the Environmental Presence of Burkholderia pseudomallei in South-Central Ghana.

Burkholderia pseudomallei is a Gram-negative soil saprophyte with the potential to cause melioidosis, an opportunistic disease with a high mortality potential. Periodic case reports of melioidosis in or imported from Africa occur in the literature dating back decades. Furthermore, statistical models suggest Western sub-Saharan Africa as a high-risk zone for the presence of B. pseudomallei. A recent case report from the United Kingdom of a returning traveler from Ghana highlights the need for environmental studies in Ghana. We examined 100 soil samples from a rice farm in south-central Ghana. Soil was subjected to selective enrichment culture for B. pseudomallei using threonine-basal salt solution with colistin (TBSS-C50) and erythritol medium, as described in the literature. Bacterial cultures were identified with standard biochemical tests, a rapid antigen detection assay, and real-time PCR specific for B. pseudomallei. Of the 100 soil samples, 55% yielded cultures consistent with B. pseudomallei on Ashdown's agar as well as by capsular polysaccharide antigen production. This is the first confirmatory report of culture-confirmed B. pseudomallei in the environment of Ghana. Our study emphasizes the need for further exploration of the burden of human melioidosis in Ghana. We recommend that local clinicians familiarize themselves with the diagnosis and clinical management of melioidosis, while laboratories develop capacity for the safe isolation and identification of B. pseudomallei. IMPORTANCE We present the first confirmation of the presence of B. pseudomallei in the environment of Ghana. This study will bring attention to a disease with the potential to cause significant morbidity and mortality in Ghana, but which has gone completely unrecognized until this point. Furthermore, this work would encourage local clinicians to familiarize themselves with the diagnosis and clinical management of melioidosis and laboratories to develop capacity for the safe isolation and identification of B. pseudomallei.

Evaluation of a Rapid Diagnostic Test for Detection of Burkholderia pseudomallei in the Lao People's Democratic Republic.

Burkholderia pseudomallei causes significant global morbidity and mortality, with the highest disease burden in parts of Asia where culture-based diagnosis is often not available. We prospectively evaluated the Active Melioidosis Detect (AMD; InBios International, USA) lateral flow immunoassay (LFI) for rapid detection of B. pseudomallei in turbid blood cultures, pus, sputum, sterile fluid, urine, and sera. The performance of this test was compared to that of B. pseudomallei detection using monoclonal antibody latex agglutination (LA) and immunofluorescence assays (IFA), with culture as the gold standard. AMD was 99% (99/100; 95% confidence interval, 94.6 to 100%) sensitive and 100% (308/308; 98.8 to 100%) specific on turbid blood culture bottles, with no difference from LA or IFA. AMD specificity was 100% on pus (122/122; 97.0 to 100%), sputum (20/20; 83.2 to 100%), and sterile fluid (44/44; 92 to 100%). Sensitivity on these samples was as follows: pus, 47.1% (8/17; 23.0 to 72.2%); sputum, 33.3% (1/3; 0.84 to 90.6%); and sterile fluid, 0% (0/2; 0 to 84.2%). For urine samples, AMD had a positive predictive value of 94% (32/34; 79.7 to 98.5%) for diagnosing melioidosis in our cohort. AMD sensitivity on stored sera, collected prospectively from melioidosis cases during this study, was 13.9% (5/36; 4.7% to 29.5%) compared to blood culture samples taken on the same day. In conclusion, AMD is an excellent tool for rapid diagnosis of melioidosis from turbid blood cultures and maintains specificity across all sample types. It is a promising tool for urinary antigen detection, which could revolutionize diagnosis of melioidosis in resource-limited settings. Further work is required to improve sensitivity on nonblood culture samples.

Thermoregulation of Biofilm Formation in Burkholderia pseudomallei Is Disrupted by Mutation of a Putative Diguanylate Cyclase.

Burkholderia pseudomallei, a tier 1 select agent and the etiological agent of melioidosis, transitions from soil and aquatic environments to infect a variety of vertebrate and invertebrate hosts. During the transition from an environmental saprophyte to a mammalian pathogen, B. pseudomallei encounters and responds to rapidly changing environmental conditions. Environmental sensing systems that control cellular levels of cyclic di-GMP promote pathogen survival in diverse environments. Cyclic di-GMP controls biofilm production, virulence factors, and motility in many bacteria. This study is an evaluation of cyclic di-GMP-associated genes that are predicted to metabolize and interact with cyclic di-GMP as identified from the annotated genome of B. pseudomallei 1026b. Mutants containing transposon disruptions in each of these genes were characterized for biofilm formation and motility at two temperatures that reflect conditions that the bacteria encounter in the environment and during the infection of a mammalian host. Mutants with transposon insertions in a known phosphodiesterase (cdpA) and a predicted hydrolase (Bp1026b_I2285) gene exhibited decreased motility regardless of temperature. In contrast, the phenotypes exhibited by mutants with transposon insertion mutations in a predicted diguanylate cyclase gene (Bp1026b_II2523) were strikingly influenced by temperature and were dependent on a conserved GG(D/E)EF motif. The transposon insertion mutant exhibited enhanced biofilm formation at 37°C but impaired biofilm formation at 30°C. These studies illustrate the importance of studying behaviors regulated by cyclic di-GMP under varied environmental conditions in order to better understand cyclic di-GMP signaling in bacterial pathogens.IMPORTANCE This report evaluates predicted cyclic di-GMP binding and metabolic proteins from Burkholderia pseudomallei 1026b, a tier 1 select agent and the etiologic agent of melioidosis. Transposon insertion mutants with disruptions in each of the genes encoding these predicted proteins were characterized in order to identify key components of the B. pseudomallei cyclic di-GMP-signaling network. A predicted hydrolase and a phosphodiesterase that modulate swimming motility were identified, in addition to a diguanylate cyclase that modulates biofilm formation and motility in response to temperature. These studies warrant further evaluation of the contribution of cyclic di-GMP to melioidosis in the context of pathogen acquisition from environmental reservoirs and subsequent colonization, dissemination, and persistence within the host.

Towards Development of Improved Serodiagnostics for Tularemia by Use of Francisella tularensis Proteome Microarrays.

Tularemia in humans is caused mainly by two subspecies of the Gram-negative facultative anaerobe Francisella tularensis: F. tularensis subsp. tularensis (type A) and F. tularensis subsp. holarctica (type B). The current serological test for tularemia is based on agglutination of whole organisms, and the reactive antigens are not well understood. Previously, we profiled the antibody responses in type A and B tularemia cases in the United States using a proteome microarray of 1,741 different proteins derived from the type A strain Schu S4. Fifteen dominant antigens able to detect antibodies to both types of infection were identified, although these were not validated in a different immunoassay format. Since type A and B subspecies are closely related, we hypothesized that Schu S4 antigens would also have utility for diagnosing type B tularemia caused by strains from other geographic locations. To test this, we probed the Schu S4 array with sera from 241 type B tularemia cases in Spain. Despite there being no type A strains in Spain, we confirmed the responses against some of the same potential serodiagnostic antigens reported previously, as well as determined the responses against additional potential serodiagnostic antigens. Five potential serodiagnostic antigens were evaluated on immunostrips, and two of these (FTT1696/GroEL and FTT0975/conserved hypothetical protein) discriminated between the Spanish tularemia cases and healthy controls. We conclude that antigens from the type A strain Schu S4 are suitable for detection of antibodies from patients with type B F. tularensis infections and that these can be used for the diagnosis of tularemia in a deployable format, such as the immunostrip.

IgG subclass and heavy chain domains contribute to binding and protection by mAbs to the poly γ-D-glutamic acid capsular antigen of Bacillus anthracis.

Bacterial capsules are common targets for antibody-mediated immunity. The capsule of Bacillus anthracis is unusual among capsules because it is composed of a polymer of poly-γ-d-glutamic acid (γdPGA). We previously generated murine IgG3 monoclonal antibodies (mAbs) to γdPGA that were protective in a murine model of pulmonary anthrax. IgG3 antibodies are characteristic of the murine response to polysaccharide antigens. The goal of the present study was to produce subclass switch variants of the γdPGA mAbs (IgG3 → IgG1 → IgG2b → IgG2a) and assess the contribution of subclass to antibody affinity and protection. Subclass switch antibodies had identical variable regions but differed in their heavy chains. The results showed that a switch from the protective IgG3 to IgG1, IgG2b or IgG2a was accompanied by i) a loss of protective activity ii) a change in mAb binding to the capsular matrix, and iii) a loss of affinity. These results identify a role for the heavy chain constant region in mAb binding. Hybrid mAbs were constructed in which the CH1, CH2 or CH3 heavy chain constant domains from a non-protective, low binding IgG2b mAb were swapped into the protective IgG3 mAb. The IgG3 mAb that contained the CH1 domain from IgG2b showed no loss of affinity or protection. In contrast, swapping the CH2 or CH3 domains from IgG2b into IgG3 produced a reduction in affinity and a loss of protection. These studies identify a role for the constant region of IgG heavy chains in affinity and protection against an encapsulated bacterial pathogen.

Fibronectin-binding protein of Borrelia hermsii expressed in the blood of mice with relapsing fever.

To identify and characterize surface proteins expressed by the relapsing fever (RF) agent Borrelia hermsii in the blood of infected mice, we used a cell-free filtrate of their blood to immunize congenic naive mice. The resultant antiserum was used for Western blotting of cell lysates, and gel slices corresponding to reactive bands were subjected to liquid chromatography-tandem mass spectrometry, followed by a search of the proteome database with the peptides. One of the immunogens was identified as the BHA007 protein, which is encoded by a 174-kb linear plasmid. BHA007 had sequence features of lipoproteins, was surface exposed by the criteria of in situ protease susceptibility and agglutination of Vtp(-) cells by anti-BHA007 antibodies, and was not essential for in vitro growth. BHA007 elicited antibodies during experimental infection of mice, but immunization with recombinant protein did not confer protection against needle-delivered infection. Open reading frames (ORFs) orthologous to BHA007 were found on large plasmids of other RF species, including the coding sequences for the CihC proteins of Borrelia duttonii and B. recurrentis, but not in Lyme disease Borrelia species. Recombinant BHA007 bound both human and bovine fibronectin with Kd (dissociation constant) values of 22 and 33 nM, respectively, and bound to C4-binding protein with less affinity. The distant homology of BHA007 and its orthologs to BBK32 proteins of Lyme disease species, as well as to previously described BBK32-like proteins in relapsing fever species, indicates that BHA007 is a member of a large family of multifunctional proteins in Borrelia species that bind to fibronectin as well as other host proteins.

Electrochemical Immunoassay for Capturing Capsular Polysaccharide of Burkholderia pseudomallei: Early Onsite Detection of Melioidosis.

This study presented the detection and quantification of capsular polysaccharide (CPS) as a biomarker for the diagnosis of melioidosis. After successfully screening four monoclonal antibodies (mAbs) previously determined to bind CPS molecules, the team developed a portable electrochemical immunosensor based on antibody-antigen interactions. The biosensor was able to detect CPS with a wide detection range from 0.1pg/mL to 1 µg/mL. The developed biosensor achieved high sensitivity for the detection of CPS spiked into both urine and serum. The developed assay platform was successfully programmed into a Windows app, and the sensor performance was evaluated with different spiked concentrations. The rapid electro-analytical device (READ) sensor showed great unprecedented sensitivity for the detection of CPS molecules in both serum and urine, and results were cross-validated with ELISA methods.

Unraveling the interaction between the LPS O-antigen of Burkholderia anthina and the 5D8 monoclonal antibody by using a multidisciplinary chemical approach, with synthesis, NMR, and molecular modeling methods.

The interaction between the O-chain from the lipopolysaccharide from Burkholderia anthina and a lipopolysaccharide-specific monoclonal antibody (5D8) has been studied at high resolution by NMR spectroscopy. In particular, the 5D8-bound epitope of the saccharide entity has been unraveled by a combination of saturation transfer difference (STD) and transferred NOESY (tr-NOESY) experiments performed on the 5D8/polysaccharide complex. To dissect the fine details of the molecular recognition events, further experiments with simpler carbohydrate ligands were carried out. Thus, experiments were also performed with ad hoc synthesized trisaccharide and hexasaccharide O-antigen repeating units. By using this multidisciplinary approach (chemical synthesis, NMR spectroscopy and molecular dynamics simulation), determination of the binding epitope and the contribution to the binding of the sugar units composing the O-chain have been determined.

Serum antibody levels to SARS-CoV-2 receptor-binding domain (RBD) in convalescent patients and vaccinated individuals of northern Nevada.

Antibodies reactive with the SARS-CoV-2 receptor-binding domain (RBD) of the spike protein are associated with viral neutralization, however low antibody titers, specifically against SARS-CoV-2 variants, may result in reduced viral immunity post naturally acquired infection. A cohort study comprised of 121 convalescent individuals from northern Nevada was conducted looking at anti-RBD antibody levels by enzyme-linked immunosorbent assay. Serum was collected from volunteers by staff at the University of Nevada, Reno School of Medicine Clinical Research Center and assessed for antibodies reactive to various SARS-CoV-2 RBD domains relevant to the time of the study (2020-2021). A nonpaired group of vaccinated individuals were assessed in parallel. The goal of the study was to identify antibody levels against the RBD subunit in convalescent and vaccinated individuals from northern Nevada.

Digital image analysis for biothreat detection via rapid centrifugal microfluidic orthogonal flow immunocapture.

We report clear proof-of-principle for centrifugally-driven, multiplexed, paper-based orthogonal flow sandwich-style immunocapture (cOFI) and colorimetric detection of Zaire Ebola virus-like particles. Capture antibodies are immobilized onto nanoporous nitrocellulose membranes that are then laminated into polymeric microfluidic discs to yield ready-to-use analytical devices. Fluid flow is controlled solely by rotational speed, obviating the need for complex pneumatic pumping systems, and providing more precise flow control than with the capillary-driven flow used in traditional lateral flow immunoassays (LFIs). Samples containing the antigen of interest and gold nanoparticle-labeled detection antibodies are pumped centrifugally through the embedded, prefunctionalized membrane where they are subsequently captured to generate a positive, colorimetric signal. When compared to the equivalent LFI counterparts, this cOFI approach generated immunochromatographic colorimetric responses that are objectively darker (saturation), more intense (grayscale), and less variable regarding total area of the color response. We also describe an image analysis approach that enables access to rich color data and area statistics without the need for a commercial 'strip reader' or custom-written image analysis algorithms. Instead, our analytical method exploits inexpensive equipment (e.g., smart phone, flatbed scanner, etc.) and freely available software (Fiji distribution of ImageJ) to permit characterization of immunochromatographic responses that includes multiple color metrics, offering insights beyond typical grayscale analysis. The findings reported here stand as clear proof-of-principle for the feasibility of disc-based, centrifugally driven orthogonal flow through a membrane with immunocapture (cOFI) and colorimetric readout of a sandwich-type immunoassay in less than 15 minutes. Once fully developed, this cOFI platform could render a faster, more accurate diagnosis, while processing multiple samples simul-taneously.

Functional assays to screen and select monoclonal antibodies that target Yersinia pestis.

Yersinia pestis is a gram-negative bacterium that causes plague in animals and humans. Depending on the route of disease transmission, the bacterium can cause an acute, often fatal disease that has a narrow window for treatment with antibiotics. Additionally, antibiotic resistant strains have been identified, emphasizing the need for novel treatments. Antibody therapy is an appealing option that can direct the immune system to clear bacterial infections. Advances in biotechnology have made both engineering and producing antibodies easier and more affordable. In this study, two screening assays were optimized to evaluate the ability of antibodies to promote phagocytosis of Y. pestis by macrophages and to induce a cytokine signature in vitro that may be predictive of protection in vivo. We evaluated a panel of 21 mouse monoclonal antibodies targeting either the anti-phagocytic capsule F1 protein or the LcrV antigen, which is part of the type 3 secretion system that facilitates translocation of virulence factors into the host cell, using two functional assays. Anti-F1 and anti-LcrV monoclonal antibodies both increased bacterial uptake by macrophages, with greater uptake observed in the presence of antibodies that were protective in the mouse pneumonic plague model. In addition, the protective anti-F1 and anti-LcrV antibodies produced unique cytokine signatures that were also associated with in vivo protection. These antibody-dependent characteristics from in vitro functional assays will be useful in down-selecting efficacious novel antibodies that can be used for treatment of plague.

Characterization of a Centrifugal Microfluidic Orthogonal Flow Platform.

To bring to bear the power of centrifugal microfluidics on vertical flow immunoassays, control of flow orthogonally through nanoporous membranes is essential. The on-disc approach described here leverages the rapid print-cut-laminate (PCL) disc fabrication and prototyping method to create a permanent seal between disc materials and embedded nanoporous membranes. Rotational forces drive fluid flow, replacing capillary action, and complex pneumatic pumping systems. Adjacent microfluidic features form a flow path that directs fluid orthogonally (vertically) through these embedded membranes during assay execution. This method for membrane incorporation circumvents the need for solvents (e.g., acetone) to create the membrane-disc bond and sidesteps issues related to undesirable bypass flow. In other recently published work, we described an orthogonal flow (OF) platform that exploited embedded membranes for automation of enzyme-linked immunosorbent assays (ELISAs). Here, we more fully characterize flow patterns and cellulosic membrane behavior within the centrifugal orthogonal flow (cOF) format. Specifically, high-speed videography studies demonstrate that sample volume, membrane pore size, and ionic composition of the sample matrix significantly impact membrane behavior, and consequently fluid drainage profiles, especially when cellulosic membranes are used. Finally, prototype discs are used to demonstrate proof-of-principle for sandwich-type antigen capture and immunodetection within the cOF system.

Detection and Quantification of the Capsular Polysaccharide of Burkholderia pseudomallei in Serum and Urine Samples from Melioidosis Patients.

Burkholderia pseudomallei is the causative agent of melioidosis, a life-threatening disease common in Southeast Asia and northern Australia. Melioidosis often presents with nonspecific symptoms and has a fatality rate of upwards of 70% when left untreated. The gold standard for diagnosis is culturing B. pseudomallei from patient samples. Bacterial culture, however, can take up to 7 days, and its sensitivity is poor, at roughly 60%. The successful administration of appropriate antibiotics is reliant on rapid and accurate diagnosis. Hence, there is a genuine need for new diagnostics for this deadly pathogen. The Active Melioidosis Detect (AMD) lateral flow immunoassay (LFI) detects the capsular polysaccharide (CPS) of B. pseudomallei. The assay is designed for use on various clinical samples, including serum and urine; however, there are limited data to support which clinical matrices are the best candidates for detecting CPS. In this study, concentrations of CPS in paired serum and urine samples from melioidosis patients were determined using a quantitative antigen capture enzyme-linked immunosorbent assay. In parallel, samples were tested with the AMD LFI, and the results of the two immunoassays were compared. Additionally, centrifugal concentration was performed on a subset of urine samples to determine if this method may improve detection when CPS levels are initially low or undetectable. The results indicate that while CPS levels varied within the two matrices, there tended to be higher concentrations in urine. The AMD LFI detected CPS in 40.5% of urine samples, compared to 6.5% of serum samples, suggesting that urine is a preferable matrix for point-of-care diagnostic assays. IMPORTANCE Melioidosis is very challenging to diagnose. There is a clear need for a point-of-care assay for the detection of B. pseudomallei antigen directly from patient samples. The Active Melioidosis Detect lateral flow immunoassay detects the capsular polysaccharide (CPS) of B. pseudomallei and is designed for use on various clinical samples, including serum and urine. However, there are limited data regarding which clinical matrix is preferable for the detection of CPS. This study addresses this question by examining quantitative CPS levels in paired serum and urine samples and relating them to clinical parameters. Additionally, centrifugal concentration was performed on a subset of urine samples to determine whether this might enable the detection of CPS in samples in which it was initially present at low or undetectable levels. These results provide valuable insights into the detection of CPS in patients with melioidosis and suggest potential ways forward in the diagnosis and treatment of this challenging disease.

Accuracy of 2 Rapid Antigen Tests During 3 Phases of SARS-CoV-2 Variants.

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.

Optimization of an Antibody Microarray Printing Process Using a Designed Experiment.

Antibody microarrays have proven useful in immunoassay-based point-of-care diagnostics for infectious diseases. Noncontact piezoelectric inkjet printing has advantages to print antibody microarrays on nitrocellulose substrates for this application due to its compatibility with sensitive solutions and substrates, simple droplet control, and potential for high-capacity printing. However, there remain real-world challenges in printing such microarrays, which motivated this study. The effects of three concentrations of capture antibody (cAb) reagents and nozzle hydrostatic pressures were chosen to investigate three responses: the number of printed membrane disks, dispensing performance, and microarray quality. Printing conditions were found to be most ideal with 5 mg/mL cAb and a nozzle hydrostatic pressure near zero, which produced 130 membrane disks in a single print versus the 10 membrane disks per print before optimization. These results serve to inform efficient printing of antibody microarrays on nitrocellulose membranes for rapid immunoassay-based detection of infectious diseases and beyond.

Comparison of a Prototype SARS-CoV-2 Lateral Flow IMMUNOASSAY with the BinaxNOWTM COVID-19 Antigen CARD.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus responsible for the COVID-19 pandemic. From the onset of the pandemic, rapid antigen tests have quickly proved themselves to be an accurate and accessible diagnostic platform. The initial (and still most commonly used antigen tests) for COVID-19 diagnosis were constructed using monoclonal antibodies (mAbs) specific to severe acute respiratory syndrome coronavirus (SARS-CoV) nucleocapsid protein (NP). These mAbs are able to bind SARS-CoV-2 NP due to high homology between the two viruses. However, since first being identified in 2019, SARS-CoV-2 has continuously mutated, and a multitude of variants have appeared. These mutations have an elevated risk of leading to possible diagnostic escape when using tests produced with SARS-CoV-derived mAbs. Here, we established a library of 18 mAbs specific to SARS-CoV-2 NP and used two of these mAbs (1CV7 and 1CV14) to generate a prototype antigen-detection lateral flow immunoassay (LFI). A side-by-side analysis of the 1CV7/1CV14 LFI and the commercially available BinaxNOWTM COVID-19 Antigen CARD was performed. Results indicated the 1CV7/1CV14 LFI outperformed the BinaxNOWTM test in the detection of BA.2, BA.2.12.1, and BA.5 Omicron sub-variants when testing remnant RT-PCR positive patient nasopharyngeal swabs diluted in viral transport media.

Development of a dual antigen lateral flow immunoassay for detecting Yersinia pestis.

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.

Evaluation of antigen-detecting and antibody-detecting diagnostic test combinations for diagnosing melioidosis.

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.

Genomic surveillance of Nevada patients revealed prevalence of unique SARS-CoV-2 variants bearing mutations in the RdRp gene.

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.

Development of Immunoassays for Detection of Francisella tularensis Lipopolysaccharide in Tularemia Patient Samples.

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.