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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.

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., (© AOAC INTERNATIONAL 2020. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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.

Melioidosis is caused by the gram-negative bacillus Burkholderia pseudomallei , endemic to northern Australia and Southeast Asia. We present a patient who traveled to Mexico, returned to the United States, and developed progressive manifestations of melioidosis, culminating as central nervous system disease. Standard therapy was contraindicated, and a prolonged intensive phase was employed.

Zika virus (ZIKV) is implicated in fetal stillbirth, microcephaly, intracranial calcifications, and ocular anomalies following vertical transmission from infected mothers. In adults, infection may trigger autoimmune inflammatory polyneuropathy. Transmission most commonly follows the bite of infected Aedes mosquitoes but may also occur through sexual intercourse or receipt of blood products. Definitive diagnosis through detection of viral RNA is possible in serum or plasma within 10 days of disease onset, in whole blood within 3 weeks of onset, and in semen for up to 3 months. Serological diagnosis is nonetheless critical because few patients have access to molecular diagnostics during the acute phase of infection and infection may be associated with only mild or inapparent disease that does not prompt molecular testing. Serological diagnosis is confounded by cross-reactivity of immune sera with other flaviviruses endemic in the areas where ZIKV has recently emerged. Accordingly, we built a high-density microarray comprising nonredundant 12-mer peptides that tile, with one-residue overlap, the proteomes of Zika, dengue, yellow fever, West Nile, Ilheus, Oropouche, and chikungunya viruses. Serological analysis enabled discovery of a ZIKV NS2B 20-residue peptide that had high sensitivity (96.0%) and specificity (95.9%) versus natural infection with or vaccination against dengue, chikungunya, yellow fever, West Nile, tick-borne encephalitis, or Japanese encephalitis virus in a microarray assay and an enzyme-linked immunosorbent assay (ELISA) of early-convalescent-phase sera (2 to 3 weeks after onset of symptomatic infection). IMPORTANCE The emergence of Zika virus (ZIKV) as a teratogen is a profound challenge to global public health. Molecular diagnosis of infection is straightforward during the 3-week period when patients are viremic. However, serological diagnosis thereafter of historical exposure has been confounded by cross-reactivity. Using high-density peptide arrays that tile the proteomes of a selection of flaviviruses to identify a ZIKV-specific peptide, we established two assays that enable sensitive and specific diagnosis of exposure to ZIKV. These assays may be useful in guiding clinical management of mothers at risk for potential exposure to ZIKV and enable insights into the epidemiology of ZIKV infections.

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 mLD 50 , respectively. BoNT/A was detected by the MBA between 1.8 and 18 mLD 50 , somewhat more sensitive than the MS method of 18 mLD 50 . 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.

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., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Spiperone (1) is a widely used pharmacological tool that acts as a potent dopamine D2, serotonin 5-HT1A, and serotonin 5-HT2A antagonist. Although spiperone also binds at 5-HT2C receptors, it is one of the very few agents that display some (ca. 1000-fold) binding selectivity for 5-HT2A versus 5-HT2C receptors and, hence, might serve as a useful template for the development of novel 5-HT2A antagonists if the impact of its various substituent groups on binding was known. In the present investigation we focused on the 1, 3,8-triazaspiro[4.5]decanone portion of spiperone and found that replacement of the N1-phenyl group with a methyl group only slightly decreased affinity for cloned rat 5-HT2A receptors. However, N1-methyl derivatives displayed significantly reduced affinity for 5-HT1A, 5-HT2C, and dopamine D2 receptors. Several representative examples were shown to behave as 5-HT2 antagonists. As such, N1-alkyl analogues of spiperone may afford entry into a novel series of 5-HT2A-selective antagonists.

Single amino acid mutations in the third intracellular loop, as well as other domains of G protein-coupled receptors, have been shown to confer drastic changes in receptor properties and have been postulated to be responsible for various disease states. To determine whether an amino acid mutation can confer dramatic alterations in the 5-hydroxytryptamine2A (5-HT2A) receptor, we mutated amino acid 322 to lysine (C322K), glutamate (C322E) or arginine (C322R). Transient expression of the mutant receptors revealed properties associated with constitutive activity. Radioligand binding studies revealed an increase in 5-HT affinity from 293 nM (native) to 86 nM (C322E), 25 nM (C322K) and 11 nM (C322R). 5-HT potency for stimulation of inositol phosphate production increased from 152 nM (native) to 61 nM (C322E) and 25 nM (C322K). Basal inositol phosphate levels in COS-7 cells expressing C322K and C322E mutant receptors were 8-fold and 4-fold higher, respectively, than cells expressing native 5-HT2A receptors. Basal levels of inositol phosphate stimulated by C322K receptors represented 48% of total inositol phosphate production stimulated by native receptors in the presence of 10 microM 5-HT. Antipsychotic drugs (chlorpromazine, clozapine, haloperidol, loxapine and risperidone) displayed inverse agonist activity by inhibiting C322K constitutive activation of phosphatidylinositol hydrolysis. These data indicate that amino acid 322 in the 5-HT2A receptor plays an important role in maintaining the inactive conformation and provide further evidence that amino acid mutations can produce profound alterations in G protein-coupled receptor activity.

Evidence from studies with phenylisopropylamine hallucinogens indicates that the 5HT2A receptor is the likely target for the initiation of events leading to hallucinogenic activity associated with LSD and related drugs. Recently, lisuride (a purported non-hallucinogenic congener of LSD) was reported to be a potent antagonist at the 5HT2C receptor and an agonist at the 5HT2A receptor. LSD exhibited agonist activity at both receptors. These data were interpreted as indicating that the 5HT2C receptor might be the initiating site of action for hallucinogens. To test this hypothesis, recombinant cells expressing 5HT2A and 5HT2C receptors were used to determine the actions of LSD and lisuride. LSD and lisuride were potent partial agonists at 5HT2A receptors with EC50 values of 7.2 nM and 17 nM, respectively. Also, LSD and lisuride were partial agonists at 5HT2C receptors with EC50 values of 27 nM and 94 nM, respectively. We conclude that lisuride and LSD have similar actions at 5HT2A and 5HT2C receptors in recombinant cells. As agonist activity at brain 5HT2A receptors has been associated with hallucinogenic activity, these results indicate that lisuride may possess hallucinogenic activity, although the psychopharmacological effects of lisuride appear to be different from the hallucinogenic effects of LSD.