Sylvatic Transmission of Chikungunya Virus among Nonhuman Primates in Myanmar.

Nonhuman primates living in proximity to humans increase risks for sylvatic arbovirus transmission. We collected serum samples from nonhuman primates in Hlawga National Park near Yangon, Myanmar, and detected antibodies against chikungunya (33%) and Japanese encephalitis (4%) viruses. Buffer zones between primate and human communities might reduce cross-species arbovirus transmission.

Validation of a Lateral Flow Test for the Presumptive Identification of the Presence of Burkholderia mallei or Burkholderia pseudomallei in Environmental Samples.

We conducted a comprehensive, multiphase laboratory evaluation of InBios Active Melioidosis Detect (AMD) rapid test, a lateral flow immunoassay designed to detect capsular polysaccharides produced by Burkholderia mallei or Burkholderia pseudomallei, used in conjunction with the Omni Array Reader (OAR) for the rapid detection of B mallei or B pseudomallei in environmental (nonclinical) samples at 2 sites. The limit of detection, using reference strains B mallei strain ATCC 23344 and B pseudomallei strain ATCC 11668, was determined to be 103 to 104 CFU/mL. In different phases of the evaluation, inclusivity strains that included geographically diverse strains of B mallei (N = 13) and B pseudomallei (N = 22), geographically diverse phylogenetic near neighbor strains (N = 66), environmental background strains (N = 64), white powder samples (N = 26), and environmental filter extracts (N = 1 pooled sample from 10 filter extracts) were also tested. A total of 1,753 tests were performed, which included positive and negative controls. Visual and OAR results showed that the AMD test detected 92.3% of B mallei and 95.5% of B pseudomallei strains. Of the 66 near-neighbor strains tested, cross-reactivity was observed with only B stabilis 2008724195 and B thailandensis 2003015869. Overall, the specificity and sensitivity were 98.8% and 98.7%, respectively. The results of this evaluation support the use of the AMD test as a rapid, qualitative assay for the presumptive detection of B mallei and B pseudomallei in suspicious environmental samples such as white powders and aerosol samples by first responders and laboratory personnel.

Rapid Presumptive Identification of Burkholderia mallei and Burkholderia pseudomallei Clinical Isolates Using a Highly Specific Lateral Flow Assay.

We conducted a comprehensive, multiphase laboratory evaluation of the InBios Active Melioidosis Detect (AMD) rapid test, a lateral flow immunoassay designed to detect capsular polysaccharides produced by Burkholderia mallei or Burkholderia pseudomallei, used in conjunction with the Omni Array Reader for the rapid identification of culture isolates of B mallei or B pseudomallei to support clinical diagnosis for response and triage during a mass casualty event, such as a biological attack. The study was conducted at 2 sites to assess the performance of the AMD test. The sensitivity, specificity, and reproducibility of the assay was determined using 5 replicates of 35 inclusivity strains and 64 clinical background strains. A total of 520 tests were performed, which included both positive and negative controls. Results obtained visually and with the Omni Array Reader showed a sensitivity of 92.3% for B mallei and 95.6% for B pseudomallei; no cross-reactivity was observed with any of the 64 clinical background organisms. The results from this study indicate that the AMD test for the presumptive identification of B mallei and B pseudomallei isolates to support clinical diagnosis is highly robust, specific, and sensitive. This evaluation supports the use of this test as a rapid, qualitative assay for the presumptive identification of B mallei and B pseudomallei in a clinical setting.

Multi-laboratory validation of the xMAP-Food Allergen Detection Assay: A multiplex, antibody-based assay for the simultaneous detection of food allergens.

The increasing prevalence of individuals with multiple food allergies and the need to distinguish between foods containing homologous, cross-reactive proteins have made the use of single-analyte antibody-based methods (e.g., ELISAs) sometimes insufficient. These issues have resulted in the need to conduct multiple analyses and sometimes employ orthogonal methods like mass spectrometry or DNA-based methods for confirmatory purposes. The xMAP Food Allergen Detection Assay (xMAP FADA) was developed to solve this problem while also providing increased throughput and a modular design suitable for adapting to changes in analytical needs. The use of built-in redundancy provides the xMAP FADA with built-in confirmatory analytical capability by including complementary antibody bead sets and secondary analytical end points (e.g., ratio analysis and multi-antibody profiling). A measure of a method's utility is its performance when employed by analysts of varying expertise in multiple laboratory environments. To gauge this aspect, a multi-laboratory validation (MLV) was conducted with 11 participants of different levels of proficiency. The MLV entailed the analysis of incurred food samples in four problematic food matrices, meat sausage, orange juice, baked muffins, and dark chocolate. Except for a couple of instances, involving two confirmatory components in the analysis of baked muffins, the allergenic foods were detected by all participants at concentrations in the analytical samples comparable to ≤ 10 µg/g in the original food sample. In addition, despite high levels of inter-lab variance in the absolute intensities of the responses, the intra-laboratory reproducibility was sufficient to support analyses based on the calibration standards and direct comparison controls (DCCs) analyzed alongside the samples. In contrast, ratio analyses displayed inter-laboratory %CV (RSDR) values < 20%; presumably because the ratios are based on inherent properties of the antigenic elements. The excellent performance of the xMAP FADA when performed by analysts of varying proficiency indicates a reliability sufficient to meet analytical needs.

Comprehensive Laboratory Evaluation of a Specific Lateral Flow Assay for the Presumptive Identification of Francisella tularensis in Suspicious White Powders and Aerosol Samples.

We conducted a comprehensive, multi-phase laboratory evaluation of the Tularemia BioThreat Alert® (BTA) test, a lateral flow assay (LFA) for the rapid detection of Francisella tularensis. The study, conducted at 2 sites, evaluated the limit of detection (LOD) of this assay using the virulent SchuS4 strain and the avirulent LVS strain of F. tularensis. In 6-phase evaluation (linear dynamic range and reproducibility, inclusivity, near-neighbor, environmental background, white powder, and environmental filter extract), 13 diverse strains of F. tularensis, 8 Francisella near neighbors, 61 environmental background organisms, 26 white powders, and a pooled aerosol extract were tested. In the 937 tests performed, the Tularemia BTA demonstrated an LOD of 107 to 108 cfu/mL, with a sensitivity of 100.00%, specificity of 98.08%, and accuracy of 98.84%. These performance data are important for accurate interpretation of qualitative results arising from screening suspicious white powders in the field.

Comprehensive Laboratory Evaluation of a Lateral Flow Assay for the Detection of Yersinia pestis.

We conducted a comprehensive, multiphase laboratory evaluation of the Plague BioThreat Alert® (BTA) test, a lateral flow immunoassay (LFA), for the rapid detection of Yersinia pestis. The study was conducted in 7 phases at 2 sites to assess the performance of the LFA. The limit of detection (LOD) was determined using both a virulent and avirulent strain of Y. pestis, CO99-3015 (105 CFU/ml) and A1122 (104 CFU/ml), respectively. In the other phases, 18 Y. pestis strains, 20 phylogenetic near-neighbor strains, 61 environmental background microorganisms, 26 white powders, and a pooled aerosol sample were also tested. A total of 1,110 LFA test results were obtained, and their analysis indicates that this LFA had a sensitivity of 97.65% and specificity of 96.57%. These performance data are important for accurate interpretation of qualitative results arising from testing suspicious white powders and aerosol samples in the field. Any positive specimen in this assay is considered presumptive positive and should be referred to the Centers for Disease Control and Prevention Laboratory Response Network for additional testing, confirmation, and characterization for an appropriate public health response.

Rapid Presumptive Identification of Bacillus anthracis Isolates Using the Tetracore RedLine Alert™ Test.

A comprehensive laboratory evaluation of the Tetracore RedLine Alert test, a lateral flow immunoassay (LFA) for the rapid presumptive identification of Bacillus anthracis, was conducted at 2 different test sites. The study evaluated the sensitivity of this assay using 16 diverse strains of B. anthracis grown on sheep blood agar (SBA) plates. In addition, 83 clinically relevant microorganisms were tested to assess the specificity of the RedLine Alert test. The results indicated that the RedLine Alert test for the presumptive identification of B. anthracis is highly robust, specific, and sensitive. RedLine Alert is a rapid test that has applicability for use in a clinical setting for ruling-in or ruling-out nonhemolytic colonies of Bacillus spp. grown on SBA medium as presumptive isolates of B. anthracis.

Botulinum Neurotoxin Detection Methods for Public Health Response and Surveillance.

Botulism outbreak due to consumption of food contaminated with botulinum neurotoxins (BoNTs) is a public health emergency. The threat of bioterrorism through deliberate distribution in food sources and/or aerosolization of BoNTs raises global public health and security concerns due to the potential for high mortality and morbidity. Rapid and reliable detection methods are necessary to support clinical diagnosis and surveillance for identifying the source of contamination, performing epidemiological analysis of the outbreak, preventing and responding to botulism outbreaks. This review considers the applicability of various BoNT detection methods and examines their fitness-for-purpose in safeguarding the public health and security goals.

Proteomic profiles by multiplex microsphere suspension array.

Advances in high-throughput proteomic approaches have provided substantial momentum to novel disease-biomarker discovery research and have augmented the quality of clinical studies. Applications based on multiplexed microsphere suspension array technology are making strong in-roads into the clinical diagnostic/prognostic practice. Conventional proteomic approaches are designed to discover a broad set of proteins that are associated with a specific medical condition. In comparison, multiplex microsphere immunoassays use quantitative measurements of selected set(s) of specific/particular molecular markers such as cytokines, chemokines, pathway signaling or disease-specific markers for detection, metabolic disorders, cancer, and infectious agents causing human, plant and animal diseases. This article provides a foundation to the multiplexed microsphere suspension array technology, with an emphasis on the improvements in the technology, data analysis approaches, and applications to translational and clinical research with implications for personalized and precision medicine.

Clinical, Serological, and Molecular Observations from a Case Series Study during the Asian Lineage Zika Virus Outbreak in Grenada during 2016.

This paper describes the spatial and temporal distribution of cases, demographic characteristics of patients, and clinical manifestations of Zika virus (ZIKV) during the 2016 outbreak in Grenada. The first reported case was recorded in St. Andrew Parish in April, and the last reported case was seen in November, with peak transmission occurring in the last week of June, based on test results. Data were collected from a total of 514 patients, of whom 207 (40%) tested positive for ZIKV. No evidence was found that testing positive for ZIKV infection was related to age, gender, or pregnancy status. Clinical presentation with rash (OR = 2.4, 95% CI = 1.5 to 3.7) or with lymphadenopathy (OR = 1.7, 95% CI = 1.0 to 2.9) were the only reported symptoms consistent with testing positive for ZIKV infection. During the Zika outbreak, the infection rate was 20 clinical cases per 10,000 in the population compared to 41 cases per 10,000 during the chikungunya outbreak in Grenada in 2014 and 17 cases per 10,000 during the dengue outbreak in 2001-2002. Even though the country has employed vector control programs, with no apparent decrease in infection rates, it appears that new abatement approaches are needed to minimize morbidity in future arbovirus outbreaks.

Comprehensive Laboratory Evaluation of a Highly Specific Lateral Flow Assay for the Presumptive Identification of Bacillus anthracis Spores in Suspicious White Powders and Environmental Samples.

We conducted a comprehensive, multiphase laboratory evaluation of the Anthrax BioThreat Alert(®) test strip, a lateral flow immunoassay (LFA) for the rapid detection of Bacillus anthracis spores. The study, conducted at 2 sites, evaluated this assay for the detection of spores from the Ames and Sterne strains of B. anthracis, as well as those from an additional 22 strains. Phylogenetic near neighbors, environmental background organisms, white powders, and environmental samples were also tested. The Anthrax LFA demonstrated a limit of detection of about 10(6) spores/mL (ca. 1.5 × 10(5) spores/assay). In this study, overall sensitivity of the LFA was 99.3%, and the specificity was 98.6%. The results indicated that the specificity, sensitivity, limit of detection, dynamic range, and repeatability of the assay support its use in the field for the purpose of qualitatively evaluating suspicious white powders and environmental samples for the presumptive presence of B. anthracis spores.

Simultaneous multiplex detection and confirmation of the proteinaceous toxins abrin, ricin, botulinum toxins, and Staphylococcus enterotoxins a, B, and C in food.

Detection of proteinaceous toxins in complex heterogeneous mixtures requires highly specific and sensitive methods. Multiplex technology employing multiple antibodies that recognize different epitopes on a toxin provides built-in confirmatory analysis as part of the initial screen and thereby increases the reliability associated with both presumptive positive and negative results. Polyclonal and monoclonal antibodies were obtained for abrin, botulinum toxins, ricin, and Staphylococcus enterotoxins A, B, and C (SEA, SEB, and SEC). Food samples were spiked with the toxins either individually or mixed and analyzed following 40-fold dilution. Abrin, botulinum toxin A complex, ricin, and SEB displayed limits of detection in the original food samples ranging from 0.03 to 1.3 microg/mL, from 0.03 to 0.07 microg/mL, from 0.01 to 0.1 microg/mL, and from <0.01 to 0.03 microg/mL, respectively. Redundancy, that is, multiple antibodies for each toxin, some recognizing different epitopes or displaying different binding affinities, provided a "fingerprint" for the presence of the toxins and built-in confirmation, thus reducing the likelihood of false-positive and false-negative results. Inclusion of internal controls, including a unique protein, helped control for variations in dilution. Paramagnetic microspheres facilitated the detection of analyte in foods containing particulate matter incompatible with the use of filter plates normally used in the wash steps of assays employing standard polystyrene microspheres.

Magnetic bead based immunoassay for autonomous detection of toxins.

We are developing an automated system for the simultaneous, rapid detection of a group of select agents and toxins in the environment. To detect toxins, we modified and automated an antibody-based approach previously developed for manual medical diagnostics that uses fluorescent eTag reporter molecules and is suitable for highly multiplexed assays. Detection is based on two antibodies binding simultaneously to a single antigen, one of which is labeled with biotin while the other is conjugated to a fluorescent eTag through a cleavable linkage. Aqueous samples are incubated with the mixture of antibodies along with streptavidin-coated magnetic beads and a photoactive porphyrin complex. In the presence of antigen, a molecular complex is formed where the cleavable linkage is held in proximity to the photoactive group. Upon excitation at 680 nm, free radicals are generated, which diffuse and cleave the linkage, releasing the eTags. Released eTags are analyzed using capillary gel electrophoresis with laser-induced fluorescence detection. Limits of detection for ovalbumin and botulinum toxoid individually were 4 (or 80 pg) and 16 ng/mL (or 320 pg), respectively, using the manual assay. In addition, we demonstrated the use of pairs of antibodies from different sources in a single assay to decrease the rate of false positives. Automation of the assay was demonstrated in a flow-through format with higher LODs of 32 ng/mL (or 640 ng) each of a mixture of ovalbumin and botulinum toxoid. This versatile assay can be easily modified with the appropriate antibodies to detect a wide range of toxins and other proteins.

Simultaneous detection of antibodies to six nonhuman-primate viruses by multiplex microbead immunoassay.

To maintain healthy nonhuman primates for use in biomedical research, animals are routinely screened for several infectious agents at most facilities. Commonly, monkey serum samples are tested by conventional immunoassays, such as enzyme-linked immunosorbent assays (ELISAs) or Western blotting, for antibodies to specific infectious agents. For testing for antibodies against multiple agents in each sample, conventional immunoassays are laborious and time-consuming. More efficient immunoassays are needed. Accordingly, we have developed a novel multiplex serodiagnostic system based on individually identifiable, fluorescent microbead sets, where each bead set is coated with antigens from a purified preparation of a specific virus. The coated bead sets are mixed to enable the detection of antibodies to multiple viruses in one serum or plasma sample. These viruses include four agents that are routinely tested for maintenance of specific-pathogen-free monkeys, namely, simian immunodeficiency virus, simian type D retrovirus, simian T-cell lymphotropic virus, and herpes B virus, as well as simian foamy virus and rhesus cytomegalovirus, both of which are commonly found in nonhuman primates. This multiplex microbead immunoassay (MMIA) enabled the simultaneous detection of antibodies to all six viruses in single serum samples as small as 1 microliter. The results obtained by MMIA analysis correlated with results of conventional ELISAs, which detect antibodies to single agents. Thus, this multiplex microbead detection system is an efficient diagnostic modality for serosurveillance of nonhuman primates.

Autonomous detection of aerosolized biological agents by multiplexed immunoassay with polymerase chain reaction confirmation.

The autonomous pathogen detection system (APDS) is an automated, podium-sized instrument that continuously monitors the air for biological threat agents (bacteria, viruses, and toxins). The system has been developed to warn of a biological attack in critical or high-traffic facilities and at special events. The APDS performs continuous aerosol collection, sample preparation, and detection using multiplexed immunoassay followed by confirmatory PCR using real-time TaqMan assays. We have integrated completely reusable flow-through devices that perform DNA extraction and PCR amplification. The fully integrated system was challenged with aerosolized Bacillus anthracis, Yersinia pestis, Bacillus globigii, and botulinum toxoid. By coupling highly selective antibody- and DNA-based assays, the probability of an APDS reporting a false positive is extremely low.

Multiplexed liquid arrays for simultaneous detection of simulants of biological warfare agents.

Liquid array-based multiplexed immunoassays designed for rapid, sensitive, specific, and simultaneous detection of multiple simulants of biological warfare agents have been developed. In both blind and standard laboratory trials, we demonstrate the simultaneous detection of four simulant agents from a single sample. The challenge agents comprise broad classes of pathogens (virus, protein toxins, bacterial spores, vegetative cells). Assay performance of each analyte was optimized, and dose-response curves and the limits of detection (LODs) for individual analytes are presented. Assay performance, including dynamic range, sensitivity, and LODs for liquid arrays and enzyme-linked immunosorbant assay were compared and are shown to be similar. Maximum assay sensitivity is obtained in approximately 1 h, and good sensitivity is achieved in as little as 30 min. Although the sample matrixes are very complex, even for highly multiplexed assays the samples do not exhibit evidence of nonspecific binding, demonstrating that the assays also have high specificity.

Autonomous detection of aerosolized Bacillus anthracis and Yersinia pestis.

We have developed and tested a fully autonomous pathogen detection system (APDS) capable of continuously monitoring the environment for airborne biological threat agents. The system is designed to provide early warning to civilians in the event of a terrorist attack. The final APDS will be completely automated, offering aerosol sampling, in-line sample preparation fluidics, multiplexed detection and identification immunoassays, and orthogonal, multiplexed PCR (nucleic acid) amplification and detection. The system performance (current capabilities include aerosol collection, multiplexed immunoassays, sample archiving, data reporting, and alarming) was evaluated in a field test conducted in a Biosafety Level 3 facility, where the system was challenged with, and detected, a series of aerosolized releases containing two live, virulent biological threat agents (Bacillus anthracis and Yersinia pestis). Results presented here represent the first autonomous, simultaneous measurement of these agents.