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Abstract: The objective of this study was to adapt and evaluate two in vitro botulinum neurotoxin (BoNT) detection methods, including the Botulinum Toxin ELISA and the Endopep MS (a mass spectrometric-based endopeptidase method), for use with drinking water samples. The method detection limits (MDL) of the ELISA and Endopep MS were 260pg/mL and 21pg/mL of BoNT/A complex toxin, respectively. Since toxin could be present in water samples at highly dilute concentrations, large volume (100-L) samples of municipal tap water from five US municipalities having distinct water compositions were dechlorinated, spiked with 5μg BoNT/A, and subjected to tangential-flow ultrafiltration (UF) using hollow fiber dialyzers. The recovery efficiency of BoNT/A using UF and quantified by ELISA ranged from 11% to 36% while efficiencies quantified by MS ranged from 26% to 55%. BoNT/A was shown to be stable in dechlorinated municipal tap water stored at 4°C for up to four weeks. In addition, toxin present in UF-concentrated water samples was also shown to be stable at 4°C for up to four weeks, allowing holding of samples prior to analysis. Finally, UF was used to concentrate a level of toxin (7pg/mL) which is below the MDL for direct analysis by both ELISA and Endopep MS. Following UF, toxin was detectable in these samples using both in vitro analysis methods. These data demonstrate that UF-concentration of toxin from large volume water samples followed by use of existing analytical methods for detection of BoNT/A can be used in support of a monitoring program for contaminants in drinking water. [Copyright &y& Elsevier]

Abstract: Botulinum neurotoxin (BoNT) producing clostridia contain genes encoding a specific neurotoxin serotype (A–G) and nontoxic associated proteins that form the toxin complex. The nontoxic nonhemagglutinin (NTNH) is a conserved component of the toxin complex in all seven toxin types. A real-time PCR assay that utilizes a locked nucleic acid hydrolysis probe to target the NTNH gene was developed to detect bacterial strains harboring the botulinum neurotoxin gene cluster. The specificity of the assay for Clostridium botulinum types A–G, Clostridium butyricum type E and Clostridium baratii type F was demonstrated using a panel of 73 BoNT producing clostridia representing all seven toxin serotypes. In addition, exclusivity of the assay was demonstrated using non-botulinum toxin producing clostridia (7 strains) and various enteric bacterial strains (n =27). Using purified DNA, the assay had a sensitivity of 4–95 genome equivalents. C. botulinum type A was detected directly in spiked stool samples at 102–103 CFU/ml. Stool spiked with 1 CFU/ml was detected when the sample was inoculated into enrichment broth and incubated for 24 h. These results indicate that the NTNH real-time PCR assay can be used to screen enrichment cultures of primary specimens at earlier time points (24 h) than by toxin detection of unknown culture supernatants (up to 5 days). [Copyright &y& Elsevier]

For nearly one hundred years, researchers have attempted to categorize botulinum neurotoxin-producing clostridia and the toxins that they produce according to biochemical characterizations, serological comparisons, and genetic analyses. Throughout this period the bacteria and their toxins have defied such attempts at categorization. Below is a description of both historic and current Clostridium botulinum strain and neurotoxin information that illustrates how each new finding has significantly added to the knowledge of the botulinum neurotoxin-containing clostridia and their diversity. [ABSTRACT FROM AUTHOR]

The majority of Legionnaires' disease (LD) cases are caused by Legionella pneumophila , a genetically heterogeneous species composed of at least 17 serogroups. Previously, it was demonstrated that L. pneumophila consists of three subspecies: pneumophila , fraseri and pascullei . During an LD outbreak investigation in 2012, we detected that representatives of both subspecies fraseri and pascullei colonized the same water system and that the outbreak-causing strain was a new member of the least represented subspecies pascullei . We used partial sequence based typing consensus patterns to mine an international database for additional representatives of fraseri and pascullei subspecies. As a result, we identified 46 sequence types (STs) belonging to subspecies fraseri and two STs belonging to subspecies pascullei . Moreover, a recent retrospective whole genome sequencing analysis of isolates from New York State LD clusters revealed the presence of a fourth L. pneumophila subspecies that we have termed raphaeli . This subspecies consists of 15 STs. Comparative analysis was conducted using the genomes of multiple members of all four L. pneumophila subspecies. Whereas each subspecies forms a distinct phylogenetic clade within the L. pneumophila species, they share more average nucleotide identity with each other than with other Legionella species. Unique genes for each subspecies were identified and could be used for rapid subspecies detection. Improved taxonomic classification of L. pneumophila strains may help identify environmental niches and virulence attributes associated with these genetically distinct subspecies. [ABSTRACT FROM AUTHOR]

Lymphogranuloma venereum (LGV) can be differentiated from non-LGV chlamydial infection using Sanger sequencing or molecular assays, including those that are commercially-available internationally. Here, we describe the performance of a rapid real-time PCR (RT-PCR)-based strategy in differentiating Chlamydia trachomatis infections associated with LGV or non-LGV serovars. One hundred three rectal swabs, previously genotyped using Sanger sequencing of the ompA gene as a reference method, were tested in the RT-PCR assays. All non-LGV specimens were correctly identified, but the RT-PCR failed to detect 1 LGV specimen, resulting in a sensitivity of 87.5% for the non-LGV/LGV RT-PCR assay. Additional performance characteristics (e.g., specificity, accuracy, and reproducibility) were all between 93% and 100% with a limit of detection ≤100 copies/reaction. Thus, this rapid RT-PCR method for LGV detection in clinical specimens is comparable to the reference method. [ABSTRACT FROM AUTHOR]

Two plasmid gene protein (Pgp3)-based serological assays, the Pgp3-ELISA and multiplex bead assay (Pgp3-MBA), were compared and used to estimate seropositivity of Chlamydia trachomatis (CT) among females 14 to 39 years old participating in the National Health and Nutrition Examination Survey between 2013–2016. Of the 2,201 specimens tested, 502 (29.5%, 95% CI 27.6–31.5) were positive using Pgp3-ELISA and 624 (28.4%, 95% CI 26.5–30.3) were positive using Pgp3-MBA. The overall agreement between the assays was 87.7%. Corresponding nucleic acid amplification test (NAAT) results were available for 1,725 specimens (from women 18–39 years old); of these, 42 (2.4%, 95% CI 1.8–3.3) were CT NAAT-positive. Most of the CT NAAT-positive specimens had corresponding positive serological assay results; 33 (78.6%, 95% CI 62.8–89.2) were Pgp3-ELISA-positive and 36 (85.7%, 95% CI 70.8–94.1) were Pgp3-MBA-positive. Although Pgp3-ELISA and Pgp3-MBA demonstrated equivalent performance in this study, an advantage of the Pgp3-MBA over Pgp3-ELISA is that it is well suited for high sample throughput applications. [ABSTRACT FROM AUTHOR]

Abstract: We describe the adaptation of a sample recovery method for botulinum neurotoxins from stainless steel. Botulinum toxin was recovered from surfaces left to dry for up to 16h and detected by either ELISA or EndoPep mass spectrometry methods. In addition, we demonstrate that this method can be used to evaluate the efficacy of surface decontamination procedures. [Copyright &y& Elsevier]