Development and Validation of Three Automated High-Throughput Molecular Tests to Detect Monkeypox Virus Infections.

BACKGROUND: The 2022 outbreak of the clade IIb monkeypox virus and subsequent global spread lead to an urgent need for the development of high-throughput, sensitive, and reproducible diagnostic tests. METHODS: We developed 3 assays to detect monkeypox virus, 2 (MPXV+ and MPXV) for m2000 RealTime and 1 (MPXV) for Alinity m platforms. Dual targets in E9L and B6R (MPXV+) and J2L and B7R (MPXV) increased mutation resistance. In silico prediction indicates MPXV+ cross-reactivity with orthopox viruses and specific monkeypox virus detection with MPXV. RESULTS: m2000 RealTime MPXV+ and MPXV assay sensitivity was determined to be 3.2 plaque-forming units/mL using a reference virus culture diluted into universal transport medium (UTM). Alinity m MPXV lower limit of detection was 200 copies/mL using monkeypox virus plasmids in pooled UTM matrix. m2000 RealTime MPXV+ and MPXV assays were validated with lesion swabs in UTM and 1:1 saliva to UTM mixtures. Commercially available and remnant clinical lesion specimens in UTM were tested with RealTime MPXV+, RealTime MPXV and Alinity m MPXV assays and demonstrated high agreement to known mpox (MPX)-positive specimens. CONCLUSIONS: RealTime MPXV+, RealTime MPXV, and Alinity MPXV are high throughput and sensitive assays used for the detection of monkeypox virus. These assays maybe useful during MPX outbreaks.

Pre-analytical considerations in the development of a prototype SARS-CoV-2 antigen ARCHITECT automated immunoassay.

OBJECTIVES: To evaluate pre-analytical challenges related to high-volume central laboratory SARS-CoV-2 antigen testing with a prototype qualitative SARS-CoV-2 antigen immunoassay run on the automated Abbott ARCHITECT instrument. METHODS: Contrived positive and negative specimens and de-identified nasal and nasopharyngeal specimens in transport media were used to evaluate specimen and reagent on-board stability, assay analytical performance and interference, and clinical performance. RESULTS: TCID50/mL values were similar for specimens in various transport media. Inactivated positive clinical specimens and viral lysate (USA-WA1/2020) were positive on the prototype immunoassay. Within-laboratory imprecision was ≤0.10 SD (<1.00 S/C) with a ≤10% CV (≥1.00 S/C). Assay reagents were stable on board the instrument for 14 days. No high-dose hook effect was observed with a SARS-CoV-2 stock of Ct 13.0 (RLU>1.0 × 106). No interference was observed from mucin, whole blood, 12 drugs, and more than 20 cross-reactants. While specimen stability was limited at room temperature for specimens with or without viral inactivation, a single freeze/thaw cycle or long-term storage (>30 days) at -20 °C did not adversely impact specimen stability or assay performance. Specificity of the prototype SARS-CoV-2 antigen immunoassay was ≥98.5% and sensitivity was ≥89.5% across two ARCHITECT instruments. Assay sensitivity was inversely correlated with Ct and was similar to that reported for the Roche Elecsys® SARS-CoV-2 Ag immunoassay. CONCLUSIONS: The prototype SARS-CoV-2 antigen ARCHITECT immunoassay is sensitive and specific for detection of SARS-CoV-2 in nasal and nasopharyngeal specimens. Endogenous proteases in mucus may degrade the target antigen, which limits specimen storage and transport times and complicates assay workflow.

Functional analysis of the exported type IV HSP40 protein PfGECO in Plasmodium falciparum gametocytes.

During Plasmodium falciparum infection, host red blood cell (RBC) remodeling is required for the parasite's survival. Such modifications are mediated by the export of parasite proteins into the RBC that alter the architecture of the RBC membrane and enable cytoadherence. It is probable that some exported proteins also play a protective role against the host defense response. This may be of particular importance for the gametocyte stage of the life cycle that is responsible for malaria transmission, since the gametocyte remains in contact with blood as it proceeds through five morphological stages (I to V) during its 12-day maturation. Using microarray analysis, we identified several genes with encoded secretory or export sequences that were differentially expressed during early gametocytogenesis. One of these, PfGECO, encodes a predicted type IV heat shock protein 40 (HSP40) that we show is expressed in gametocyte stages I to IV and is exported to the RBC cytoplasm. HSPs are traditionally induced under stressful conditions to maintain homeostasis, but PfGECO expression was not increased upon heat shock, suggesting an alternate function. Targeted disruption of PfGECO indicated that the gene is not essential for gametocytogenesis in vitro, and quantitative reverse transcriptase PCR (RT-PCR) showed that there was no compensatory expression of the other type IV HSP40 genes. Although P. falciparum HSP40 members are implicated in the trafficking of proteins to the RBC surface, removal of PfGECO did not affect the targeting of other exported gametocyte proteins. This work has expanded the repertoire of known gametocyte-exported proteins to include a type IV HSP40, PfGECO.

Development of a new automated IL-6 immunoassay.

OBJECTIVES: Quantitative detection of interleukin-6 (IL-6) in serum and plasma can help monitor immune responses and the development of acute inflammation to guide patient management. We developed an IL-6 immunoassay for use with the automated ARCHITECT system for detecting an increase in the inflammatory response. METHODS: Immunized mouse sera were tested and selected B-cells were harvested for fusion with myeloma cells. A panel of monoclonal antibodies were produced, from which capture and detection monoclonal antibodies for the prototype IL-6 immunoassay were selected and screened on the ARCHITECT instrument. The antibody pair that most effectively captured and detected IL-6 was selected to develop a prototype IL-6 immunoassay. Calibrator and panel preparations using an internal recombinant IL-6 standard were compared to serum panels prepared with the IL-6 International Standard 89/548. Assay specificity and spike recovery were determined, and assay sensitivity was compared with the Roche EUA Elecsys IL-6 assay run on the cobas analyzer. RESULTS: Twenty-one antibodies in 441 antibody pairs were screened. The prototype IL-6 assay showed high sensitivity with an estimated limit of detection of 0.317 pg/mL and limit of quantitation of <1.27. Spike recovery was 90%-110% in serum and plasma. The internal recombinant human IL-6 calibrator showed excellent stability for 63 days at 2-8 °C. The prototype IL-6 immunoassay was specific for IL-6, exhibited no cross reactivity to related cytokines and interleukins, and was 10-fold more sensitive than the Elecsys IL-6 assay. CONCLUSIONS: The prototype ARCHITECT IL-6 automated immunoassay is a reliable and robust method for the quantitative determination of IL-6 in human serum and plasma.