Lab Matters Fall 2023 | Page 92

APHL 2023 POSTER ABSTRACTS

Successful Identification and Consensus Assembly of Mpox Genome using Direct DNA Sequencing Approach

B . Schwem , C . Barnes , N . Palmateer , K . Patel , L . Schlitt , A . Kowalsky , E . Acheampong , S . Verma , D . Woell , R . Siderits , T . Kirn , B . Jeong ; New Jersey Department of Health Public Health and Environmental Laboratories

Mpox virus is a member of the poxvirus family which can infect humans typically causing a skin rash and may cause death in rare cases . Historically , most mpox cases were confined to Africa , but in 2022 , a significant number of cases have been reported in the US . As of Jan 4 , 2023 , there have been 29,913 cases in the US , of which 764 cases occurred in New Jersey . As a part of New Jersey mpox surveillance , the NJ PHEL has received Mpox specimens and performed confirmatory qPCR testing and whole genome sequencing employing amplicon-based sequencing method . In addition , as a part of the NJ PHEL sequencing capability expansion , we also performed exploratory no-PCR direct DNA sequencing using the Oxford Nanopore Technology ( ONT ) sequencing platform , taking advantage of its long-read sequencing capability . Genome sequencing was conducted according to institutional SOPs . Briefly , the extracted DNA was subjected to a standard qPCR test for Mpox detection and the determination of Ct values and then sequenced using both PCR-amplicon and direct-DNA sequencing methods . Six Mpox-positive specimens with a range of Ct values between 15 and 25 were selected for the comparison of amplicon-based and DNA-based sequencing methods . Generated raw reads were subjected to a series of bioinformatic analysis tools for comparison . Our results show that all six samples were successfully sequenced and assembled to > 87 % genome coverage from the amplicon-based approach on Illumina platform whereas two of the samples with Ct ~ 25 failed to generate sufficient mpox-specific reads in direct-DNA sequencing approach on ONT platform . Assembly length in direct DNA method showed a correlation between genome coverage and qPCR Ct values . A near complete genome ( Mpox reference genome used is ~ 197kb ) was assembled from the samples with Ct ~ 15 in direct-DNA sequencing . As expected , the PCR ampliconbased method produced more complete assemblies in all 6 samples whereas direct-DNA sequencing generated high coverage assemblies in only 2 samples with the lowest Ct values . A taxonomic identification of the reads using Kraken2 showed that only 0.33- 0.47 % of the trimmed reads were classified as Mpox ( taxonomy id = 10244 ). A clade and lineage assignment by Nextclade also showed good agreement between the amplicon- and direct-DNAsequencing methods . Despite being more error prone , the direct DNA sequencing approach was able to successfully identify clade and the pathogen present . In summary , although further refinement in direct DNA sequencing is necessary , our exploratory work demonstrated that it is possible to assemble a target species using the native genomic DNA sequencing ( without PCR ) approach from a clinical specimen . This opens the possibility that this approach may be used for identification and assembly of potential pathogenic genome ( s ) presented in an unknown clinical sample using ONT platform ’ s long-read capability .

Presenter : Brian Schwem , brian . schwem @ doh . nj . gov

Validation of the bioMérieux EMAG ® Nucleic Acid Extraction Instrument for Non-variola Orthopox Real-time PCR

S . Veleker , A . Bateman , E . Hanson , R . Griesser ; Wisconsin State Laboratory of Hygiene

Mpox virus is a member of the genus Orthopoxvirus within the family Poxviridae . Infection with mpox presents clinically similar to other pox virus infections causing the formation of rash and pimple like lesions . Mpox is endemic to West and Central Africa and is a zoonotic disease . Rodents are believed to be the primary carrier or reservoir . May 2022 marked the beginning of a large number of positive cases of mpox clade II virus being reported in nonmpox endemic countries including the United States . On July 23 , 2022 the magnitude of this outbreak was realized and the WHO declared mpox a public health emergency . The primary test used in the U . S . for mpox response is the CDC non-Variola Orthopoxvirus ( NVO ) real-time PCR assay . This CDC developed test has FDA 510 ( k ) clearance and is used for the presumptive detection of non-Variola Orthopoxviruses , including Vaccinia , Cowpox , mpox , and Ectromelia viruses . Samples positive by the NVO test at state public health laboratories were sent to CDC for confirmation with a monkeypoxspecific PCR test . In summer 2022 the FDA provided enforcement discretion on a number of NVO-related issues , including allowing additional extraction supplies and instruments . However , the bioMérieux EMAG ® extraction instrument , the preferred extraction instrument at WSLH , was not included in this enforcement discretion . Benefits of the EMAG ® include its familiarity within the virus team , sensitive extraction ability on all viruses tested at WSLH to date , and ease of use within existing testing workflows . Therefore , following FDA EUA guidance , we validated the EMAG ® as a modification of the NVO assay , and to date we are the only laboratory in the country to perform a modification of an FDAcleared assay for mpox . To validate the EMAG ®, we evaluated the LOD , accuracy , specificity , precision , and reproducibility data of the NVO PCR test after EMAG ® extraction . Performance characteristics showed that the EMAG is acceptable for use with the NVO PCR assay . The accuracy panel performed resulted in 100 % concordance with known results and no carry-over of positive samples into negative samples . The limit of detection was calculated to be 39 copies / reaction . Taken together , the data show that the bioMérieux EMAG ® nucleic acid extraction instrument has comparable performance characteristics to Qiagen spin columns , and the EMAG ® is acceptable for extraction prior to NVO real-time PCR .

Presenter : Sylvia Veleker , sveleker93 @ gmail . com

Wastewater-based Epidemiology for Human Influenza A and B , Parainfluenza , Metapneumovirus , RSV , Seasonal Coronaviruses , Rhinovirus , Norovirus , Mpox , and SARS- CoV-2

A . Boehm 1 , M . Wolfe 2 , B . White 3 , A . Lockwood 3 , B . Hughes 3 , D . Duong 3 , V . Chan-Herur 3 ; 1 Stanford University , 2 Emory University ,

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Verily Life Sciences LLC

In order to provide more complete data , especially representing individuals who do not seek healthcare and may not be included in traditional surveillance systems , additional methods for understanding respiratory , enteric , and emerging , novel disease dynamics are needed . Enhanced data can be used to better inform