Whole-Blood PCR Preferred for Timely Diagnosis of Neuroinvasive West Nile Virus Infections: Lessons From the 2021 Arizona Outbreak.

Background: In 2021, the state of Arizona experienced the largest focal outbreak of West Nile virus (WNV) in US history. Timely and accurate diagnostic testing remains a challenge for WNV due to transient viremia and limited immunoassay specificity. Recent studies have identified whole blood (WB) and urine as more sensitive specimen types for the detection of WNV RNA. Methods: We evaluated ordering practices, test performance, and patient characteristics of probable and confirmed cases. In total, we identified 190 probable and proven cases, including 127 patients (66.8%) with neuroinvasive disease. Results: Among all cases, only 29.5% had WNV polymerase chain reaction (PCR) testing ordered on WB, of which 80.3% resulted as positive, including 7 cases in which WNV serologic testing was negative and 5 cases for which serologic testing was not ordered. In comparison, only 23.7% of cases that had cerebrospinal fluid (CSF) PCR ordered had a positive result, including 3 cases that were negative by PCR on WB. In contrast, WNV PCR on WB detected 12 neuroinvasive cases that were CSF PCR negative. WNV PCR testing in urine was only ordered on 2 patients, both of whom were positive. Crossing cycle threshold (Ct) values were not significantly different between WB and CSF specimen types, nor was there a correlation between Ct value and days from symptom onset at the time of sample collection; all specimen types and time points had Ct values, with 98% above 30. WB was positive by WNV PCR in several patients for >7 days (range, 7-25 days) after symptom onset, as was the CSF PCR. Conclusions: Taken together, these findings indicate that WNV PCR testing on WB may be the best initial test for timely diagnosis of WNV infection, irrespective of clinical manifestation; however, if negative in patients with suspected neuroinvasive disease, WNV PCR testing on CSF should be ordered.

Development of a rapid lateral flow assay for detection of anti-coccidioidal antibodies.

Coccidioides spp. are dimorphic fungi that are capable of infecting human and non-human mammals and can cause diverse manifestations of coccidioidomycosis or Valley fever (VF). In combination with clinical symptoms and radiographic findings, antibody-based diagnostic tests are often used to diagnose and monitor patients with VF. Chitinase 1 (CTS1) has previously been identified as the seroreactive antigen used in these diagnostic assays to detect anticoccidial IgG. Here, an indirect enzyme-linked immunosorbent assay to detect IgG to CTS1 demonstrated 165 of 178 (92.7%) patients with a positive result by immunodiffusion (ID) and/or complement fixation (CF) had antibodies to the single antigen CTS1. We then developed a rapid antibody lateral flow assay (LFA) to detect anti-CTS1 antibodies. Out of 143 samples tested, the LFA showed 92.9% positive percent agreement [95% confidence interval (CI), 84.3%-96.9%] and 97.7% negative percent agreement (95% CI, 87.9%-99.6%) with ID and CF assays. Serum or plasma from canines, macaques, and dolphins was also tested by the CTS1 LFA. Test line densities of the CTS1 LFA correlated in a linear manner with the reported CF and ID titers for human and non-human samples, respectively. This 10-min point-of-care test for the rapid detection of anti-coccidioidal antibodies could help to inform healthcare providers in real-time, potentially improving the efficiency of healthcare delivery.

Thawed plasma (TP) as a substitute for intravenous immune globulin (IVIG) to prevent hypogammaglobulinemia post-therapeutic plasma exchange.

Intravenous immune globulin (IVIG) is a common treatment given after plasma exchange procedures to either prevent secondary hypogammaglobulinemia or as an adjunctive treatment for organ transplant rejection. However, side-effects are relatively common with this medication during and after infusion. This case-report describes our alternative to IVIG infusions post-plasma exchange. We hypothesize that in patients unable to tolerate IVIG, using thawed plasma as a replacement fluid provides a suitable increase in the patients post procedure immunoglobulin G (IgG) levels for patients with secondary hypogammaglobulinemia that are unable to tolerate IVIG infusions.

Clinical Laboratory Utility of a Humanized Antibody in Commercially Available Enzyme Immunoassays for Coccidioidomycosis.

Coccidioidomycosis, also called valley fever (VF), is a fungal infection with endemicity in desert regions of the western United States as well as certain arid regions of Central and South America. Laboratory-based diagnosis of VF often relies on the composite results from three serologic-based diagnostics, complement fixation, immunodiffusion, and enzyme immunoassay (EIA). EIA is commonly performed in clinical laboratories because results can be obtained in a few hours. Two commercially available EIAs, IMMY clarus Coccidioides antibody and Meridian Premier Coccidioides, look for the presence of anticoccidioidal IgG and IgM in patient sera that are diluted 1:441. Per regulatory requirements, this dilution step must be verified with a dilution step control despite not being provided as a reagent in either FDA-approved EIA kit. Therefore, clinical laboratories collect and reuse patient sera in subsequent tests that had a positive result in a previous test. This is a nonstandard process, reinforcing the need for a consistent and reliable dilution control. Here, we evaluate the performance of a humanized IgG and IgM antibody as a dilution control in both EIA kits. Both humanized IgG and IgM work well in each EIA and meet the appropriate threshold for positivity. IMPORTANCE In southwestern and western regions of the United States, at least half a million diagnostic tests for coccidioidomycosis (valley fever) are run annually. Enzyme immunoassays (EIAs) are blood tests which require precise dilution of patient serum prior to testing. To ensure patient serum is properly diluted, there is a regulatory requirement to ensure the dilution step is accurate. Two FDA-approved EIAs used to aid in the diagnosis of coccidioidomycosis do not contain controls for this dilution step, leaving clinical laboratories with the only option of using previously positive patient sera, which may not react in a reliable or predictable manner. Here, we evaluate a humanized monoclonal antibody against a coccidioidal antigen and its utility as a dilution control in both available commercial EIAs. The use of a humanized monoclonal antibody provides a standardized and well-characterized dilution control for use in serological assays that aid in diagnosis of coccidioidomycosis.

Third COVID-19 vaccine dose boosts neutralizing antibodies in poor responders.

Background: While evaluating COVID-19 vaccine responses using a rapid neutralizing antibody (NAb) test, we observed that 25% of mRNA vaccine recipients did not neutralize >50%. We termed this group "vaccine poor responders" (VPRs). The objective of this study was to determine if individuals who neutralized <50% would remain VPRs, or if a third dose would elicit high levels of NAbs. Methods: 269 healthy individuals ranging in age from 19 to 80 (Average age = 51; 165 females and 104 males) who received either BNT162b2 (Pfizer) or mRNA-1273 (Moderna) vaccines were evaluated. NAb levels were measured: (i) 2-4 weeks after a second vaccine dose, (ii) 2-4 months after the second dose, (iii) within 1-2 weeks prior to a third dose and (iv) 2-4 weeks after a third mRNA vaccine dose. Results: Analysis of vaccine recipients reveals that 25% did not neutralize above 50% (Median neutralization = 21%, titers <1:80) within a month after their second dose. Twenty-three of these VPRs obtained a third dose of either BNT162b2 or mRNA-1273 vaccine 1-8 months (average = 5 months) after their second dose. Within a month after their third dose, VPRs show an average 5.4-fold increase in NAb levels (range: 46-99%). Conclusions: The results suggest that VPRs are not permanently poor responders; they can generate high NAb levels with an additional vaccine dose. Although it is not known what levels of NAbs protect from infection or disease, those in high-risk professions may wish to keep peripheral NAb levels high, limiting infection, and potential transmission.

Accuracy of Case-Based Seroprevalence of SARS-CoV-2 Antibodies in Maricopa County, Arizona.

We conducted a community seroprevalence survey in Arizona, from September 12 to October 1, 2020, to determine the presence of antibodies to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We used the seroprevalence estimate to predict SARS-CoV-2 infections in the jurisdiction by applying the adjusted seroprevalence to the county's population. The estimated community seroprevalence of SARS-CoV-2 infections was 4.3 times greater (95% confidence interval = 2.2, 7.5) than the number of reported cases. Field surveys with representative sampling provide data that may help fill in gaps in traditional public health reporting. (Am J Public Health. 2022;112(1):38-42. https://doi.org/10.2105/AJPH.2021.306568).

Development of a rapid point-of-care test that measures neutralizing antibodies to SARS-CoV-2.

BACKGROUND: After receiving a COVID-19 vaccine, most recipients want to know if they are protected from infection and for how long. Since neutralizing antibodies are a correlate of protection, we developed a lateral flow assay (LFA) that measures levels of neutralizing antibodies from a drop of blood. The LFA is based on the principle that neutralizing antibodies block binding of the receptor-binding domain (RBD) to angiotensin-converting enzyme 2 (ACE2). METHODS: The ability of the LFA was assessed to correctly measure neutralization of sera, plasma or whole blood from patients with COVID-19 using SARS-CoV-2 microneutralization assays. We also determined if the LFA distinguished patients with seasonal respiratory viruses from patients with COVID-19. To demonstrate the usefulness of the LFA, we tested previously infected and non-infected COVID-19 vaccine recipients at baseline and after first and second vaccine doses. RESULTS: The LFA compared favorably with SARS-CoV-2 microneutralization assays with an area under the ROC curve of 98%. Sera obtained from patients with seasonal coronaviruses did not show neutralizing activity in the LFA. After a single mRNA vaccine dose, 87% of previously infected individuals demonstrated high levels of neutralizing antibodies. However, if individuals were not previously infected, only 24% demonstrated high levels of neutralizing antibodies after one vaccine dose. A second dose boosted neutralizing antibody levels just 8% higher in previously infected individuals, but over 63% higher in non-infected individuals. CONCLUSIONS: A rapid, semi-quantitative, highly portable and inexpensive neutralizing antibody test might be useful for monitoring rise and fall in vaccine-induced neutralizing antibodies to COVID-19.

Updated ferritin reference intervals for the Roche Elecsys® immunoassay.

Reference intervals (RI) for ferritin are the subject of some controversy, with indications that changes in lifestyle and demographics (e.g., obesity) have limited the validity of RIs established decades ago. Package insert RIs for the Roche Elecsys® immunoassay do not include expected values for pediatric (<17-20 years) or geriatric (>60 years) individuals; furthermore the female ranges were established in mostly premenopausal volunteers. To establish more robust RIs, we utilized 5 years of retrospective patient data from physician-ordered ferritin measurements and excluded results from patients with diagnoses known to affect ferritin concentrations. Ferritin results from 1438 unique patients aged 7 months to 91 years were included in the study. Continuous RIs were fitted for females (n = 951) and males (n = 487) as a function of age; these were then divided into clinically relevant sex-specific age breaks. RIs were established for pre-adolescent (<10 years), adolescent (10-17 years) and adult males, and for pediatric (<18 years), adult (18-50 years) and older (>50 years) females. Established RIs were verified using specimens obtained from healthy donors.

Retrospective clinical evaluation of 4 lateral flow assays for the detection of SARS-CoV-2 IgG.

In a Clinical Laboratory Improvement Amendments laboratory setting, we evaluated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) IgG detection with 4 lateral flow immunoassays [LFIAs; 2 iterations from BTNX Inc. (n = 457) and 1 each from ACON Laboratories (n = 200) and SD BIOSENSOR (n = 155)]. In a cohort of primarily hospitalized, reverse-transcription polymerase chain reaction-confirmed coronavirus disease 2019 cases, sensitivity at ≥14 days from symptom onset was: BTNX kit 1, 95%; BTNX kit 2, 91%; ACON, 95%; and SD, 92%. All assays showed good concordance with the Abbott SARS-CoV-2 IgG assay at ≥14 days from symptom onset: BTNX kit 1, 99%; BTNX kit 2, 94%; ACON, 99%; and SD, 100%. Specificity, measured using specimens collected prior to SARS-CoV-2 circulation in the United States and "cross-reactivity challenge" specimens, was 98% for BTNX kit 1 and ACON and 100% for BTNX kit 2 and SD. These results suggest that LFIAs may provide adequate results for rapid detection of SARS-CoV-2.

Evaluating Tiered Hemolysis Thresholds to Facilitate Rapid Reporting of High-Sensitivity Cardiac Troponin.

BACKGROUND: Cardiac troponin (cTn) assays are used for the diagnosis of acute myocardial infarction and frequently require serial measurements. Recollection of unacceptable specimens for hemolysis can delay results and disrupt timing of serial measurements. This study was designed to assess the influence of hemolysis on a high-sensitivity cTnT assay in granular detail at clinically important concentrations. These were used in consultation with the clinical practice to evaluate risk-based thresholds for acceptable amounts of hemolysis. METHODS: Plasma pools ranging from <10 to >100 ng/L cTnT were spiked with hemolysate to various degrees of hemolysis and measured using the Elecsys Troponin T Gen 5 STAT assay. The impact of accepting expanded hemolysis thresholds was completed using retrospective data of 12225 serial cTnT results and an additional 4651 baseline cTnT results. RESULTS: The mean percent change in cTnT was consistent for a given degree of hemolysis regardless of the initial nonhemolyzed cTnT concentration. Tiered hemolysis thresholds were evaluated for low-risk patients (apparent cTnT ≤8 ng/L), intermediate-risk patients (thresholds for 9-37 ng/L, 38-66 ng/L, and 67-99 ng/L cTnT), and high-risk patients (≥100 ng/L cTnT). The influence of tiered hemolysis thresholds was calculated for patients with serial (0 and 2 h) cTnT results, which demonstrated that the majority of 2-h delta interpretations were unchanged. Implementation of tiered thresholds dramatically decreased recollections for hemolyzed cTnT samples. CONCLUSION: Tiered hemolysis cutoffs minimized disruption to patient care for low- and high-risk patients, while maintaining the integrity of serial measurements for those with intermediate cTnT concentrations.

False-positive amphetamine results on several drug screening platforms due to mexiletine.

OBJECTIVE: False-positive urine drug of abuse screening (UDS) results can have serious implications in clinical practice, particularly when confirmation assay results are not immediately available to providers making medical decisions. Often it is not possible to identify the specific medication or other interfering compound that is responsible for the false-positive UDS result. Even when a potential interference is reported in the literature or package insert for one assay, the applicability to other UDS platforms/assays is often unknown. Mexiletine has been suggested as a cause of false-positive amphetamine results, but never confirmed as the causative agent in previous reports. The goal of this study was to confirm this drug as a cross-reacting compound in amphetamine screening tests. METHODS: We evaluated several amphetamine screening assays: the Syva EMIT II Plus and the Roche KIMS automated immunoassays, along with the Noble Split-Specimen and Synchron QuikScreen point-of-care assays. RESULTS: Urine samples from two patients treated with mexiletine were positive on all amphetamine screens but confirmed negative by mass spectrometry. Drug-free urine spiked with mexiletine caused positive results on all assays, although the EMIT II Plus and KIMS assays cross-reacted at lower mexiletine concentrations than the point-of-care assays. CONCLUSION: This report confirms that mexiletine can cross-react on several amphetamine screening assays. Assay manufacturers are encouraged to evaluate mexiletine cross-reactivity, and providers and laboratories should be aware of the potential for false-positive amphetamine screening results in patients taking mexiletine.

Genetic variation in statin intolerance and a possible protective role for UGT1A1.

The etiology of statin intolerance is hypothesized to be due to genetic variants that impact statin disposition and clearance. We sought to determine whether genetic variants were associated to statin intolerance. The studied cohort consisted of hyperlipidemic participants (n = 90) clinically diagnosed with statin intolerance by a cardiologist and matched controls without statin intolerance. Creatine kinase activity, lipid profiles and genetic analyses were performed on genes involved in statin metabolism and included UGT1A1 and UGT1A3 sequencing and targeted analyses of CYP3A4*22, CYP3A5*3, SLCO1B1*5 and *1b, ABCB1 c.3435C>T, ABCG2 c.421C>A and GATM rs9806699. Although lipids were higher in cases, genetic variant minor allele frequencies were similar between cases and controls, except for UGT1A1*28, which was less prevalent in cases than controls.

Neonatal Intensive Care Unit Quality Initiative: Identifying Preanalytical Variables Contributing to Specimen Hemolysis and Measuring the Impact of Evidence-Based Practice Interventions.

OBJECTIVES: Blood specimen hemolysis is a major cause of sample recollection in the neonatal intensive care unit. We aimed to reduce the hemolysis rate from 6.3% at baseline to less than 4% within the 9 months' duration of the study. METHODS: Intravenous infusion of lipid emulsion during sample collection, sample collection site, and blood sample transportation methods were investigated as possible contributors to hemolysis. Subsequently, two practice improvements were implemented: pausing lipid emulsion infusion prior to collection and slowing withdrawal rates through arterial catheters. RESULTS: Samples were more likely to be hemolyzed if they were collected during lipid infusion and subsequently transported by pneumatic tube or collected through an arterial catheter. Retrospective analysis demonstrated a decreased number of tests cancelled due to specimen hemolysis (3.5%) after our interventions. CONCLUSIONS: We identified three variables contributing to hemolysis and instituted two clinical practice interventions to significantly reduce test cancellations due to hemolysis.

Simultaneous Quantitation of 78 Drugs and Metabolites in Urine with a Dilute-And-Shoot LC-MS-MS Assay.

A novel LC-MS-MS assay that simultaneously detects and quantitates 78 drugs and metabolites was developed and validated for chronic pain management. Urine specimen was diluted and mixed with internal standards (ISs) before injected into LC-MS-MS. Seventy-two analytes were detected with positive electrospray ionization mode and the remaining six analytes with negative mode. Two separate gradient elution chromatographic programs were established with the same mobile phases on the same bi-phenyl HPLC column. The assay was linear for all analytes with linear regression coefficient ranging 0.994-1.000. The intra-assay precision was between 1.7 and 8.8% and inter-assay precision between 1.9 and 12.2%, with bias <20% for all but six analytes. All analytes in urine specimens were stable for 7 days at 4°C, and no significant matrix effect or carryover was observed. A suboptimal recovery rate (60.0-156.8%) was observed for six analytes, potentially due to the lack of available deuterated ISs, requiring comparison to a chemically different IS. Method comparison using patient and proficiency testing samples demonstrated that this assay was sensitive and accurate. The assay improves on currently existing assays by including glucuronide conjugates, allowing direct detection of metabolites that might otherwise be missed by existing methods.

Label-free detection and identification of protein ligands captured by receptors in a polymerized planar lipid bilayer using MALDI-TOF MS.

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) coupled with affinity capture is a well-established method to extract biological analytes from complex samples followed by label-free detection and identification. Many bioanalytes of interest bind to membrane-associated receptors; however, the matrices and high-vacuum conditions inherent to MALDI-TOF MS make it largely incompatible with the use of artificial lipid membranes with incorporated receptors as platforms for detection of captured proteins and peptides. Here we show that cross-linking polymerization of a planar supported lipid bilayer (PSLB) provides the stability needed for MALDI-TOF MS analysis of proteins captured by receptors embedded in the membrane. PSLBs composed of poly(bis-sorbylphosphatidylcholine) (poly(bis-SorbPC)) and doped with the ganglioside receptors GM1 and GD1a were used for affinity capture of the B subunits of cholera toxin, heat-labile enterotoxin, and pertussis toxin. The three toxins were captured simultaneously, then detected and identified by MS on the basis of differences in their molecular weights. Poly(bis-SorbPC) PSLBs are inherently resistant to nonspecific protein adsorption, which allowed selective toxin detection to be achieved in complex matrices (bovine serum and shrimp extract). Using GM1-cholera toxin subunit B as a model receptor-ligand pair, we estimated the minimal detectable concentration of toxin to be 4 nM. On-plate tryptic digestion of bound cholera toxin subunit B followed by MS/MS analysis of digested peptides was performed successfully, demonstrating the feasibility of using the PSLB-based affinity capture platform for identification of unknown, membrane-associated proteins. Overall, this work demonstrates that combining a poly(lipid) affinity capture platform with MALDI-TOF MS detection is a viable approach for capture and proteomic characterization of membrane-associated proteins in a label-free manner.

Quantitation of 1α,25-dihydroxyvitamin D by LC-MS/MS using solid-phase extraction and fixed-charge derivitization in comparison to immunoextraction.

BACKGROUND: The objective of this study was to present analysis of 1α,25-dihydroxyvitamin D (DHVD) by solid-phase extraction (SPE) using fixed-charge derivitization extraction to enhance ionization for liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) in comparison to traditional immunoextraction (IE) techniques. METHODS: Full analytical validation of both a SPE and IE LC-MS/MS assay was performed, and included accuracy, intra- and inter-assay precision, limit of detection and limit of quantitation. Performance of these two assays was compared with reference laboratory IE LC-MS/MS testing. RESULTS: This SPE LC-MS/MS assay demonstrated similar performance to the IE LC-MS/MS assay validated simultaneously. Intra-assay precision for low (12 pg/mL), medium (25 pg/mL) and high (60 pg/mL) control samples was 7.2%, 13.7% and 11.3% for DHVD2, respectively, and 9.1%, 5.9% and 8.9% for DHVD3. The inter-assay precision was 11.6%, 10.3% and 3.9% for DHVD2 and 10.6%, 7.0% and 5.6% for DHVD3. The limit of detection was 1.9 and 2.7 pg/mL for DHVD2 and DHVD3, and limit of quantitation was 4 pg/mL for both DHVD2 and DHVD3. Comparison to a reference LC-MS/MS assay showed excellent correlation (slope 0.936, R2=0.996, -0.2% bias). CONCLUSIONS: The study demonstrated comparability of the SPE-LC-MS/MS assay for analysis of DHVD and offers an attractive option for assessment of vitamin D status as an alternative to traditional IE techniques.

Management structure: establishing a laboratory utilization program and tools for utilization management.

As laboratories are challenged to do more with fewer resources, the pathologist and laboratory director will play a greater role in improving the effectiveness of the laboratory, as well as addressing the overuse, misuse and underuse of laboratory testing. We describe the necessary characteristics for pathologists and laboratory directors to successfully lead utilization efforts, as well as key leadership tools and essential steps in creating a utilization management program. When we established a laboratory test utilization program de novo, it became clear how important the laboratory director was in guiding those initiatives by working with stakeholders outside of the laboratory, particularly clinicians, nurses and administrators.