Development of a novel serological assay for the detection of mpox infection in vaccinated populations.

In 2022 the World Health Organization declared a Public Health Emergency for an outbreak of mpox, the zoonotic Orthopoxvirus (OPV) affecting at least 104 nonendemic locations worldwide. Serologic detection of mpox infection is problematic, however, due to considerable antigenic and serologic cross-reactivity among OPVs and smallpox-vaccinated individuals. In this report, we developed a high-throughput multiplex microsphere immunoassay using a combination of mpox-specific peptides and cross-reactive OPV proteins that results in the specific serologic detection of mpox infection with 93% sensitivity and 98% specificity. The New York State Non-Vaccinia Orthopoxvirus Microsphere Immunoassay is an important tool to detect subclinical mpox infection and understand the extent of mpox spread in the community through retrospective analysis.

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.

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.

A novel chemical lysis method for maximum release of DNA from difficult-to-lyse bacteria.

Molecular detection of microorganisms requires releasing DNA from cells. However, since certain microbial organisms are refractory to lysis by chemical or enzymatic methods, mechanical lysis by bead-beating is typically employed to disrupt difficult-to-lyse microbes. A newly developed chemical lysis method called sporeLYSE enables release of DNA from difficult-to-lyse microbes without bead-beating. The sporeLYSE method was compared to bead-beating and an alkaline/detergent lysis solution for releasing DNA from microbes grown in vitro, including surrogates of Category A bioterrorism agents. sporeLYSE released 83% to 100% of DNA from Mycobacterium smegmatis, Francisella philomiragia, Yersinia enterocolitica, Bacillus thuringiensis, Pseudomonas aeruginosa, Moraxella catarrhalis and Klebsiella pneumoniae. qPCR results indicated that sporeLYSE extracted an equal or greater amount of DNA than either bead-beating or alkaline/detergent lysis from Gram-positive and Gram-negative bacteria. When sporeLYSE was used to extract DNA from saliva and sputum spiked with M. smegmatis and M. tuberculosis, respectively, the qPCR Ct values were 4-8 cycles lower than those for extractions via alkaline/detergent lysis and heat. Mean Ct values for sporesLYSE extractions from spores of Clostridium difficile and C. botulinum were approximately two cycles lower than those of MagNA Pure DNA extractions. Our results suggest that sporeLYSE is an easy-to-use liquid reagent that can efficiently release large amounts of DNA from a variety of bacteria, including spores.

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.

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.

Two cases of adult botulism caused by botulinum neurotoxin producing Clostridium baratii.

Type F botulism occurs rarely in clinical cases. Two cases of type F botulism in elderly patients that were clustered in time and space are described. Clostridium baratii producing type F botulinum neurotoxin was isolated from both patients; molecular typing of these isolates revealed that they were unrelated strains.

Development of a Novel Serological Assay for the Detection of Mpox Infection in Vaccinated Populations.

In 2022 the World Health Organization declared a Public Health Emergency for an outbreak of mpox, the zoonotic Orthopoxvirus (OPV) affecting at least 103 non-endemic locations world-wide. Serologic detection of mpox infection is problematic, however, due to considerable antigenic and serologic cross-reactivity among OPVs and smallpox-vaccinated individuals. In this report, we developed a high-throughput multiplex microsphere immunoassay (MIA) using a combination of mpox-specific peptides and cross-reactive OPV proteins that results in the specific serologic detection of mpox infection with 93% sensitivity and 98% specificity. The New York State Non-Vaccinia Orthopoxvirus Microsphere Immunoassay is an important diagnostic tool to detect subclinical mpox infection and understand the extent of mpox spread in the community through retrospective analysis.

Secondary and tertiary transmission of vaccinia virus from US military service member.

During February and March 2010, the New York State Department of Health investigated secondary and tertiary vaccinia contact transmission from a military vaccinee to 4 close contacts. Identification of these cases underscores the need for strict adherence to postvaccination infection control guidance to avoid transmission of the live virus.

Different substrate recognition requirements for cleavage of synaptobrevin-2 by Clostridium baratii and Clostridium botulinum type F neurotoxins.

Botulinum neurotoxins (BoNTs) cause botulism, which can be fatal if it is untreated. BoNTs cleave proteins necessary for nerve transmission, resulting in paralysis. The in vivo protein target has been reported for all seven serotypes of BoNT, i.e., serotypes A to G. Knowledge of the cleavage sites has led to the development of several assays to detect BoNT based on its ability to cleave a peptide substrate derived from its in vivo protein target. Most serotypes of BoNT can be subdivided into subtypes, and previously, we demonstrated that three of the currently known subtypes of BoNT/F cleave a peptide substrate, a shortened version of synaptobrevin-2, between Q58 and K59. However, our research indicated that Clostridium baratii type F toxin did not cleave this peptide. In this study, we detail experiments demonstrating that Clostridium baratii type F toxin cleaves recombinant synaptobrevin-2 in the same location as that cleaved by proteolytic F toxin. In addition, we demonstrate that Clostridium baratii type F toxin can cleave a peptide substrate based on the sequence of synaptobrevin-2. This peptide substrate is an N-terminal extension of the original peptide substrate used for detection of other BoNT/F toxins and can be used to detect four of the currently known BoNT/F subtypes by mass spectrometry.

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.

Diagnosis of Zika Virus Infection by Peptide Array and Enzyme-Linked Immunosorbent Assay.

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.

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.

Development and evaluation of a 4-target multiplex real-time polymerase chain reaction assay for the detection and characterization of Yersinia pestis.

A multiplexed, 4-target real-time polymerase chain reaction (PCR) assay for the detection and characterization of Yersinia pestis was designed and optimized for respiratory and environmental samples. The target sequences include the entF3 gene of the chromosome, pla (plasminogen activator) on the pPCP1 virulence plasmid, caf1 (F1 capsule antigen) on the pMT1 virulence plasmid, and a region located on the pCD1 plasmid. The sensitivity of this assay was determined to be less than 85 CFU per reaction for each specimen type analyzed. This assay was also determined to be 100% specific with strains of Y. pestis, 9 additional Yersinia species, and related enteric and respiratory organisms. The results show that this multiplex real-time PCR assay using TaqMan(R) (Roche Molecular Systems, Inc., Alameda, CA) chemistry is sensitive and specific, requires minimal sample input, and can yield results in approximately 4 h. This assay is the first 4-target multiplex real-time PCR assay for Y. pestis in which detection and virulence assessment of Y. pestis can occur in one reaction, from clinical and environmental samples.

The CODE RED Solution: biothreat response training for first responders.

The terrorist events of 2001 brought to light the need for a close working relationship between the first responder communities and the public health laboratories in New York State (NYS). Since 2002, the Wadsworth Center's Biodefense Laboratory (BDL) has been providing outreach training to first responders in New York, to enable them to respond safely, correctly, and confidently to biothreat events. A pocket trifold was developed, titled "CODE RED," which describes sampling protocols, risk analysis criteria, and important contact information for use during an emergency response to a potential bioterrorism situation. In addition, the BDL has provided training to more than 1,000 first responders in the basic knowledge of biothreat agents, routes of dissemination, sampling and decontamination methods, contamination control protocols, biothreat risk assessment, and legal chain of custody procedures. The training methods have been established for use by first responders wearing personal protective equipment (PPE). All states can benefit from highly trained first responders who are capable of efficient, safe, and effective biothreat response, resulting in increased safety of the first responders and laboratorians, as well as decreased turnaround times for laboratory results. The CODE RED trifold provides a working model for training first responders at the state and county levels for emergency biothreat response.

Multiplex diagnostic platforms for detection of biothreat agents.

The availability of rapid, sensitive and cost-effective diagnostic methods is paramount to the success of a comprehensive national health security system in the USA. The national networks that were established to safeguard US infrastructures (e.g., public health, livestock, agriculture and water supply) have developed sufficient capability and capacity for monitoring. However, additional advanced methods will be required to maintain operational readiness. Currently available methods, although sensitive and specific, are generally costly and not amenable to high-throughput analyses. Critical to the success of biothreat surveillance is the ability to screen for and detect multiple agents rapidly in a single reaction and with minimal sample processing. This review will examine currently available diagnostic platforms (i.e., PCR-, immuno- and array-based) and biosensors that can detect multiple biothreat analytes in a single reaction (i.e., multiplex assays). The maturity, benefits and limitations of each platform will be described and a prospective view, from current to future state of the art, will be proposed.

Sylvatic typhus associated with flying squirrels (Glaucomys volans) in New York State, United States.

Sylvatic typhus is an infrequent, potentially life-threatening emerging zoonotic disease. In January of 2009, the New York State Department of Health was notified of a familial cluster of two suspected cases. Due to the paucity of typhus cases in New York, epidemiologic and environmental investigations were conducted to establish rickettsial etiology and determine potential sources of infection. Patients presented with symptoms consistent with typhus, and serologic testing of each patient confirmed infection with typhus group rickettsiae. Serologic analysis of blood obtained from southern flying squirrels (Glaucomys volans) captured from the attic crawlspace above an enclosed front porch of the cases' residence indicated evidence of infection with Rickettsia prowazekii, with 100% seroprevalence (n=11). Both patients reported spending significant time on the porch and hearing animal activity above the ceiling prior to onset of illness, implicating these flying squirrels as the likely source of infection.