Taqman PACMAN: a simple molecular approach for positive rapid antigen test confirmation during periods of low prevalence.

Antigen-based rapid diagnostic tests (Ag-RDTs) were widely deployed to enhance SARS-CoV-2 testing capacity during the COVID-19 pandemic. Consistent with national guidance for low prevalence settings, positive Ag-RDTs were confirmed using nucleic acid amplification tests (NAATs) to avoid false positive results. However, increasing demands for positive Ag-RDT confirmation competed with other testing priorities in clinical laboratories. This work hypothesized that real-time RT-PCR without nucleic acid extraction (NAE) would be sufficiently sensitive to support positive Ag-RDT confirmation. Ag-RDT and NAAT results from community-based asymptomatic testing sites prior to the omicron variant wave were compared to calculate the weekly false positive rate (FPR) and false detection rate (FDR). Real-time RT-PCR was compared with and without NAE using 752 specimens previously tested positive for SARS-CoV-2 using commercial NAATs and 344 specimens from Ag-RDT-positive individuals. The impact of SARS-CoV-2 prevalence on laboratory resources required to sustain Ag-RDT confirmation was modeled for the RT-PCR with and without NAE. Overall, FPR was low [0.07% (222/330,763)] in asymptomatic testing sites, but FDR was high [30.7% (222/724)]. When RT-PCR was compared with and without NAE, 100% concordance was obtained with NAAT-positive specimens, including those from Ag-RDT-positive individuals. NAE-free RT-PCR significantly reduced time to results, human resources, and overall costs. A 30.7% FDR reaffirms the need for NAAT-based confirmation of positive Ag-RDT results during low SARS-CoV-2 prevalence. NAE-free RT-PCR was shown to be a simple and cost-sparing NAAT-based solution for positive Ag-RDT confirmation, and its implementation supported data-driven broader Ag-RDT deployment into communities, workplaces, and households. IMPORTANCE: Rapid antigen testing for SARS-CoV-2 was widely deployed during the COVID-19 pandemic. In settings of low prevalence, national guidance recommends that positive antigen test results be confirmed with molecular testing. Given the high testing burden on clinical laboratories during the COVID-19 pandemic, the high volume of positive antigen tests submitted for confirmatory testing posed challenges for laboratory workflow. This study demonstrated that a simple PCR method without prior nucleic acid purification is an accurate and cost-effective solution for positive rapid antigen test confirmation. Implementing this method allowed molecular confirmatory testing for positive antigen tests to be sustained as antigen testing was expanded into large populations such as workplaces, schools, and households.

Exploring alternative swabs for use in SARS-CoV-2 detection from the oropharynx and anterior nares.

The COVID-19 pandemic has led to a worldwide shortage of nasopharyngeal swabs and universal transport media. This study evaluated a combined oropharynx/nares (OP/Na) sample collection using two readily-available non-flocked swabs, transported in phosphate-buffered saline, and demonstrates equivalent performance in SARS-CoV-2 detection compared to a previously-validated OP/Na collection kit.

A combined oropharyngeal/nares swab is a suitable alternative to nasopharyngeal swabs for the detection of SARS-CoV-2.

Given the global shortage of nasopharyngeal (NP) swabs typically used for respiratory virus detection, alternative collection methods were evaluated during the COVID-19 pandemic. This study showed that a combined oropharyngeal/nares swab is a suitable alternative to NP swabs for the detection of SARS-CoV-2, with sensitivities of 91.7% and 94.4%, respectively.

Comparison of two automated instruments for Epstein-Barr virus serology in a large adult hospital and implementation of an Epstein-Barr virus nuclear antigen-based testing algorithm.

Serology remains the mainstay for diagnosis of Epstein-Barr virus (EBV) infection. This study compared two automated platforms (BioPlex 2200 and Architect i2000SR) to test three EBV serological markers: viral capsid antigen (VCA) immunoglobulins of class M (IgM), VCA immunoglobulins of class G (IgG) and EBV nuclear antigen-1 (EBNA-1) IgG. Using sera from 65 patients at various stages of EBV disease, BioPlex demonstrated near-perfect agreement for all EBV markers compared to a consensus reference. The agreement for Architect was near-perfect for VCA IgG and EBNA-1 IgG, and substantial for VCA IgM despite five equivocal results. Since the majority of testing in our hospital was from adults with EBNA-1 IgG positive results, post-implementation analysis of an EBNA-based algorithm showed advantages over parallel testing of the three serologic markers. This small verification demonstrated that both automated systems for EBV serology had good performance for all EBV markers, and an EBNA-based testing algorithm is ideal for an adult hospital.

Cross-reactivity between Lyme and syphilis screening assays: Lyme disease does not cause false-positive syphilis screens.

Increased rates of Lyme disease and syphilis in the same geographic area prompted an assessment of screening test cross-reactivity. This study supports the previously described cross-reactivity of Lyme screening among syphilis-positive sera and reports evidence against the possibility of false-positive syphilis screening tests resulting from previous Borrelia burgdorferi infection.

Pushing the limits of chemistry point-of-care testing for the management of patients under investigation for Ebola virus disease.

BACKGROUND: With the recent outbreak in West Africa, hospitals worldwide have been developing protocols for suspect of cases of Ebola virus disease. Patients with Ebola virus disease present with a severe gastroenteritis leading to dehydration and electrolyte abnormalities and as such, routine chemistry analysis is essential for patient management. While point-of-care testing can be used with additional precautions for rapid chemistry analyses in a laboratory setting, significant delays could ensue before specimens arrive to the laboratory. This study evaluated the stability of eight chemistry analytes up to 4 h post-collection. METHODS: Blood was collected by venipuncture from 20 healthy volunteers and tested at times 0, 30, 60, 90, 120 and 240 h. Approximately 100 µl of blood was dispensed into a CHEM 8+Cartridge and processed on a model 300 i-STAT 1 Analyzer (Abbott Point of Care Inc.) and ANOVA was used to assess statistical significant difference from the initial time point. RESULTS: While the manufacturer recommends testing within 30 min of collection, no significant variation was observed for most analytes with time points extending up to 4 h. In contrast, glucose concentrations decreased significantly (P < 0.0001) over time at an average rate of 0.0032 mmol/L per min. CONCLUSIONS: This study provides supporting data suggesting that delays up to 4 h can be tolerated, giving ample time for collection and transport of specimens to the clinical laboratory. For glucose, POC testing could still be used, taking into account the collection time and the average rate of decrease.

Molecular detection of varicella zoster virus while keeping an eye on the budget.

Varicella zoster virus (VZV) PCR is highly sensitive compared to traditional detection methods like culture and direct fluorescent antibody testing (DFA); however, the high cost of commercial assays prohibits their use in many clinical laboratories. Major contributors to cost are the nucleic acid extraction and the PCR reagents. This study evaluated an "in-house" qualitative real-time PCR where the nucleic acid extraction was replaced by a crude extraction, homogenization and heat treatment. Three methods were compared: virus culture and DFA and real-time PCR following each extraction methods. The real-time PCR was highly specific for VZV, and the analytical sensitivity was equivalent following both extraction methods. In contrast, virus culture and DFA was approximately 10,000-fold less sensitive. Using 200 clinical specimens, the sensitivity for the real-time PCR following nucleic acid extraction or homogenization and heat treatment was essentially equivalent at 100% and 97.2%, respectively; whereas, virus culture and DFA was significantly less sensitive at 54.8%. Overall, homogenization and heat treatment combined with a qualitative in-house real-time PCR is a rapid, accurate and cost effective method for the detection of VZV.

Homogenization with heat treatment: A cost effective alternative to nucleic acid extraction for herpes simplex virus real-time PCR from viral swabs.

Most laboratories use expensive commercial kits to purify nucleic acids and remove PCR inhibitors that may be present in clinical specimens. In this study a simple homogenization with heat treatment of herpes simplex virus types 1 and 2 (HSV-1/2) was shown to be equivalent to commercial kit-based nucleic acid extraction methods. With a cost of less than $1 USD per extraction, this method provides an economical, rapid, and effective method to recover HSV-1/2 DNA from swabs suitable for real-time HSV PCR.