Pharmacological Profiling of Antifentanyl Monoclonal Antibodies in Combination with Naloxone in Pre- and Postexposure Models of Fentanyl Toxicity.

The incidence of fatal drug overdoses in the United States is an alarming public health threat that has been exacerbated by the COVID-19 pandemic, resulting in over 100,000 deaths between April 2020 and April 2021. A significant portion of this is attributable to widespread access to fentanyl and other synthetic opioids, alone or in combination with heroin or psychostimulants, such as cocaine or methamphetamine. Monoclonal antibodies (mAb) offer prophylactic and therapeutic interventions against opioid overdose by binding opioids in serum, reducing distribution of drug to the brain and other organs. Here, we investigated the efficacy of a leading antifentanyl mAb, clone HY6-F9, in reversal and prevention of fentanyl-induced toxicity compared with the opioid receptor antagonist naloxone (NLX) in rats. In postexposure models, rats were challenged with fentanyl, followed by HY6-F9, NLX, or both. HY6-F9 reversed fentanyl-induced antinociception, respiratory depression, and bradycardia, and rats retained protection against additional challenges for at least 1 week. Although intravenous NLX reversed fentanyl-induced respiratory depression more rapidly than mAb alone, kinetics of reversal by intravenous mAb were similar to subcutaneous NLX. Coadministration of mAb and NLX provided greater protection than individual treatments against high doses of fentanyl. Prophylactic administration of mAb reduced the ED50 of NLX approximately twofold against 2.25 mg/kg of fentanyl. Finally, mAb sequestered fentanyl and its metabolite norfentanyl in serum and reduced brain concentrations of fentanyl. These results support the translation of mAb as medical interventions alone or in combination with NLX to prevent and reverse fentanyl-related overdose. SIGNIFICANCE STATEMENT: Fentanyl-related overdoses have increased dramatically in the US and worldwide. Currently, approved pharmacotherapies for treatment of opioid use disorder and reversal of overdose are not sufficient to curb the incidence of opioid-related deaths. Additionally, fentanyl and its potent analogs present a potential risk from use in deliberate poisoning or chemical attacks. This study demonstrates the use of monoclonal antibodies as a countermeasure to fentanyl-induced toxicity in pre- and postexposure scenarios, supporting their use in combination with the opioid antagonist naloxone.

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.

Critical Comparison between Large and Mini Vertical Flow Immunoassay Platforms for Yersinia Pestis Detection.

Yersinia pestis is a Gram-negative bacterium that is the causative agent of plague and is widely recognized as a potential biological weapon. Due to the high fatality rate of plague when diagnosis is delayed, the development of rapid, sensitive, specific, and cost-effective methods is needed for its diagnosis. The Y. pestis low calcium response V (LcrV) protein has been identified as a potential microbial biomarker for the diagnosis of plague. In this paper, we present a highly sensitive, paper-based, vertical flow immunoassay (VFI) prototype for the detection of LcrV and the diagnosis of plague. An antigen-capture assay using monoclonal antibodies is employed to capture and detect the LcrV protein, using a colorimetric approach. In addition, the effect of miniaturizing the VFI device is explored based on two different sizes of VFI platforms, denoted as "large VFI" and "mini VFI." Also, a comparative analysis is performed between the VFI platform and a lateral flow immunoassay (LFI) platform to exhibit the improved assay sensitivity suitable for point-of-care (POC) diagnostics. The analytical sensitivity or limit of detection (LOD) in the mini VFI is approximately 0.025 ng/mL, that is, 10 times better than that of the large VFI platform or 80 times over a standard lateral flow configuration. The low LOD of the LcrV VFI appears to be highly suitable for testing clinical samples and potentially diagnosing plague at earlier time points. In addition, optimization of the gold nanoparticle (AuNP) concentration, nanomaterial plasmonic properties, and flow velocity analysis could improve the performance of the VFI. Furthermore, we developed automated image analysis software that shows potential for integrating the diagnostic system into a smartphone. These methods and findings demonstrate that the VFI platform is a highly sensitive device for detecting the LcrV and potentially many other biomarkers.

Monoclonal Antibodies Counteract Opioid-Induced Behavioral and Toxic Effects in Mice and Rats.

Monoclonal antibodies (mAbs) and vaccines have been proposed as medical countermeasures to treat opioid use disorder (OUD) and prevent opioid overdose. In contrast to current pharmacotherapies (e.g., methadone, buprenorphine, naltrexone, and naloxone) for OUD and overdose, which target brain opioid receptors, mAbs and vaccine-generated polyclonal antibodies sequester the target opioid in the serum and reduce drug distribution to the brain. Furthermore, mAbs offer several potential clinical benefits over approved medications, such as longer serum half-life, higher selectivity, reduced side effects, and no abuse liability. Using magnetic enrichment to isolate opioid-specific B cell lymphocytes prior to fusion with myeloma partners, this study identified a series of murine hybridoma cell lines expressing mAbs with high affinity for opioids of clinical interest, including oxycodone, heroin and its active metabolites, and fentanyl. In mice, passive immunization with lead mAbs against oxycodone, heroin, and fentanyl reduced drug-induced antinociception and the distribution of the target opioid to the brain. In mice and rats, mAb pretreatment reduced fentanyl-induced respiratory depression and bradycardia, two risk factors for opioid-related overdose fatality. Overall, these results support use of mAbs to counteract toxic effects of opioids and other chemical threats. SIGNIFICANCE STATEMENT: The incidence of fatal overdoses due to the widespread access to heroin, prescription opioids, and fentanyl suggests that current Food and Drug Administration-approved countermeasures are not sufficient to mitigate the opioid epidemic. Monoclonal antibodies (mAbs) may provide acute protection from overdose by binding to circulating opioids in serum. Use of mAbs prophylactically, or after exposure in combination with naloxone, may reduce hospitalization and increase survival.

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.

Melioidosis diagnostic workshop, 2013.

Melioidosis is a severe disease that can be difficult to diagnose because of its diverse clinical manifestations and a lack of adequate diagnostic capabilities for suspected cases. There is broad interest in improving detection and diagnosis of this disease not only in melioidosis-endemic regions but also outside these regions because melioidosis may be underreported and poses a potential bioterrorism challenge for public health authorities. Therefore, a workshop of academic, government, and private sector personnel from around the world was convened to discuss the current state of melioidosis diagnostics, diagnostic needs, and future directions.

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.

Alkaline surface treatment and time-resolved reading of mn-doped nanocrystal signal transducer for enhanced bioassay sensitivity.

Recently, Mn-doped semiconductor nanocrystals (NCs) with high brightness, long lifetimes, and low-energy excitation are emerging for time-resolved luminescence biosensing/imaging. Following our previous work on Mn-doped NCs, in this work we developed poly(styrene-co-maleic anhydride) (PSMA)-encapsulated Mn-doped AgZnInS/ZnS NCs as signal transducers for immunoassay of capsular polysaccharide (CPS), a surface antigen and also a biomarker of Burkholderia pseudomallei which causes a fatal disease called melioidosis. To enhance the assay sensitivity, a surface treatment for PSMA-encapsulated NCs (NC-probes) was performed to promote the presence of carboxyl groups that help conjugate more anti-CPS antibodies to the surface of NC-probes and thus enhance bioassay signals. Meanwhile, time-resolved reading on the luminescence of NC-probes was adopted to minimize the assay background autofluorescence. Both strategies essentially enhance the assay signal-to-background ratio (or equivalently the assay sensitivity) by increasing the signal and decreasing the background, respectively. Through performing and comparing immunoassays with different NC-probes (with and without surface treatment) and different signal reading methods (time-resolved reading and non-time-resolved reading), it was proven that the immunoassay adopting surface-treated NC-probes and time-resolved reading achieved a lower limit-of-detection (LOD) than the ones adopting non-surface-treated NC-probes or non-time-resolved reading. Moreover, the achieved LOD is comparable to the LOD of immunoassay using enzyme horseradish peroxidase as a signal transducer.

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.

Point-of-care vertical flow immunoassay system for ultra-sensitive multiplex biothreat-agent detection in biological fluids.

This paper presents simple, fast, and sensitive detection of multiple biothreat agents by paper-based vertical flow colorimetric sandwich immunoassay for detection of Yersinia pestis (LcrV and F1) and Francisella tularensis (lipopolysaccharide; LPS) antigens using a vertical flow immunoassay (VFI) prototype with portable syringe pump and a new membrane holder. The capture antibody (cAb) printing onto nitrocellulose membrane and gold-labelled detection antibody (dAb) were optimized to enhance the assay sensitivity and specificity. Even though the paper pore size was relaxed from previous 0.1 µm to the current 0.45 µm for serum samples, detection limits as low as 0.050 ng/mL for LcrV and F1, and 0.100 ng/mL for FtLPS have been achieved in buffer and similarly in diluted serum (with LcrV and F1 LODs remained the same and LPS LOD reduced to 0.250 ng/mL). These were 40, 80, and 50X (20X for LPS in serum) better than those from lateral flow configuration. Furthermore, the comparison of multiplex format demonstrated low cross-reactivity and equal sensitivity to that of the singleplex assay. The optimized VFI platform thus provides a portable and rapid on-site monitoring system for multiplex biothreat detection with the potential for high sensitivity, specificity, reproducibility, and multiplexing capability, supporting its utility in remote and resource-limited settings.

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.

A Compact and Sensitive Time-Resolved-Optical-Reader for Bioassay Using Low-Energy Excitable and Long-Lived-Fluorescence Nanolabels.

Time-resolved fluorescence measurement, a technique utilized to improve sensitivity for fluorescence detection, is becoming more accessible due to the recent development of bright nanolabels with long fluorescence lifetimes and low-energy excitation. Currently, instruments taking advantage of this technique for broader applications (in-field or point-of-care testing) are lagging behind in their development. In this work, a low-cost, compact, and sensitive time-resolved-optical-reader was developed aiming to advance such a technique for wider applications.

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.