Comparison of a dual antibody and antigen HCV immunoassay to standard of care algorithmic testing.

The Centers for Disease Control and Prevention (CDC) guidelines for hepatitis C virus (HCV) testing, although effective, may miss crucial diagnostic opportunities. The goal of this study was to assess the utility of an antibody (Ab) and antigen (Ag) combination immunoassay as an alternative to traditional HCV screening. Remnant specimens from 1,341 patients with concurrent third-generation serologic (Roche anti-HCV-II) and nucleic acid amplification testing (NAAT) were assessed using the HCV Duo Ab/Ag immunoassay (Roche). Patient demographics, risk factors, and standard of care (SOC) laboratory results from the medical records were recorded. Overall, 99.0% (197/199) of the HCV Duo Ab+/Ag+specimens accurately identified active infections as confirmed by NAAT, and 99.9% (670/671) Ab-/Ag- samples corresponded to those without HCV infections. Individually, the HCV Duo Ab component demonstrated a 95.6% positive percent agreement (PPA) (95% CI = 93.8-96.9) and 99.1% negative percent agreement (NPA) (98.8-99.6) compared with SOC anti-HCV II Ab assay. The HCV Duo Ag had a 73.5% PPA (67.9-78.4) and 99.8% NPA (99.3-100) with NAAT. Among RNA+ specimens, 73.4% (197/267) were HCV Duo Ag+, and 265/267 (99.3%) were successfully detected on the HCV Duo Ab component. Notably, 5/7 (71.4%) Ab-/RNA +specimens were detected by HCV Duo, which would have been missed by traditional algorithmic testing. Fourth generation HCV Duo Ab/Ag assay demonstrated comparable performance to SOC testing and shortens the diagnostic window but does not eliminate the need for NAAT in all patients. Ab/Ag testing identified several Ab-/RNA+ cases, a subgroup often undiagnosed by current algorithmic testing, demonstrating promise for improved diagnostic efficiency and accuracy in HCV detection.IMPORTANCEThis study highlights the potential of a combined hepatitis C virus (HCV) Duo antibody (Ab) and antigen (Ag) immunoassay to improve early detection of HCV infections. Traditional Ab-only screening methods recommended by the Centers for Disease Control and Prevention may miss early-stage infections. The HCV Duo assay showed high accuracy, detecting nearly all active infections confirmed by nucleic acid amplification testing. Dual detection of HCV Ab and Ag shortens the diagnostic window, enabling intervention and treatment in a single visit, which is crucial for improving patient outcomes and reducing HCV transmission, especially in areas with limited access to confirmatory molecular testing.

Evaluation of a point-of-care rapid HIV antibody test with insights into acute HIV symptomatology in a population with low prevalence.

Many emergency departments (ED) use rapid human immunodeficiency virus (HIV) antibody tests as screening tools, despite limited sensitivity for detecting acute HIV infections. In a 4-year retrospective analysis of 1,192 patients, we evaluated the performance of a third-generation rapid HIV antibody assay tested at point-of-care (POC, Chembio Sure Check HIV 1/2) against in-lab fourth-generation screening (Abbott Architect Ag/Ab Combo). Compared to complete algorithmic testing, the POC test demonstrated a 92.5% sensitivity (95% CI = 84.6-96.5), 98.1% specificity (95% CI = 97.1-98.8), 99.5% negative predictive value (NPV; 95% CI = 98.8-99.8), and a 77.9% positive predictive value (PPV; 95% CI = 68.6-85.1). Notably, the POC test failed to detect 100% (3/3) of acute HIV infections (defined as Fiebig stage 2) and 3.8% (2/52) established HIV infections, where viral loads were 5.9, 6.7, and >7 log10 copies/mL. Symptoms such as fever, nausea/vomiting, malaise, headache, and photophobia were significantly associated with acute HIV infections diagnosed in the ED. The rapid HIV antibody test demonstrated high sensitivity, specificity, and NPV in our study population, reaffirming its effectiveness as a valuable screening tool. However, the low PPV and 100% failure to detect acute HIV infections underscore the importance of prioritizing in-lab fourth-generation HIV antigen/antibody combination immunoassays in cases of suspected acute HIV infection to ensure a timely and accurate diagnosis.

Multiplatform Assessment of Saliva for SARS-CoV-2 Molecular Detection in Symptomatic Healthcare Personnel and Patients Presenting to the Emergency Department.

BACKGROUND: Saliva has garnered great interest as an alternative specimen type for molecular detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Data are limited on the relative performance of different molecular methods using saliva specimens and the relative sensitivity of saliva to nasopharyngeal (NP) swabs. METHODS: To address the gap in knowledge, we enrolled symptomatic healthcare personnel (n = 250) from Barnes-Jewish Hospital/Washington University Medical Center and patients presenting to the Emergency Department with clinical symptoms compatible with coronavirus disease 2019 (COVID-19; n = 292). We collected paired saliva specimens and NP swabs. The Lyra SARS-CoV-2 assay (Quidel) was evaluated on paired saliva and NP samples. Subsequently we compared the Simplexa COVID-19 Direct Kit (Diasorin) and a modified SalivaDirect (Yale) assay on a subset of positive and negative saliva specimens. RESULTS: The positive percent agreement (PPA) between saliva and NP samples using the Lyra SARS-CoV-2 assay was 63.2%. Saliva samples had higher SARS-CoV-2 cycle threshold values compared to NP swabs (P < 0.0001). We found a 76.47% (26/34) PPA for Simplexa COVID-19 Direct Kit on saliva and a 67.6% (23/34) PPA for SalivaDirect compared to NP swab results. CONCLUSION: These data demonstrate molecular assays have variability in performance for detection of SARS-CoV-2 in saliva.

Comparison of 6 SARS-CoV-2 Molecular Methods and Correlation With the Cycle Threshold Distribution in Clinical Specimens.

BACKGROUND: The detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in patient samples is of critical importance in the management of patients and monitoring transmission in the population. However, data on the analytical performance characteristics for detection of SARS-CoV-2 in clinical specimens between individual targets within the same platform, and among different analytical platforms, are limited. METHODS: Here we evaluated the performance of 6 different sample-to-answer SARS-CoV-2 detection methods-Roche cobas 6800, Cepheid GeneXpert, Diasorin Simplexa, Luminex Aries emergency use authorization (EUA), Luminex Aries research use only (RUO), and bioMérieux BioFire-in clinical specimens with a range of viral loads. RESULTS: The positive percentage agreement between the Roche cobas 6800 and GeneXpert was 100%, Diasorin 95%, Aries EUA 74%, Aries RUO 83%, and BioFire 97%. Notably, in samples with cycle threshold (Ct) values below 30 for the E gene on the Roche cobas 6800 platform, we found 100% positive agreement among all platforms. Given these results, we examined the distribution of over 10 000 Ct values of all positive specimens from individuals at our institution on the Roche cobas platform. Nearly 60% of specimens from asymptomatic individuals had a PCR Ct value >30 as measured using the cobas 6800 assay E gene. CONCLUSIONS: Our results demonstrate performance characteristics between different platforms by Ct value and provide data regarding the distribution of viral RNA present in positive specimens.

Evaluation of PCR cycle threshold values by patient population with the quidel lyra SARS-CoV-2 assay.

The Lyra SARS-CoV-2 assay was the primary method for molecular testing performed at Barnes-Jewish Healthcare System in St. Louis, Missouri during the initial COVID-19 surge from mid-March to late-April 2020. We performed a retrospective analysis of 1,043 positive Lyra SARS-CoV-2 results during these 36 days to investigate associations between cycle threshold (CT)  value and patient characteristics. Total RNA were extracted from NP or OP swabs using either the EasyMag or KingFisher automated extraction systems and quantified with RotorGene Q (Qiagen) or Applied Biosystems 7500 Fast Dx thermocyclers respectively. Notably, we found lower a significant median lower CT for samples tested on the KingFisher-ABI 7500 fastDX (KF/ABI) system compared to the EasyMag/RotorGene (EM/RGQ) platform. Since 77.5% of our tests were ran on the EM/RGQ pipeline we then perform additional analysis on these values and found that C T values in outpatient care settings compared to samples obtained in the emergency department or inpatient had significantly lower C T values. These collective findings suggests a difference in viral load amongst various patient populations.

The Effects of "Dry Swab" Incubation on SARS-CoV-2 Molecular Testing.

BACKGROUND: Widespread testing of SARS-CoV-2 has resulted in shortages of collection devices and transport media. We evaluated the stability of flocked swabs inoculated with SARS-CoV-2-containing specimen incubated dry (i.e., without transport medium) at room temperature. METHODS: A pool of SARS-CoV-2 positive specimen was used to inoculate flocked swabs. Five swabs were placed immediately into universal transport media (UTM) following inoculation, and tested immediately (day 0). Fifteen of the swabs were placed into sterile 15-mL conical tubes and incubated at room temperature for 1, 2, or 7 days. Following incubation, swabs were hydrated in separate vials of UTM and tested. This protocol was repeated for viral transport media (VTM) and saline. As a comparison, a series of swabs was prepared and tested in parallel, but stored in the corresponding liquid transport media (UTM, VTM, or saline) and incubated at room temperature. Testing was performed at 1, 2, and 7 days postinoculation in duplicate. All molecular testing was performed using the Roche cobas SARS-CoV-2 assay. RESULTS: All dry swabs tested on days 1, 2, and 7 provided results that were within 2 cycle thresholds (CTs) of the average CT values for swabs hydrated in the same media and tested on day 0. There was no statistical difference in CT values between swabs incubated in liquid media versus dry swabs incubated at room temperature prior to hydration in liquid media. CONCLUSIONS: The utilization of "dry swabs" may simplify specimen collection, negate the need for liquid transport media, and mitigate safety risks while preserving the accuracy of testing.

Evaluation of Infectious Disease Test Ordering and Positivity Rates in Illicit Fentanyl Users.

BACKGROUND: The emergence of illicit fentanyl use has resulted in considerable morbidity and mortality. Although illicit use of other opioids has been associated with transmission of viral and bacterial infections, limited data exist for the prevalence of infectious diseases among illicit fentanyl users. The purpose of this study was to assess the likelihood of infectious disease testing and infection prevalence among illicit fentanyl users. METHODS: Results from urine drug screens (UDSs) performed from August 13, 2019, to October 16, 2019, were obtained from the laboratory information system with concurrent microbial testing. Patients were categorized based on UDS results, and illicit drug use was inferred from physician encounter notes in the electronic medical record. RESULTS: Suspected illicit drugs users with fentanyl detected by UDS were more likely to be screened [odds ratio (OR): 1.7; 95% CI, 1.26-2.4] and test positive for hepatitis C virus (HCV) by immunoassay (OR: 5.89; 95% CI, 2.93-11.31) than patients without drugs detected. Patients with suspected illicit fentanyl use who were discharged from the emergency department (ED) were less likely to be tested for HCV than patients in outpatient settings (OR: 3.47; 95% CI, 1.05-10.4) and inpatient settings (OR: 17.43; 95% CI, 6.53-45.88). Patients with suspected illicit fentanyl use were more likely to have infected abscesses or wounds (OR: 5.12; 95% CI, 2.07-13.7) and Staphylococcus aureus infections (OR: 4.5; 95% CI, 1.59-12.28) than patients without drugs detected. CONCLUSIONS: Patients with a positive UDS for fentanyl and suspected illicit use were more likely to test positive for HCV, were rarely screened for HCV in the ED, and had an increased risk of invasive S. aureus wound or abscess infection. These findings may represent considerable barriers to care for patients who use fentanyl illicitly.

Association between SARS-CoV-2 Neutralizing Antibodies and Commercial Serological Assays.

BACKGROUND: Commercially available SARS-CoV-2 serological assays based on different viral antigens have been approved for the qualitative determination of anti-SARS-CoV-2 antibodies. However, there are limited published data associating the results from commercial assays with neutralizing antibodies. METHODS: Sixty-six specimens from 48 patients with PCR-confirmed COVID-19 and a positive result by the Roche Elecsys Anti-SARS-CoV-2, Abbott SARS-CoV-2 IgG, or EUROIMMUN SARS-CoV-2 IgG assays and 5 control specimens were analyzed for the presence of neutralizing antibodies to SARS-CoV-2. Correlation, concordance, positive percent agreement (PPA), and negative percent agreement (NPA) were calculated at several cutoffs. Results were compared in patients categorized by clinical outcomes. RESULTS: The correlation between SARS-CoV-2 neutralizing titer (EC50) and the Roche, Abbott, and EUROIMMUN assays was 0.29, 0.47, and 0.46, respectively. At an EC50 of 1:32, the concordance kappa with Roche was 0.49 (95% CI; 0.23-0.75), with Abbott was 0.52 (0.28-0.77), and with EUROIMMUN was 0.61 (0.4-0.82). At the same neutralizing titer, the PPA and NPA for the Roche was 100% (94-100) and 56% (30-80); Abbott was 96% (88-99) and 69% (44-86); and EUROIMMUN was 91% (80-96) and 81% (57-93) for distinguishing neutralizing antibodies. Patients who were intubated, had cardiac injury, or acute kidney injury from COVID-19 infection had higher neutralizing titers relative to those with mild symptoms. CONCLUSIONS: COVID-19 patients generate an antibody response to multiple viral proteins such that the calibrator ratios on the Roche, Abbott, and EUROIMMUN assays are all associated with SARS-CoV-2 neutralization. Nevertheless, commercial serological assays have poor NPA for SARS-CoV-2 neutralization, making them imperfect proxies for neutralization.

Repeat Molecular Testing for Respiratory Pathogens: Diagnostic Gain or Diminishing Returns?

BACKGROUND: Upper respiratory tract infections are common, and the ability to accurately and rapidly diagnose the causative pathogen has important implications for patient management. METHODS: We evaluated the test-ordering practices for 2 commonly utilized nucleic acid amplification tests (NAATs) for the detection of respiratory pathogens: the Xpert Flu Assay for influenza A/B (Flu assay) and the Biofire FilmArray respiratory panel assay (RP assay), which detects 20 different targets. Our study examined repeat testing; that is, testing within 7 days from an initial test. RESULTS: Our study found that repeat testing is common for each of the individual assays: 3.0% of all Flu assays and 10.0% of all RP assays were repeat testing. Of repeat testing, 8/293 (2.7%) of repeat Flu assays and 75/1257 (6.0%) of RP assays resulted diagnostic gains, i.e., new detections. However, for the RP assay, these new detections were not always clinically actionable. The most frequently discrepant organisms were rhinovirus/enterovirus (28/102, 27.5%), followed by respiratory syncytial virus (12/102, 11.8%) and coronavirus OC43 (11/102, 10.8%). Furthermore, there were 3,336 instances in which a patient was tested using both a Flu assay and RP assay, of which only 44 (1.3%) had discrepant influenza results. CONCLUSIONS: Our findings suggest opportunities exist to better guide ordering practices for respiratory pathogen testing, including limiting repeat testing, with the goal of optimization of clinical yield, and diagnostic stewardship.

Clinical Performance of Two SARS-CoV-2 Serologic Assays.

BACKGROUND: The recent emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in a rapid proliferation of serologic assays. However, little is known about their clinical performance. Here, we compared two commercial SARS-CoV-2 IgG assays. METHODS: 103 specimens from 48 patients with PCR-confirmed SARS-CoV-2 infections and 153 control specimens were analyzed using SARS-CoV-2 serologic assays by Abbott and EUROIMMUN (EI). Duration from symptom onset was determined by medical record review. Diagnostic sensitivity, specificity, and concordance were calculated. RESULTS: The Abbott SARS-CoV-2 assay had a diagnostic specificity of 99.4% (95% CI; 96.41-99.98%), and sensitivity of 0.0% (95% CI; 0.00-26.47%) at <3 days post symptom onset, 30.0% (95% CI; 11.89-54.28) at 3-7d, 47.8% (95% CI; 26.82-69.41) at 8-13d and 93.8% (95% CI; 82.80-98.69) at ≥14d. Diagnostic specificity on the EI assay was 94.8% (95% CI; 89.96-97.72) if borderline results were considered positive and 96.7% (95% CI; 92.54-98.93) if borderline results were considered negative. The diagnostic sensitivity was 0.0% (95% CI; 0.00-26.47%) at <3d, 25.0% (95% CI; 8.66-49.10) at 3-7d, 56.5% (95% CI; 34.49-76.81) at 3-7d and 85.4% (95% CI; 72.24-93.93) at ≥14d if borderline results were considered positive. The qualitative concordance between the assays was 0.83 (95% CI; 0.75-0.91). CONCLUSION: The Abbott SARS-CoV-2 assay had fewer false positive and false negative results than the EI assay. However, diagnostic sensitivity was poor in both assays during the first 14 days of symptoms.

Evaluation of the BioFire FilmArray Pneumonia Panel for Detection of Viral and Bacterial Pathogens in Lower Respiratory Tract Specimens in the Setting of a Tertiary Care Academic Medical Center.

Our objective was to evaluate the diagnostic yield and accuracy of the BioFire FilmArray pneumonia panel (BFPP) for identification of pathogens in lower respiratory tract specimens (n = 200) from emergency department (ED) and intensive care unit (ICU) patients at a tertiary care academic medical center. Specimens were collected between January and November 2018, from patients ≥18 years of age, and culture was performed as part of standard-of-care testing. The BFPP identified a viral or bacterial target in 117/200 (58.5%) samples, including Staphylococcus aureus in 22% of samples and Haemophilus influenzae in 14%, and both a viral and bacterial target in 4% of samples. The most common viruses detected by BFPP were rhinovirus/enterovirus (4.5%), influenza A virus (3%), and respiratory syncytial virus (RSV) (2%). Overall, there was strong correlation between BFPP and standard methods for detection of viruses (99.2%) and bacteria (96.8%). Most bacteria (60/61 [98.4%]) detected by standard methods were also identified by BFPP, and 92 additional bacteria were identified by BFPP alone, including 22/92 (23.9%) additional S. aureus isolates and 25/92 (27.2%) H. influenzae isolates, which were more frequently discordant when detected at low concentrations (S. aureus, P < 0.001; H. influenzae, P < 0.0001) and in sputum-type specimens (S. aureus, P < 0.05). A potential limitation of the BFPP assay is the absence of fungal targets and Stenotrophomonas maltophilia, which were detected in 26 and 4 of 200 specimens, respectively. Real-time specimen analysis with BFPP has the potential to identify bacterial pathogens and resistance markers 44.2 and 56.3 h faster than culture-based methods. The BFPP is a rapid and accurate method for detection of pathogens from lower respiratory tract infections.