Lab Matters Fall 2024 | Page 68

APHL 2024 POSTER ABSTRACTS

A collaborative effort is currently underway between the US Centers for Disease Control and Prevention , Association of Public Health Laboratories , the Johns Hopkins University Applied Physics Laboratory and several state public health laboratories to develop and optimize laboratory workflows and a bioinformatics pipeline for pathogen agnostic detection using direct-from-specimen metagenomic sequencing of various specimen types . These workflows will provide public health laboratories the ability to screen specimens for a wide variety of pathogens using platforms already available in most public health labs .

In addition to the lab-based specimen workflows , this project also includes development of a standardized , open-sourced metagenomics pipeline for public health laboratory use for identification of pathogens using metagenomic sequencing techniques . Bioinformaticians are in high demand and development of a bioinformatics pipeline ( multiple software scripts / packages run serially or in tandem to quality control and process raw sequence data ) will allow public health laboratories to better leverage directfrom-specimen next-generation sequencing methods while reducing need for dedicated bioinformatics staff . Additionally , implementation flexibilities ( e . g ., cloud-based or local , Linux or Windows ) will allow for wider adoption and capabilities without requiring local highpower computing resources .

These methods can be leveraged by public health laboratories for a variety of use cases ( e . g ., facilitating outbreak investigations , serving as a stop-gap diagnostic for novel pathogens , characterizing genomes of novel or modified organisms ). Additionally , these and other methods ( e . g ., massively multiplexed assays , targeted sequencing panels ) can help augment existing or yet-tobe developed surveillance and early warning systems to improve frontline detection capabilities for new emerging pathogens for which no current tests exist .

Presenter : Tyler Wolford , tyler . wolford @ aphl . org

Development and Evaluation of a Multiplex Microsphere Immunoassay for the Detection of Antibodies Consistent with Travel-associated Arboviral Infections

K . Howard , L . Jones , K . Carson , A . Ciota , A . Dupuis , K . Kulas , D . Hunt , T . Lamson , J . Yates , W . Lee , New York State Department of Health-Wadsworth Center

The Diagnostic Immunology Laboratory ( DIL ) at the Wadsworth Center ( New York State Department of Health ) performs serology testing for a wide range of infectious diseases , including multiple vector-borne infections . Owing to the large number of travelers and immigrants in New York , the Wadsworth Center test catalogue offers a wide range of molecular and serologic testing for infectious diseases associated with travel . For example , in 2023 , the DIL received 297 requests for clinical or confirmatory serology testing for travel-associated infections , primarily arboviruses such as , Dengue viruses ( DENV ), Zika virus ( ZIKV ) and Chikungunya virus ( CHIKV ). Commonly , testing requests come with no specific pathogen request , but with associated vague symptomology ( e . g ., fever , joint pain , etc .) or encephalitic symptoms and a recent history of travel . Further , testing for these viral infections , as well as testing for endemic arboviruses ( e . g ., West Nile virus , Powassan virus ) may be confounded by prior vaccination for Yellow Fever virus ( YFV ). To facilitate arboviral serology testing , the DIL has developed a broad multiplexed microsphere immunoassay ( MIA ) screen for travelassociated virus using a Luminex™ platform . Recombinant protein antigens derived from ZIKV ( E , NS1 ), DENV 1-4 ( NS1 ), YFV ( NS1 ) and CHIKV ( E1 and E2 ) were coupled to magnetic microspheres to form the solid phase of the MIA . Each antigen-microsphere complex had a unique fluorescence signature making the antigens individually identifiable in the assay . Using minimal patient serum diluted 1:00 and a phycoerythrin ( PE ) -conjugated anti-human Ig ( detecting human IgG , IgM and IgA ), specific antibodies to each analyte could be evaluated using a Luminex FlexMap 3D™ . Results , expressed as median fluorescence intensity ( MFI ), are based upon the degree of PE fluorescence associated with each antigenmicrobead complex . Cutoffs for each antigen-microsphere were determined using a panel of 94 normal ( noninfected ) human sera and a defined set of known positive sera , determined by polymerase chain reaction or a plaque reduction neutralization assay ( PRNT ) for the given analyte , followed by the generation of receiver operating characteristic ( ROC ) curves . The MFIs of the specimen-antigenmicrobead complex were related to the cutoff values to determine Reactivity / Non-reactivity . As there is considerable cross-reactive among arboviruses , particularly among the flaviviruses in this assay ( DENV1-4 , ZIKV , YFV ), an algorithm was developed to guide interpretations . The algorithm largely relied upon MFIs associated with non-envelope antigens , where there is markedly less crossreactivity . The performance characteristics for each individual pathogen were determined using a panel of positive , negative and infectious ( non-arboviral , including autoimmune ) sera . In the overall DIL testing scheme , the MIA screen helps to inform reflexed capture IgM ELISA testing , requests for convalescent serum specimens and confirmation by plaque reduction neutralization testing .

Presenter : Kelly Howard , kelly . howard @ health . ny . gov

Development and Validation of a Multiplex Real-Time PCR Assay for Rapid Detection of Blastomyces dermatitidis , Blastomyces gilchristii and Histoplasma capsulatum

B . O ’ Brien , Y . Zhu , S . Chaturvedi , Wadsworth Center , New York State Department of Health

Background : Blastomycosis due to Blastomyces dermatitidis ( Bd ) and B . gilchristii ( Bg ) and histoplasmosis due to Histoplasma capsulatum ( Hc ) are significant cause of respiratory mycosis in North America with occasional reported outbreaks . The rapid identification systems are key for patient care as these fungi require an extended time to grow in culture . Therefore , the aim of this investigation is to develop a multiplex TaqMan-based real-time PCR assay for culture identification and direct detection of Bd , Bg and Hc from primary specimens .

Methods : We used our previously published primers and probes targeting BAD1 gene for Bd and Bg . The primers and probe targeting Msp gene for Hc were designed in this investigation . The Bg probe was tagged with FAM , Bd probe was tagged with Cy5 and Hc probe was tagged with HEX ( VIC ). One strain each of Bd , Bg and Hc was used for initial standardization of multiplex rt-PCR assay followed by assay sensitivity , specificity and reproducibility . Sensitivity was done by testing serial dilutions of DNA of each organism over six orders of magnitude to determine the limit of detection ( LOD ). Reproducibility was assessed by testing three DNA dilutions comprising low , medium and high of each organism in triplicate over three separate