Mpox Viral Lineage Analysis and Technique Development Using Next-generation Sequencing Approach.

BACKGROUND: In response to Mpox endemic and public health emergency, DCHHS aimed to develop NGS based techniques to streamline Mpox viral clade and lineage analysis. METHODS: The Mpox sequencing workflow started with DNA extraction and adapted Illumina's COVIDSeq assay using hMpox primer pools from Yale School of Public Health. Sequencing steps included cDNA amplification, tagmentation, PCR indexing, pooling libraries, sequencing on MiSeq, data analysis, and report generation. The bioinformatic analysis comprised read assembly and consensus sequence mapping to reference genomes and variant identification, and utilized pipelines including Illumina BaseSpace, NextClade, CLC Workbench, Terra.bio for data quality control (QC) and validation. RESULTS: In total, 171 mpox samples were sequenced using modified COVIDSeq workflow and QC metrics were assessed for read quality, depth, and coverage. Multiple analysis pipelines identified the West African clade IIb as the only clade during peak Mpox infection from July through October 2022. Analyses also indicated lineage B.1.2 as the dominant variant comprising the majority of Mpox viral genomes (77.7%), implying its geographical distribution in the United States. Viral sequences were uploaded to GISAID EpiPox. CONCLUSIONS: We developed NGS workflows to precisely detect and analyze mpox viral clade and lineages aiding in public health genomic surveillance.

Comparison of an ID NOW COVID-19 Assay Used at the Point of Care to Laboratory-Based Nucleic Acid Amplification Tests.

The emergence of a novel coronavirus, namely, SARS-CoV-2, necessitated the use of rapid, accurate diagnostics to quickly diagnose COVID-19. This need has increased with the emergence of new variants and continued waves of COVID-19 cases. The ID NOW COVID-19 assay is a rapid nucleic acid amplification test (NAAT) that is used by hospitals, urgent care facilities, medical clinics, and public health laboratories for rapid molecular SARS-CoV-2 testing at the point of care. The District of Columbia Department of Forensic Sciences Public Health Laboratory Division (DC DFS PHL) implemented ID NOW COVID-19 testing in nontraditional laboratory settings, including a mobile testing unit, health clinic, and emergency department, to assist with rapid identification and isolation for populations at high risk of SARS-CoV-2 transmission in the District of Columbia. The DC DFS PHL provided these nontraditional laboratories with safety risk assessment, assay training, competency assessment, and quality control monitoring as parts of a comprehensive quality management system (QMS). We assessed the accuracy of the ID NOW COVID-19 assay when operated in the context of these trainings and systems. This was done by comparing results from 9,518 paired tests, and strong agreement (κ = 0.88, OPA = 98.3%) was found between the ID NOW COVID-19 assay and laboratory-based NAATs. These findings indicate that the ID NOW COVID-19 assay can be used to detect SARS-CoV-2 in nontraditional laboratory settings when used within the context of a comprehensive QMS.

Genuine and counterfeit prescription pill surveillance in Washington, D.C.

For the first time in Washington, D.C., an in-depth analysis of counterfeit pills has been performed as part of a larger initiative to understand the city's illicit drug supply. Over a 56-month period, 567 pills that were physically identified as a pharmaceutical were analyzed using gas chromatography mass spectrometry (GC-MS) and gas chromatography flame ionization detection (GC-FID). Out of the 567 pills submitted to our laboratory, 119 were confirmed to be counterfeit. Beginning in 2018, an increase in counterfeit pills was observed in suspected pharmaceutical submissions. By 2021, 62.5% of all pill exhibits were determined to be counterfeit. Most of the counterfeit pills submitted during this time frame had a '30M' imprint with blue coloring, consistent with the physical identification of a 30 mg Oxycodone tablet. Fentanyl was the number one identified psychoactive substance detected in counterfeit pills (75.4%), however, other opioids, precursors, and a novel benzodiazepine were also identified. This preliminary research hopes to illustrate counterfeit pill trends in Washington, D.C. and highlight the importance of analyzing pharmaceuticals in addition to suspected illicit substances. This surveillance is ongoing and collaboration with neighboring jurisdictions is anticipated in the future.

Photoemission ambient pressure ionization (PAPI) with an ultraviolet light emitting diode and detection of organic compounds.

The development of compact, rugged and low-power ion sources is critical for the further advancement of handheld mass analyzers. Further, there is a need to replace the common (63)Ni source used at atmospheric pressure with a non-radioactive substitute. We present here a description of a light emitting diode (LED) photoemission ionization source for use in mass spectrometry for the detection of volatile organic compounds. This technique relies upon the generation of photoelectrons from a low-work function metal via low-energy ultraviolet (UV) light (280 or 240 nm) generated by a single LED in air at atmospheric pressure. These low-energy photoelectrons result in either direct electron capture by the analyte or chemical ionization. Currently, only negative ions are demonstrated due to operation at atmospheric pressure. Ion generation occurs without use of high electric fields such as those found in corona discharge or electrospray ionization. This source is effective for measuring organic vapors from gases, liquids and surface residues via atmospheric pressure chemical ionization, initiated by photoemission off a conductive surface. Several classes of organic vapors are analyzed and found to be effectively detected, including compounds that ionize via electron attachment, dissociative electron capture, proton abstraction, adduct formation and replacement ionization.

Analysis of drug residue in needle-exchange syringes in Washington, D.C.

For the first time in Washington, D.C., an analysis of drug residue from used needle-exchange syringes has been performed. This analysis is part of a larger initiative to understand the District of Columbia's illicit drug supply and its intravenous (IV) user's consumption trends as our nation faces the opioid epidemic. The goal of this study is to develop a more comprehensive monitoring program that provides real-time analysis necessary for public health organizations, in addition to providing initial observations of drugs detected. A total of 1187 syringes were analyzed over a period of nine months. Of these, 732 syringes (61.7%) were confirmed to contain a controlled dangerous substance (CDS). Fentanyl was detected in 490 syringes, the most observed CDS in all syringes analyzed. Heroin was the second most detected CDS, observed in 192 syringes. The third most detected CDS was cocaine, which was observed in 132 syringes, followed by the fourth most detected CDS, methamphetamine, observed in 82 syringes. Novel findings of this study include the first reported detections of methamphetamine, synthetic cathinones, and synthetic cannabinoids in used syringes in D.C. Ninety-seven syringes that contained no CDS contained a non-controlled substance of interest, such as diphenhydramine, xylazine, and etizolam. One limitation of this study is that this method cannot determine whether mixtures present in syringes stem from mixtures present prior to injection, back-to-back usage, or sharing of needles. This preliminary study illustrates the strength of surveillance to monitor drug trends and can be used to detect emerging novel dangerous substances in the future.

APPI-MS: effects of mobile phases and VUV lamps on the detection of PAH compounds.

The technique of atmospheric pressure photoionization (APPI) has several advantages over electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI), including efficient ionization of nonpolar or low charge affinity compounds, reduced susceptibility to ion suppression, high sensitivity, and large linear dynamic range. These benefits are greatest at low flow rates (i.e.,

Liquid chromatography-atmospheric pressure photoionization-mass spectrometry analysis of triacylglycerol lipids--effects of mobile phases on sensitivity.

In this work, we evaluate the performance of liquid chromatography-atmospheric pressure photoionization-mass spectrometry (LC-APPI-MS) for non-aqueous reversed phase analysis of six triacylglycerol model compounds using six binary mobile phases including MeOH/iPrOH, MeOH/CHCl(3), MeOH/CH(2)Cl(2), CH(3)CN/iPrOH, CH(3)CN/CHCl(3), and CH(3)CN/CH(2)Cl(2). All mobile phases give comparably good separation performance on a Gemini C(18) column with carefully adjusted gradient elution programs. APPI sensitivity varies from one mobile phase to the other without dopants; however use of dopants brings sensitivity to comparable levels for all mobile phases. MeOH/iPrOH offers high sensitivity without dopants due to self-doping effect and dopants are not necessary for this mobile phase. Dopants enhance analyte sensitivity to a varying degree for each of the mobile phases tested. Photo-induced chemical ionization (PCI) of solvent may play a significant role in achieving high sensitivity. Two critical parameters affecting sensitivity are photoabsorption cross-sections and ionization potentials of mobile phase solvents. How these mobile phase solvents affect APPI sensitivity and their dependency on dopant use are discussed. All six mobile phases offer comparable overall limits of detection for the analytes tested. These results indicate that LC-APPI-MS is a successful tool for neutral lipid analysis, giving high sensitivity with a variety of non-aqueous mobile phases.

Simultaneous measurement of nitric oxide (NO) and nitrogen dioxide (NO2) in simulated automobile exhaust using medium pressure ionization-mass spectrometry.

In previous papers we have demonstrated two different, two-color resonance-enhanced multiphoton ionization (REMPI) schemes for the simultaneous measurement of trace amounts (ppbV to pptV) of nitrogen monoxide (NO) and nitrogen dioxide (NO(2)). The goal of this study is to provide a laser ionization-mass spectrometric scheme capable of measuring ppmV to ppthV concentrations of NO and NO(2) within vehicle exhaust containing up to ppthV of aromatic hydrocarbons and a time frame of seconds. Two ionization schemes are used here to measure NO and NO(2) in simulated automobile exhaust with three different sources. REMPI Scheme 1 uses broad-bandwidth light and an effusive source to measure NO (limit of detection (LOD) 300 ppmV), NO(2) (LOD 100 ppmV), and aromatic hydrocarbons (via photoionization) along with fragments (via electron impact). REMPI Scheme 2 uses narrow-bandwidth light and a medium pressure laser ionization (MPLI) source to measure NO (LOD 60 ppmV), NO(2) (LOD 3 ppmV), and fragments (via electron impact). The LOD is determined using 10-second sampling times. A newly developed delayed-ion extraction technique for MPLI is then applied to REMPI Scheme 2, dramatically reducing the electron impact signal, so that only NO and NO(2) are observed. We conclude that Scheme 2 with delayed-electron extraction is best suited for measuring in situ NO and NO(2) within engine exhaust.

Real-time analysis of exhaled breath via resonance-enhanced multiphoton ionization-mass spectrometry with a medium pressure laser ionization source: observed nitric oxide profile.

An elevated concentration of nitric oxide (NO) in alveolar ventilation is indicative of inflammatory stress within the lung. We present here the first description of time-resolved measurement of NO in breath using photoionization mass spectrometry, providing new capabilities for the medical investigator, such as isotopic tracing. Here we use resonance-enhanced multiphoton ionization (REMPI) with time-of-flight mass spectrometry (TOF-MS) coupled with a medium pressure laser ionization (MPLI) source for the selective detection of NO in breath. To demonstrate this technology, a single male subject breathes NO-free air for several minutes, and then the exhaled breath is monitored. The ability of REMPI to differentiate among three different isotopomers of NO is demonstrated, and then the concentration profile of NO in exhaled breath is measured. A similar time-dependence concentration is found as observed by previous techniques. The advantages of this approach compared to other techniques are: (1) parts-per-billion by volume (ppbV) mixing ratios of NO can be measured on a sub-second time scale, (2) since the technique operates optically as well as mass-resolved, isotopomers of NO are discernable, permitting the use of isotopic tracing, and (3) other biologically significant gas molecules can be measured via REMPI.

Development of medium pressure laser ionization, MPLI. Description of the MPLI ion source.

A novel pulsed valve/ion source combination capable of time-resolved sampling from atmospheric pressure has been developed for use with laser ionization time of flight mass spectrometry. The source allows ionization extremely close to the nozzle of the pulsed valve, enabling ultra-sensitive detection of a number of compounds, e.g., NO, at mixing ratios <1 pptV. Furthermore, at analyte mixing ratios in the ppbV range, the temporal resolution of the system is in the sub-second regime, allowing time-resolved monitoring of highly dynamic and complex mixtures, e.g., human breath or reacting chemical mixtures in atmospheric smog chamber experiments. Rotational temperatures of approximately 50 K have been observed for analytes seeded in the supersonic jet expansion at a distance of 1 mm downstream of the nozzle orifice. The refinement of the original ion source has drastically reduced the impact of reflected laser light and the resultant electron impact signals previously observed. The general applicability of this technique is demonstrated here by coupling the source to commercially available as well as home-built time-of-flight mass spectrometers. Finally, we discuss the MPLI technique in view of the very recently introduced atmospheric pressure laser ionization (APLI) as well as the traditional jet-REMPI approach.

Electrospray photoionization (ESPI) liquid chromatography/mass spectrometry for the simultaneous analysis of cyclodextrin and pharmaceuticals and their binding interactions.

We report on the use of a multimode electrospray ionization/atmospheric pressure photoionization source (ESI/APPI or ESPI for short) with liquid chromatography/mass spectrometry (LC/MS) to measure all components of a mixed-polarity liquid sample containing: (1) low-polarity component (hormone, pharmaceutical or sterol), (2) polar component (cyclodextrin substrate), and (3) bound polar complex. The ESPI source has several advantages over both single ESI and multimode electrospray ionization/chemical ionization (ESCI) analysis, including an enhanced bound-complex detection and better performance at lower solvent flow rates. Relative binding constants are determined with (i) ESI mode, resulting in relative R(ESI-MS) values, and (ii) both ESI and APPI modes, providing relative K(D) values. We find that low molecular-substitution (Ms) values of cyclodextrin, i.e., Ms = 0.4, preferentially bind to the low-polarity compounds tested. This investigation is intended to demonstrate the feasibility of ESPI as an additional tool for investigating mixed-polarity binding systems, providing mass-specific data for all solution components, both polar and non-polar.

Electrospray ionization/atmospheric pressure photoionization multimode source for low-flow liquid chromatography/mass spectrometric analysis.

Analysis of several polar and non-polar compounds is performed with a newly developed dual electrospray ionization/atmospheric pressure photoionization (ESI/APPI) or ESPI source. Several variables are considered in the source, such as ESI probe heater temperature, solvent flow, dopant effects, repeller plate voltage, source geometry and photon energy (Kr vs. Ar lamp). Direct photoionization resulting in a molecular radical cation [M](*+) dominates at high temperatures (>400 degrees C) and low flow rates (<200 microL/min). Indirect photo-induced chemical ionization (PCI) involving solvent molecules becomes important at lower temperatures and higher solvent flow rates. Indirect PCI is enhanced using an Ar lamp, which yields comparable [M+H](+) signal but poorer [M](*+) signal than the Kr lamp at lower temperatures and higher flow rates. This is in support of our recent finding that the Ar lamp results in a solvent-dependent enhancement of analyte molecules via PCI. Analysis of 12 compounds in methanol under low-flow conditions (10 microL/min) demonstrates that the dual ESPI source performs favorably for most compounds versus the standard ESCI source, and significantly better than ESCI for the analysis of unstable drugs, like flurbiprofen. Several factors contributing to the benefits of the ESPI source are the shared optimal geometry for ESI and APPI sources and soft ionization of APPI versus APCI.

Selective measurement of HCHO in urine using direct liquid-phase fluorimetric analysis.

Quantification of formaldehyde (HCHO) in urine was recently shown to be a promising tool in the investigation of cancer, particularly bladder cancer. Development of a low-maintenance, inexpensive and rapid analyzer for HCHO in urine would greatly facilitate future research and the potential diagnosis of bladder cancer. We examine here the application of an off-the-shelf system, originally designed for gas-phase atmospheric monitoring of HCHO, for the quantification of HCHO in urine. Under strict dietary protocols, e.g., avoidance of foods rich in free or chemically bound HCHO, an increase in HCHO in urine is an indirect indicator of cancer in the urogenital system. The concentration of HCHO in urine samples from an individual over a several-month period was determined, with a range from 39 to 1400 microM and a mean of 600 microM. The limit of detection for the present method was 0.1 microM. The proposed technique provides a direct, low-cost and greatly simplified analytical method for the quantification of HCHO in urine compared to other available techniques.