Current Developments in Diagnostic Assays for Laboratory Confirmation and Investigation of Botulism.

Detection of botulinum neurotoxin or isolation of the toxin-producing organism is required for the laboratory confirmation of botulism in clinical specimens. In an effort to reduce animal testing required by the gold standard method of botulinum neurotoxin detection, the mouse bioassay, many technologies have been developed to detect and characterize the causative agent of botulism. Recent advancements in these technologies have led to improvements in technical performance of diagnostic assays; however, many emerging assays have not been validated for the detection of all serotypes in complex clinical and environmental matrices. Improvements to culture protocols, endopeptidase-based assays, and a variety of immunological and molecular methods have provided laboratories with a variety of testing options to evaluate and incorporate into their testing algorithms. While significant advances have been made to improve these assays, additional work is necessary to evaluate these methods in various clinical matrices and to establish standardized criteria for data analysis and interpretation.

SENTINEL COYOTE PATHOGEN SURVEY TO ASSESS DECLINING BLACK-FOOTED FERRET (MUSTELA NIGRIPES) POPULATION IN SOUTH DAKOTA, USA.

As part of the national recovery effort, endangered black-footed ferrets (Mustela nigripes) were reintroduced to the Cheyenne River Sioux Reservation in South Dakota, US in 2000. Despite an encouraging start, numbers of ferrets at the site have declined. In an effort to determine possible causes of the population decline, we undertook a pathogen survey in 2012 to detect exposure to West Nile virus (WNV), canine distemper virus (CDV), plague (Yersinia pestis), tularemia (Francisella tularensis), and heartworm (Dirofilaria immitis) using coyotes (Canis latrans) as a sentinel animal. The highest seroprevalence was for WNV with 71% (20/28) of coyotes testing antibody-positive. Seroprevalence of CDV and plague were lower, 27% and 13%, respectively. No evidence of active infection with tularemia or heartworm was seen in the coyotes sampled. As this study did not sample black-footed ferrets themselves, the definitive cause for the decline of this population cannot be determined. However, the presence of coyotes seropositive for two diseases, plague and CDV, lethal to black-footed ferrets, indicated the potential for exposure and infection. The high seroprevalence of WNV in the coyotes indicated a wide exposure to the virus; therefore, exposure of black-footed ferrets to the virus is also likely. Due to the ability of WNV to cause fatal disease in other species, studies may be useful to elucidate the impact that WNV could have on the success of reintroduced black-footed ferrets as well as factors influencing the spread and incidence of the disease in a prairie ecosystem.

Utilization of Standard Method Performance Requirements for the Detection of Coxiella burnetii in Environmental Samples.

BACKGROUND: Coxiella burnetii, the causative agent of Q fever, is a long-standing public health problem. Infected animals shed the organism, resulting in aerosol transmission to humans. This organism can potentially be used as a bioterrorism weapon and is on the Department of Health and Human Service Select Agent List. Assay development for detecting C. burnetii in environmental samples has been limited. OBJECTIVE: We describe the use of Standard Method Performance Requirements (SMPR®) 2015.011 to detect Coxiella in air filters and liquids to validate additional environmental samples. METHOD: SMPR 2015.011 was used to validate a real-time polymerase chain reaction (rtPCR) assay developed to detect C. burnetii DNA in powder samples submitted to the public health laboratory for biothreat analysis. RESULTS: Our laboratory developed an assay to detect the icd gene of C. burnetii. The LOD for the assay was 33 gene copies per rtPCR reaction in buffer and 260 in each of the three separate powdered samples. CONCLUSIONS: The SMPR 2015.011 allowed validation of an assay to detect Coxiella nucleic acid in an environmental sample. The assay was sensitive, robust, specific, and able to detect this select agent in powders. HIGHLIGHTS: Development of detection assays for agents that are difficult to culture and have limited validation material available can be problematic for manufacturers. Using the SMPR 2015.011 developed for the detection of Coxiella as well as the SMPR 2016.012 for the detection of Variola, we demonstrated that assays can be appropriately validated using alternative approaches.

Outbreak of botulism type A in dairy cows detected by MALDI-TOF mass spectrometry.

Twenty-eight lactating dairy cattle in New York State were exposed to botulism toxin; 12 died and 16 recovered but never returned to full productivity. Pieces of a raccoon carcass were found in the total mixed ration on the first day of the outbreak. Clinical signs included anorexia, decreased milk production, decreased tongue tone, profound weakness, and recumbency. Clostridium botulinum type A (BoNT/A) was detected in rumen contents from 2 deceased cows via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). In addition, C. botulinum type C was cultured from the liver of a third cow, and C. botulinum neurotoxin-producing type C gene (bont/C) was detected via real-time PCR. On postmortem examination, 4 cows had findings suggestive of toxic myopathy, but the cause and significance of these lesions is unknown given that botulism is typically not associated with gross or histologic lesions. This outbreak of BoNT/A in cattle in North America was diagnosed via MALDI-TOF MS, a rapid and sensitive modality for detection of botulinum preformed neurotoxin.

Brucella Exposure Risk Events in 10 Clinical Laboratories, New York City, USA, 2015 to 2017.

From 2015 to 2017, 11 confirmed brucellosis cases were reported in New York City, leading to 10 Brucella exposure risk events (Brucella events) in 7 clinical laboratories (CLs). Most patients had traveled to countries where brucellosis is endemic and presented with histories and findings consistent with brucellosis. CLs were not notified that specimens might yield a hazardous organism, as the clinicians did not consider brucellosis until they were notified that bacteremia with Brucella was suspected. In 3 Brucella events, the CLs did not suspect that slow-growing, small Gram-negative bacteria might be harmful. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), which has a limited capacity to identify biological threat agents (BTAs), was used during 4 Brucella events, which accounted for 84% of exposures. In 3 of these incidents, initial staining of liquid media showed Gram-positive rods or cocci, including some cocci in chains, suggesting streptococci. Over 200 occupational exposures occurred when the unknown isolates were manipulated and/or tested on open benches, including by procedures that could generate infectious aerosols. During 3 Brucella events, the CLs examined and/or manipulated isolates in a biological safety cabinet (BSC); in each CL, the CL had previously isolated Brucella Centers for Disease Control and Prevention recommendations to prevent laboratory-acquired brucellosis (LAB) were followed; no seroconversions or LAB cases occurred. Laboratory assessments were conducted after the Brucella events to identify facility-specific risks and mitigations. With increasing MALDI-TOF MS use, CLs are well-advised to adhere strictly to safe work practices, such as handling and manipulating all slow-growing organisms in BSCs and not using MALDI-TOF MS for identification until BTAs have been ruled out.

One-step pentaplex real-time polymerase chain reaction assay for detection of zika, dengue, chikungunya, West nile viruses and a human housekeeping gene.

BACKGROUND: Recent emergence of Zika virus (ZIKV), and the global spread of dengue (DENV), chikungunya (CHIKV) and West Nile viruses (WNV) raised urgent need of accurate and affordable molecular diagnosis of these clinically indistinguishable arboviral infections. OBJECTIVES: We established a pentaplex real-time reverse transcription PCR (rRT-PCR) assay (CII-ArboViroPlex rRT-PCR) for specific and sensitive detection of the African and American genotypes of ZIKV, all four serotypes of DENV, CHIKV, WNV and a housekeeping gene as internal control in single reaction. STUDY DESIGN: Specific primers and probe sets were designed for ZIKV, DENV, CHIKV, WNV and RNase P (housekeeping gene) and tested for in-vitro transcribed RNA standards, virus cultures, clinical samples positive for ZIKV, DENV, CHIKV and WNV and limit of detection (LOD) were determined for each. Results Using ten-fold serially diluted in-vitro transcribed RNA, CII- ArboViroPlex rRT-PCR assay has LOD of 100 RNA copies/reaction (Rn) for ZIKV in serum or urine, 100 RNA copies/Rn for DENV in serum, and 10 RNA copies/Rn for CHIKV and WNV in serum. LODs from sera spiked with quantitated viral stocks were 2.6 × 102 GEQ/Rn for ZIKV, 2.2 × 101 GEQ/Rn for DENV-1, 9.4 × 100 GEQ/Rn for DENV-2, 2.3 × 102 GEQ/Rn for DENV-3, 1.4 × 103 GEQ/Rn for DENV-4, 2.7 × 102 GEQ/Rn for CHIKV, and 1.05 × 101 GEQ/Rn for WNV. CONCLUSIONS: The CII-ArboViroPlex rRT-PCR assay is a quantitative one-step pentaplex rRT-PCR assay for the molecular detection and differential diagnosis of ZIKV, DENV, CHIKV, WNV and a human housekeeping gene control in a single- PCR reaction.

Molecular Characterization of Clostridium botulinum Harboring the bont/B7 Gene.

Clostridium botulinum produces botulinum neurotoxin (BoNT), which is the causative agent of botulism, a rare but serious disease that can result in death if not treated. Infant botulism occurs when C. botulinum colonizes the intestinal tract of infants and produces BoNT. It has been proposed that infants under the age of 1 year are uniquely susceptible to colonization by C. botulinum as their intestinal microbiota is not fully developed and provides little competition, allowing C. botulinum to thrive and produce BoNT in the gut. There are seven well-characterized serotypes (A-G) of BoNT identified by the ability of specific antitoxins to neutralize BoNTs. Molecular technology has allowed researchers to narrow these further into subtypes based on nucleic acid sequences of the botulinum toxin (bont) gene. One of the most recently recognized subtypes for bont/B is subtype bont/B7. We identified through whole genome sequencing five C. botulinum isolates harboring bont/B7 from CDC's strain collection, including patient isolates and an epidemiologically linked isolate from an opened infant formula container. In this study, we report the results of whole genome sequencing analysis of these C. botulinum subtype bont/B7 isolates. Average nucleotide identity and high quality single nucleotide polymorphism (hqSNP) analysis resulted in two major clades. The epidemiologically linked isolates differed from each other by 2-6 hqSNPs, and this clade separated from the other isolates by 95-119 hqSNPs, corroborating available epidemiological evidence.

Safety and Accuracy of Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Identification of Highly Pathogenic Organisms.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) sample preparation methods, including the direct, on-plate formic acid, and ethanol/formic acid tube extraction methods, were evaluated for their ability to render highly pathogenic organisms nonviable and safe for handling in a biosafety level 2 laboratory. Of these, the tube extraction procedure was the most successful, with none of the tested strains surviving this sample preparation method. Tube extracts from several agents of bioterrorism and their near neighbors were analyzed in an eight-laboratory study to examine the utility of the Bruker Biotyper and Vitek MS MALDI-TOF MS systems and their in vitro diagnostic (IVD), research-use-only, and Security-Relevant databases, as applicable, to accurately identify these agents. Forty-six distinct strains of Bacillus anthracis, Yersinia pestis, Francisella tularensis, Burkholderia mallei, Burkholderia pseudomallei, Clostridium botulinum, Brucella melitensis, Brucella abortus, Brucella suis, and Brucella canis were extracted and distributed to participating laboratories for analysis. A total of 35 near-neighbor isolates were also analyzed.

Implementing the Bruker MALDI Biotyper in the Public Health Laboratory for C. botulinum Neurotoxin Detection.

Currently, the gold standard method for active botulinum neurotoxin (BoNT) detection is the mouse bioassay (MBA). A Centers for Disease Control and Prevention-developed mass spectrometry (MS)-based assay that detects active BoNT was successfully validated and implemented in a public health laboratory in clinical matrices using the Bruker MALDI-TOF MS (Matrix-assisted laser desorption ionization-time of flight mass spectrometry) Biotyper. For the first time, a direct comparison with the MBA was performed to determine MS-based assay sensitivity using the Bruker MALDI Biotyper. Mice were injected with BoNT/A, /B, /E, and /F at concentrations surrounding the established MS assay limit of detection (LOD) and analyzed simultaneously. For BoNT/B, /E, and /F, MS assay sensitivity was equivalent or better than the MBA at 25, 0.3, and 8.8 mLD50, respectively. BoNT/A was detected by the MBA between 1.8 and 18 mLD50, somewhat more sensitive than the MS method of 18 mLD50. Studies were performed to compare assay performance in clinical specimens. For all tested specimens, the MS method rapidly detected BoNT activity and serotype in agreement with, or in the absence of, results from the MBA. We demonstrate that the MS assay can generate reliable, rapid results while eliminating the need for animal testing.

Detection of Staphylococcus aureus enterotoxin production genes from patient samples using an automated extraction platform and multiplex real-time PCR.

To minimize specimen volume, handling and testing time, we have developed two TaqMan(®) multiplex real-time PCR (rtPCR) assays to detect staphylococcal enterotoxins A-E and Toxic Shock Syndrome Toxin production genes directly from clinical patient stool specimens utilizing a novel lysis extraction process in parallel with the Roche MagNA Pure Compact. These assays are specific, sensitive and reliable for the detection of the staphylococcal enterotoxin encoding genes and the tst1 gene from known toxin producing strains of Staphylococcus aureus. Specificity was determined by testing a total of 47 microorganism strains, including 8 previously characterized staphylococcal enterotoxin producing strains against each rtPCR target. Sensitivity for these assays range from 1 to 25 cfu per rtPCR reaction for cultured isolates and 8-20 cfu per rtPCR for the clinical stool matrix.

Skeletal muscle fiber types in the ghost crab, Ocypode quadrata: implications for running performance.

Ghost crabs possess rapid running capabilities, which make them good candidates for comparing invertebrate exercise physiology with that of more extensively studied vertebrates. While a number of studies have examined various aspects of running physiology and biomechanics in terrestrial crabs, none to date have defined the basic skeletal muscle fiber types that power locomotion. In the current study, we investigated skeletal muscle fiber types comprising the extensor and flexor carpopodite muscles in relation to running performance in the ghost crab. We used kinematic analyses to determine stride frequency and muscle shortening velocity and found that both parameters are similar to those of comparably sized mammals but slower than those observed in running lizards. Using several complementary methods, we found that the muscles are divided into two primary fiber types: those of the proximal and distal regions possess long sarcomeres (6.2+/-2.3 microm) observed in crustacean slow fibers and have characteristics of aerobic fibers whereas those of the muscle mid-region have short sarcomeres (3.5+/-0.4 microm) characteristic of fast fibers and appear to be glycolytic. Each fiber type is characterized by several different myofibrillar protein isoforms including multiple isoforms of myosin heavy chain (MHC), troponin I (TnI), troponin T (TnT) and a crustacean fast muscle protein, P75. Three different isoforms of MHC are differentially expressed in the muscles, with fibers of the mid-region always co-expressing two isoforms at a 1:1 ratio within single fibers. Based on our analyses, we propose that these muscles are functionally divided into a two-geared system, with the aerobic fibers used for slow sustained activities and the glycolytic mid-region fibers being reserved for explosive sprints. Finally, we identified subtle differences in myofibrillar isoform expression correlated with crab body size, which changes by several orders of magnitude during an animal's lifetime.