Laboratory-Developed Tests Account for a Small Minority of Tests Ordered in an Academic Hospital System.

OBJECTIVES: To determine the frequency of use of laboratory-developed tests (LDTs) in an academic medical center system. METHODS: Retrospective analysis of 2021 test order data from an academic medical center (hospital, outpatient clinics, and cancer center) was done. Measures included assay type, assay methodology, regulatory status, test order volume, inpatient vs outpatient setting, and provider medical specialty. RESULTS: Of the 3,016,928 tests ordered in 2021, 2,831,489 (93.9%) were tests cleared, approved, and/or authorized by the US Food and Drug Administration (FDA); 116,583 (3.9%) were LDTs; and 68,856 (2.3%) were standard methods. These test orders were performed using a total of 1,954 distinct assays. Of these, 983 (50.3%) were FDA assays, 880 (45.0%) were LDTs, and 91 (4.7%) were standard methods. Laboratory-developed tests were more commonly ordered in the outpatient vs inpatient setting and represented a higher proportion of the test volume at the cancer center compared with the university hospital (5.6% vs 3.6%, respectively). The top 167 LDT assays accounted for 90% of the LDT volume (104,996 orders). Among the 20 most frequently ordered LDTs were mass spectrometry assays and tests used in the care of immunocompromised patients. Internal/family medicine placed the greatest number of orders (1,044,642) and ordered one of the lowest proportions of LDTs (3.2%). CONCLUSIONS: Laboratory-developed tests made up a small percentage of the total laboratory tests ordered within the academic health system studied.

In support of interoperability: A laboratory perspective.

Healthcare in the United States has become increasingly digital since the passage of the HITECH Act in 2009. As a result, there is a growing need to optimize healthcare IT to allow for the interoperable exchange of data. As a result, the Office of the National Coordinator for Health IT has implemented their Final Rule for the 21st Century Cures Act. This requires certified health IT systems to use modernized messaging standards for the safe and secure exchange of data within health information networks and also requires the use of terminology standards including LOINC, SNOMED CT, and UCUM for coding clinical and laboratory data. Given the critical importance of laboratory results in the delivery of healthcare, laboratorians must become familiar with these principles of interoperability. Their clinical laboratory expertise is needed to appropriately structure and code test results to safeguard against improper aggregation or misinterpretation by downstream users and systems.

Development of an international glossary for clinical guidelines collaboration.

OBJECTIVES: Clinical practice guidelines (CPGs) are often created through collaboration among organizations. The use of inconsistent terminology may cause poor communication and delays. This study aimed to develop a glossary of terms related to collaboration in guideline development. STUDY DESIGN AND SETTING: A literature review of collaborative guidelines was performed to develop an initial list of terms related to guideline collaboration. The list of terms was presented to the members of the Guideline International Network Guidelines Collaboration Working Group, who provided presumptive definitions for each term and proposed additional terms to be included. The revised list was subsequently reviewed by an international, multidisciplinary panel of expert stakeholders. Recommendations received during this pre-Delphi review were implemented to augment an initial draft glossary. The glossary was then critically evaluated and refined through two rounds of Delphi surveys and a virtual consensus meeting with all panel members as Delphi participants. RESULTS: Forty-nine experts participated in the pre-Delphi survey, and 44 participated in the two-round Delphi process. Consensus was reached for 37 terms and definitions. CONCLUSION: Uptake and utilization of this guideline collaboration glossary by key organizations and stakeholder groups may facilitate collaboration among guideline-producing organizations by improving communication, minimizing conflicts, and increasing guideline development efficiency.

Evaluation of a Novel Multiplex Platform for Simultaneous Detection of IgG Antibodies Against the 4 Main SARS-CoV-2 Antigens.

BACKGROUND: Numerous serology assays are available for detection of SARS-CoV-2 antibodies but are limited in that only 1 or 2 target antigen(s) can be tested at a time. Here, we describe a novel multiplex assay that simultaneously detects and quantifies IgG antibodies to SARS-CoV-2 antigens, spike (S), nucleocapsid (N), receptor-binding domain (RBD), and N-terminal domain (NTD) in a single well. METHODS: Sensitivity was determined using samples (n = 124) from confirmed SARS-CoV-2 RT-PCR positive individuals. Prepandemic (n = 100) and non-COVID respiratory infection positive samples (n = 100) were used to evaluate specificity. Samples were analyzed using COVID-19 IgG multiplex serology assay from Meso Scale Discovery (MSD) and using commercial platforms from Abbott, EUROIMMUN, and Siemens. RESULTS: At >14 days post-PCR, MSD assay displayed >98.0% sensitivity [S 100% (95% CI 98.0%-100.0%); N 98.0% (95% CI 97.2%-98.9%); RBD 94.1% (95% CI 92.6%-95.6%); NTD 98.0% (95% CI, 97.2%-98.9%)] and 99% specificity (95% CI 99.3%-99.7%) for antibodies to all 4 antigens. Parallel assessment of antibodies to more than 1 antigen improved the sensitivity to 100% (95% CI 98.0%-100.0%) while maintaining 98% (95% CI 97.6%-98.4%) specificity regardless of the combinations used. When AU/mL concentrations of IgG antibodies from the MSD assay were compared against the corresponding IgG signals acquired from the single target commercial assays, the following correlations were observed: Abbott (vs MSD N, R2 = 0.73), Siemens (vs MSD RBD, R2 = 0.92), and EUROIMMUN (vs MSD S, R2 = 0.82). CONCLUSION: MSD assay offers an accurate and a comprehensive assessment of SARS-CoV-2 antibodies with higher sensitivity and equivalent specificity compared to the commercial IgG serology assays.

Evaluation of a Surrogate Enzyme-Linked Immunosorbent Assay-Based Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) cPass Neutralization Antibody Detection Assay and Correlation With Immunoglobulin G Commercial Serology Assays.

CONTEXT.­: Emerging evidence shows correlation between the presence of neutralization antibodies (nAbs) and protective immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Currently available commercial serology assays lack the ability to specifically identify nAbs. An enzyme-linked immunosorbent assay-based nAb assay (GenScript cPass neutralization antibody assay) has recently received emergency use authorization from the Food and Drug Administration. OBJECTIVE.­: To evaluate the performance characteristics of this assay and compare and correlate it with the commercial assays that detect SARS-CoV-2-specific immunoglobulin G (IgG). DESIGN.­: Specimens from SARS-COV-2 infected patients (n = 124), healthy donors obtained prepandemic (n = 100), and patients with non-coronavirus disease 2019 (COVID-19) respiratory infections (n = 92) were analyzed using this assay. Samples with residual volume were also tested on 3 commercial serology platforms (Abbott, Euroimmun, Siemens). Twenty-eight randomly selected specimens from patients with COVID-19 and 10 healthy controls were subjected to a plaque reduction neutralization test. RESULTS.­: The cPass assay exhibited 96.1% (95% CI, 94.9%-97.3%) sensitivity (at >14 days post-positive PCR), 100% (95% CI, 98.0%-100.0%) specificity, and zero cross-reactivity for the presence of non-COVID-19 respiratory infections. When compared with the plaque reduction assay, 97.4% (95% CI, 96.2%-98.5%) qualitative agreement and a positive correlation (R2 = 0.76) was observed. Comparison of IgG signals from each of the commercial assays with the nAb results from plaque reduction neutralization test/cPass assays displayed greater than 94.7% qualitative agreement and correlations with R2 = 0.43/0.68 (Abbott), R2 = 0.57/0.85 (Euroimmun), and R2 = 0.39/0.63 (Siemens), respectively. CONCLUSIONS.­: The combined data support the use of cPass assay for accurate detection of the nAb response. Positive IgG results from commercial assays associated reasonably with nAbs presence and can serve as a substitute.

Neutralization assay with SARS-CoV-1 and SARS-CoV-2 spike pseudotyped murine leukemia virions.

BACKGROUND: Virus neutralization by antibodies is an important prognostic factor in many viral diseases. To easily and rapidly measure titers of neutralizing antibodies in serum or plasma, we developed pseudovirion particles composed of the spike glycoprotein of SARS-CoV-2 incorporated onto murine leukemia virus capsids and a modified minimal murine leukemia virus genome encoding firefly luciferase. This assay design is intended for use in laboratories with biocontainment level 2 and therefore circumvents the need for the biocontainment level 3 that would be required for replication-competent SARS-CoV-2 virus. To validate the pseudovirion assay, we set up comparisons with other available antibody tests including those from Abbott, Euroimmun and Siemens, using archived, known samples. RESULTS: 11 out of 12 SARS-CoV-2-infected patient serum samples showed neutralizing activity against SARS-CoV-2-spike pseudotyped MLV viruses, with neutralizing titers-50 (NT50) that ranged from 1:25 to 1:1,417. Five historical samples from patients hospitalized for severe influenza infection in 2016 tested negative in the neutralization assay (NT50 < 25). Three serum samples with high neutralizing activity against SARS-CoV-2/MLV pseudoviruses showed no detectable neutralizing activity (NT50 < 25) against SARS-CoV-1/MLV pseudovirions. We also compared the semiquantitative Siemens SARS-CoV-2 IgG test, which measures binding of IgG to recombinantly expressed receptor binding domain of SARS-CoV-2 spike glycoprotein with the neutralization titers obtained in the pseudovirion assay and the results show high concordance between the two tests (R2 = 0.9344). CONCLUSIONS: SARS-CoV-2 spike/MLV pseudovirions provide a practical means of assessing neutralizing activity of antibodies in serum or plasma from infected patients under laboratory conditions consistent with biocontainment level 2. This assay offers promise also in evaluating immunogenicity of spike glycoprotein-based candidate vaccines in the near future.

Evaluation of 3 SARS-CoV-2 IgG Antibody Assays and Correlation with Neutralizing Antibodies.

BACKGROUND: As serologic assays for SARS-CoV-2 become more widely utilized, it is important to understand their performance characteristics and correlation with neutralizing antibodies. We evaluated 3 commonly used SARS-CoV-2 IgG assays (Abbott, DiaSorin, and EUROIMMUN) for clinical sensitivity, specificity, and correlation with neutralizing antibodies, and then compared antibody kinetics during the acute phase of infection. METHODS: Three panels of samples were tested on every assay. Sensitivity was assessed using a panel of 35 specimens serially collected from 7 patients with RT-PCR-confirmed COVID-19. Specificity was determined using 100 sera samples collected in 2018 from healthy individuals prior to the outbreak. Analytical specificity was determined using a panel of 37 samples from individuals with respiratory illnesses other than COVID-19. RESULTS: Clinical sensitivity was 91.43% (95% CI 76.94-98.20%) for Abbott, and 88.57% (95% CI 73.26-96.80%) for both DiaSorin and EUROIMMUN. Clinical specificity was 99.00% (95% CI 94.55-99.97%) for Abbott and DiaSorin and 94.00% (95% CI 87.40-97.77%) for EUROIMMUN. The IgG assays demonstrated good qualitative agreement (minimum of 94%) and good correlation between the quantitative result for each combination of assays (r2 ≥ 0.90). The neutralizing antibody response did not necessarily follow the same temporal kinetics as the IgG response and did not necessarily correlate with IgG values. CONCLUSION: The 3 IgG antibody assays demonstrated comparable performance characteristics. Importantly, a qualitative positive IgG result obtained with any of the assays was associated with the presence of neutralizing antibodies; however, neutralizing antibody concentrations did not correlate well with signal to cutoff ratios.

Neutralization Assay with SARS-CoV-1 and SARS-CoV-2 Spike Pseudotyped Murine Leukemia Virions.

Antibody neutralization is an important prognostic factor in many viral diseases. To easily and rapidly measure titers of neutralizing antibodies in serum or plasma, we developed pseudovirion particles composed of the spike glycoprotein of SARS-CoV-2 incorporated onto murine leukemia virus capsids and a modified minimal MLV genome encoding firefly luciferase. These pseudovirions provide a practical means of assessing immune responses under laboratory conditions consistent with biocontainment level 2.

Fecal Host Transcriptomics for Non-Invasive Human Mucosal Immune Profiling: Proof of Concept in Clostridium Difficile Infection.

Background: Host factors play an important role in pathogenesis and disease outcome in Clostridium difficile infection (CDI), and characterization of these responses could uncover potential host biomarkers to complement existing microbe-based diagnostics. Methods: We extracted RNA from fecal samples of patients with CDI and profiled human mRNA using amplicon-based next-generation sequencing (NGS). We compared the fecal host mRNA transcript expression profiles of patients with CDI to controls with non-CDI diarrhea. Results: We found that the ratio of human actin gamma 1 (ACTG1) to 16S ribosomal RNA (rRNA) was highly correlated with NGS quality as measured by percentage of reads on target. Patients with CDI could be differentiated from those with non-CDI diarrhea based on their fecal mRNA expression profiles using principal component analysis. Among the most differentially expressed genes were ones related to immune response (IL23A, IL34) and actin-cytoskeleton function (TNNT1, MYL4, SMTN, MYBPC3, all adjusted P-values < 1 × 10-3). Conclusions: In this proof-of-concept study, we used host fecal transcriptomics for non-invasive profiling of the mucosal immune response in CDI. We identified differentially expressed genes with biological plausibility based on animal and cell culture models. This demonstrates the potential of fecal transcriptomics to uncover host-based biomarkers for enteric infections.

Evaluation of the Vitek MS v3.0 Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry System for Identification of Mycobacterium and Nocardia Species.

This multicenter study was designed to assess the accuracy and reproducibility of the Vitek MS v3.0 matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry system for identification of Mycobacterium and Nocardia species compared to DNA sequencing. A total of 963 clinical isolates representing 51 taxa were evaluated. In all, 663 isolates were correctly identified to the species level (69%), with another 231 (24%) correctly identified to the complex or group level. Fifty-five isolates (6%) could not be identified despite repeat testing. All of the tuberculous mycobacteria (45/45; 100%) and most of the nontuberculous mycobacteria (569/606; 94%) were correctly identified at least to the group or complex level. However, not all species or subspecies within the M. tuberculosis, M. abscessus, and M. avium complexes and within the M. fortuitum and M. mucogenicum groups could be differentiated. Among the 312 Nocardia isolates tested, 236 (76%) were correctly identified to the species level, with an additional 44 (14%) correctly identified to the complex level. Species within the N. nova and N. transvalensis complexes could not always be differentiated. Eleven percent of the isolates (103/963) underwent repeat testing in order to get a final result. Identification of a representative set of Mycobacterium and Nocardia species was highly reproducible, with 297 of 300 (99%) replicates correctly identified using multiple kit lots, instruments, analysts, and sites. These findings demonstrate that the system is robust and has utility for the routine identification of mycobacteria and Nocardia in clinical practice.

Multicenter Evaluation of the Vitek MS v3.0 System for the Identification of Filamentous Fungi.

Invasive fungal infections are an important cause of morbidity and mortality affecting primarily immunocompromised patients. While fungal identification to the species level is critical to providing appropriate therapy, it can be slow and laborious and often relies on subjective morphological criteria. The use of matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry has the potential to speed up and improve the accuracy of identification. In this multicenter study, we evaluated the accuracy of the Vitek MS v3.0 system in identifying 1,601 clinical mold isolates compared to identification by DNA sequence analysis and supported by morphological and phenotypic testing. Among the 1,519 isolates representing organisms in the v3.0 database, 91% (n = 1,387) were correctly identified to the species level. An additional 27 isolates (2%) were correctly identified to the genus level. Fifteen isolates were incorrectly identified, due to either a single incorrect identification (n = 13) or multiple identifications from different genera (n = 2). In those cases, when a single identification was provided that was not correct, the misidentification was within the same genus. The Vitek MS v3.0 was unable to identify 91 (6%) isolates, despite repeat testing. These isolates were distributed among all the genera. When considering all isolates tested, even those that were not represented in the database, the Vitek MS v3.0 provided a single correct identification 98% of the time. These findings demonstrate that the Vitek MS v3.0 system is highly accurate for the identification of common molds encountered in the clinical mycology laboratory.

Immunogenicity of the Bivalent Oral Cholera Vaccine Shanchol in Haitian Adults With HIV Infection.

We evaluated immune responses following bivalent oral cholera vaccination (Shanchol [Shantha Biotechnics]; BivWC) in a cohort of 25 human immunodeficiency virus (HIV)-infected adults in Haiti. Compared with adults without HIV infection, vaccination in HIV-infected individuals resulted in lower vibriocidal responses against Vibrio cholerae O1, and there was a positive relationship between the CD4(+) T-cell count and vibriocidal responses following vaccination. Nevertheless, seroconversion occurred at a rate of 65% against the Ogawa serotype and 74% against the Inaba serotype in adults with HIV infection. These results suggest that the vaccine retains substantial immunogenicity in adults with HIV infection and may benefit this population by protecting against cholera.

Evaluation of matrix-assisted laser desorption ionization-time of flight mass spectrometry for identification of Vibrio cholerae.

We evaluated the use of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MS) for the identification of Vibrio cholerae. MS identified all 42 isolates of V. cholerae O1 and O139 and 7 of 9 non-O1/O139 isolates. MS correctly discriminated between all Aeromonas and V. cholerae isolates. Overall, MS performed as well as or better than biochemical methods.

Multicenter comparison of laboratory performance in cytomegalovirus and Epstein-Barr virus viral load testing using international standards.

BACKGROUND: Infections with cytomegalovirus (CMV) and Epstein-Barr virus (EBV) remain important in solid organ transplantation. Quantitative viral nucleic acid testing is a major advance to patient management. These assays are limited by a lack of standardization, resulting in viral load measurements that differ among clinical laboratories. The variability in viral load measurements makes interpretation of multicenter clinical trials data difficult. This study compares the current practices in CMV and EBV viral load testing at four large transplant centers participating in multicenter Clinical Trials in Organ Transplantation and the Clinical Trials in Organ Transplantation in Children (CTOT and CTOTC). METHODS: Viral load testing was performed on well-defined viral preparations according to standard operating procedures at each site. RESULTS: Among centers, CMV viral load testing was accurate compared to WHO International Standards and within acceptable variation for this testing method. Epstein-Barr virus viral load data were more variable and less accurate despite the use of international standards. CONCLUSIONS: These data suggest that comparison of CMV, but not EBV, viral load measurements at these sites is possible using current assays and control standards. Standardization of these assays is facilitated using the WHO International Standards and will allow comparison of viral load results among transplant centers. Assay standardization must be performed prior to initiation of multicenter trials.

Tumor necrosis factor α is associated with viral control and early disease progression in patients with HIV type 1 infection.

Inflammation in early human immunodeficiency virus type 1 (HIV-1) disease progression is not well characterized. Ninety patients with untreated primary HIV-1 infection were studied to determine associations of inflammatory proteins with early disease progression. High plasma tumor necrosis factor α (TNF-α) levels (≥8.5 pg/mL) were significantly associated with an increased viral load set point and shorter times to reaching a CD4(+) T-cell count of <500 cells/mm(3) and initiating antiretroviral therapy. The increased risk of reaching a CD4(+) T-cell count of <500 cells/mm(3) in the group with high TNF-α levels was driven by viral load but was independent of concurrent CD4(+) T-cell count. Thus, TNF-α appears to be an important mediator of inflammation in patients with poor viral control and early HIV-1 disease progression.

Multicenter validation of the VITEK MS v2.0 MALDI-TOF mass spectrometry system for the identification of fastidious gram-negative bacteria.

The VITEK MS v2.0 MALDI-TOF mass spectrometry system's performance in identifying fastidious gram-negative bacteria was evaluated in a multicenter study. Compared with the reference method (DNA sequencing), the VITEK MS system provided an accurate, species-level identification for 96% of 226 isolates; an additional 1% were accurately identified to the genus level.

Temporal effect of HLA-B*57 on viral control during primary HIV-1 infection.

BACKGROUND: HLA-B alleles are associated with viral control in chronic HIV-1 infection, however, their role in primary HIV-1 disease is unclear. This study sought to determine the role of HLA-B alleles in viral control during the acute phase of HIV-1 infection and establishment of the early viral load set point (VLSP). FINDINGS: Individuals identified during primary HIV-1 infection were HLA class I typed and followed longitudinally. Associations between HLA-B alleles and HIV-1 viral replication during acute infection and VLSP were analyzed in untreated subjects. The results showed that neither HLA-B*57 nor HLA-B*27 were significantly associated with viral control during acute HIV-1 infection (Fiebig stage I-IV, n=171). HLA-B*57 was however significantly associated with a subsequent lower VLSP (p<0.001, n=135) with nearly 1 log10 less median viral load. Analysis of a known polymorphism at position 97 of HLA-B showed significant associations with both lower initial viral load (p<0.01) and lower VLSP (p<0.05). However, this association was dependent on different amino acids at this position for each endpoint. CONCLUSIONS: The effect of HLA-B*57 on viral control is more pronounced during the later stages of primary HIV-1 infection, which suggests the underlying mechanism of control occurs at a critical period in the first several months after HIV-1 acquisition. The risk profile of polymorphisms at position 97 of HLA-B are more broadly associated with HIV-1 viral load during primary infection and may serve as a focal point in further studies of HLA-B function.