Barriers to implementation of rapid identification and antimicrobial susceptibility testing technologies in the clinical microbiology laboratory: an American perspective.

Clinical microbiology laboratories are responsible for confirming the aetiology of infectious diseases and providing antimicrobial susceptibility testing results. Traditional culture-based testing can be augmented by more rapid testing modalities to provide clinically actionable information as quickly as possible. Despite improvements in patient outcomes, many clinical microbiology laboratories are facing challenges to in-sourcing these technologies.

A program for real-time surveillance of SARS-CoV-2 genetics.

The COVID-19 pandemic brought forth an urgent need for widespread genomic surveillance for rapid detection and monitoring of emerging SARS-CoV-2 variants. It necessitated design, development, and deployment of a nationwide infrastructure designed for sequestration, consolidation, and characterization of patient samples that disseminates de-identified information to public authorities in tight turnaround times. Here, we describe our development of such an infrastructure, which sequenced 594,832 high coverage SARS-CoV-2 genomes from isolates we collected in the United States (U.

Feasibility of using real-world free thyroxine data from the US and Europe to enable fast and efficient transfer of reference intervals from one population to another.

Objectives: The direct approach for determining reference intervals (RIs) is not always practical. This study aimed to generate evidence that a real-world data (RWD) approach could be applied to transfer free thyroxine RIs determined in one population to a second population, presenting an alternative to performing multiple RI determinations.

Design And Methods: Two datasets (US, n = 10,000; Europe, n = 10,000) were created from existing RWD.

Cheaper, faster, simpler trypsin digestion for high-throughput targeted protein quantification.

Introduction: LC-MS-based methods for protein quantification have a stigma of being relatively expensive and low-throughput. This is partly due to the cost and speed of trypsin digestion, which has primarily focused on advancements in research-based biomarker discovery applications that rely on protein/peptide identifications rather than clinical biomarker quantification. However, there is a need for simple, fast, and reproducibly efficient surrogate peptide recovery in clinical biomarker quantification.

Maximizing Microsampling: Measurement of Comprehensive Metabolic and Lipid Panels Using a Novel Capillary Blood Collection Device.

Background: Demand continues to grow for patient-centric sampling solutions that enable collection of small volumes of blood outside of healthcare facilities. Various technologies have been developed to facilitate sample collection but gaps in knowledge remain, preventing these technologies from replacing standard venipuncture.

Methods: A novel blood collection device, Touch Activated Phlebotomy (TAP) II® from YourBio Health, and standard fingerstick collection using a BD Microtainer® were utilized to collect capillary serum samples.

Evaluation of a Compact, Portable Centrifuge for Separating Microvolume Blood Samples at the Point of Collection.

Background: The increased demand for decentralized blood sample collection presents numerous operational challenges for diagnostics providers. Sample degradation including sample hemolysis due to time, temperature, and handling between collection and laboratory analysis leads to limited test menus and unreliable results. Here we introduce the lightweight, portable Labcorp TrueSpin™ for rapid point-of-care blood separation using commercially available microvolume blood collection tubes.

The pipetting Olympics: Propagating proper pipetting in clinical LC-MS/MS analysis.

Introduction: Engaging pipetting events were developed to assess and challenge technicians' practical sample handling using matrices common to the clinical laboratory. As correct pipetting stands as a prerequisite for accurate clinical laboratory testing, this helped to understand sources of imprecision and bias attributed to the underlying step of aspirating and dispensing patient samples and internal standard in clinical LC-MS/MS assays while highlighting the importance for the clinical laboratory to evaluate this source of variability on an on-going basis and mitigate its impact.

Methods: The events involved pipetting water, methanol, serum, and whole blood.

Pre-analytical conditions influencing analysis of folate in dried plasma microsamples.

Introduction: Determination of folate insufficiency is of considerable interest given its importance in fetal development and red blood cell formation; however, access to blood tests may be limited due to the requirement for phlebotomy as well as controlled temperature shipping of blood specimens to laboratories for testing due to the inherent instability of folate and its vitamers.

Methods: An LC-MS/MS test was developed and validated for the measurement of 5-methyltetrahydrofolate (5MTHF) in dried plasma specimens collected from fingerstick blood using a laminar flow blood separation device, as well as liquid venous plasma for comparison. Two pre-analytical factors investigated influencing the measurement of 5MTHF in dried plasma were hemolysis of the fingerstick blood during collection and storage/shipment of the dried plasma.

Clinical irrelevance of lower titer thyroglobulin autoantibodies in patients with differentiated thyroid carcinoma.

Objective: Thyroglobulin (Tg) is an established tumor marker for differentiated thyroid carcinoma (DTC) patients. However, Tg immunoassays can be subject to Tg autoantibody (TgAb) interference resulting in incorrect Tg values. Therefore, Tg measurement with liquid chromatography-tandem mass spectrometry (LC-MS/MS) could be promising in patients with TgAbs.

Monitoring of SARS-CoV-2 antibodies using dried blood spot for at-home collection.

The utilization of vaccines to fight the spread of SARS-CoV-2 has led to a growing need for expansive serological testing. To address this, an EUA approved immunoassay for detection of antibodies to SARS-CoV-2 in venous serum samples was investigated for use with dried blood spot (DBS) samples. Results from self-collected DBS samples demonstrated a 98.

Chlamydia trachomatis Variants Escaping Detection in the Aptima Combo 2 Assay in the United States.

Background: The Aptima Combo 2 (AC2) assay manufactured by Hologic, Inc., detects Neisseria gonorrhoeae and/or Chlamydia trachomatis (CT) in urogenital and extragenital specimens by targeting either a 16S rRNA (N. gonorrhoeae) or 23S rRNA (CT) region.

Biochemical Diagnosis of Acute Hepatic Porphyria: Updated Expert Recommendations for Primary Care Physicians.

Acute hepatic porphyria (AHP) is a group of rare, metabolic diseases where patients can experience acute neurovisceral attacks, chronic symptoms, and long-term complications. Diagnostic biochemical testing is widely available and effective, but a substantial time from symptom onset to diagnosis often delays treatment and increases morbidity. A panel of laboratory scientists and clinical AHP specialists collaborated to produce recommendations on how to enhance biochemical diagnosis of AHP in the USA.

Clinical risk assessment of biotin interference with a high-sensitivity cardiac troponin T assay.

Objectives Biotin >20.0 ng/mL (81.8 nmol/L) can reduce Elecsys® Troponin T Gen 5 (TnT Gen 5; Roche Diagnostics) assay recovery, potentially leading to false-negative results in patients with suspected acute myocardial infarction (AMI).

Empiricism in Microsampling: Utilizing a Novel Lateral Flow Device and Intrinsic Normalization to Provide Accurate and Precise Clinical Analysis from a Finger Stick.

Background: Phlebotomy plays a key role in clinical laboratory medicine but poses certain challenges for the patient and the laboratory. Dried blood spots simplify collection and stabilize specimens effectively, but clinical reference intervals are based primarily on serum or plasma. We evaluated use of dried separated blood plasma specimens to simplify plasma sample collection via finger stick; however, this sampling technique posed substantial analytical challenges.

Quality over quantity: A qualitative, targeted bottom-up proteomics approach to genotyping apolipoprotein L1.

A targeted, bottom-up proteomic assay was developed for the qualitative detection of apolipoprotein L1 (ApoL1) protein variants (G0, G1, and G2) in blood plasma for identification of high and low renal risk genotypes. Following trypsin digestion of liquid or dry plasma, surrogate peptides specific to each ApoL1 variant were detected by liquid chromatography-tandem mass spectrometry along with two surrogate peptides common among all variants which served as internal (positive) controls to verify correct sample processing. Using isotopically labeled peptide internal standards, the presence or absence of each surrogate peptide was determined using multiple objective metrics including: 1) retention time confirmation relative to its internal standard, 2) comparison of the internal standard-normalized response relative to pre-established thresholds for confident detection, and 3) ion ratio monitoring.

Prevalence of Mycoplasma genitalium infection in women with bacterial vaginosis.

Background: Bacterial vaginosis (BV) is a common condition in reproductive-age women and is known to be positively associated with risk of acquisition of sexually transmitted infections (STI) such as chlamydia and gonorrhea. Mycoplasma genitalium is an emerging STI that has been linked to increased risk of pelvic inflammatory disease, adverse pregnancy outcomes and infertility. In the present study we sought to examine whether women diagnosed with symptomatic BV were at increased risk of having concurrent infection with Mycoplasma genitalium.

More sensitivity is always better: Measuring sub-clinical levels of serum thyroglobulin on a µLC-MS/MS system.

Although liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays for thyroglobulin (Tg) are resistant to autoantibody (TgAb) interference, recent studies have demonstrated approximately 40% of TgAb-positive individuals with recurrent thyroid cancer have Tg concentrations below the lower limit of quantification (LLOQ) of the LC-MS/MS assays described to date (i.e., <0.

Syphilis Testing among Men Who Have Had Rectal Gonorrhea and Chlamydia Tests, United States.

The Centers for Disease Control and Prevention (CDC) recommends syphilis screening at least annually for sexually active men who have sex with men (MSM). The objective of this study is to assess the frequency of MSM testing for syphilis and how syphilis test results compared with results of rectal gonorrhea and chlamydia tests. In collaboration with a large US commercial laboratory, we identified men aged 15-60 years who had rectal chlamydia or gonorrhea tests during 09/01/2013-09/30/2015 as presumed MSM.

Validation of apixaban anti-factor Xa assay and impact of body weight.

Background: Direct oral anticoagulants (DOACs) are widely used as therapies for venous thromboembolism and other cardiovascular diseases. However, routine coagulation monitoring is not required, but may be clinically indicated in high risk populations including obese patients.

Objectives: The aims of this study were two fold; to validate a chromogenic assay for anti-factor Xa measurement in patients taking apixaban, and correlate it with PT/INR and PTT, and to measure anti-factor Xa levels in patients who weighed >120 kg.

Health care utilization and costs following amplified versus non-amplified molecular probe testing for symptomatic patients with suspected vulvovaginitis: a US commercial payer population.

Background: Vulvovaginitis (VV) is a common reason women seek medical attention in the USA. Both the non-specific clinical presentation and risk of preterm labor or delivery necessitate accurate identification of the causative agents to guide appropriate therapy. The diagnostic accuracy of amplified molecular probe testing (AMP) has been shown to exceed that of non-amplified molecular probe (NAMP) by 20%-25%.

Sensitization profiles to hazelnut allergens across the United States.

Background: Measurement of IgE antibody to hazelnut components can aid in the prediction of allergic responses to the food.

Objective: To investigate the association between patient demographics (age, location) and patterns of allergic sensitization to hazelnut components across the United States and to investigate the degree of correlation between hazelnut sensitization with sensitization to other tree nuts, peanuts, and their components.

Methods: Serum samples from 10,503 individuals with hazelnut extract specific IgE (sIgE) levels of 0.

Multicenter study establishing the clinical validity of a nucleic-acid amplification-based assay for the diagnosis of bacterial vaginosis.

The present study sought to validate the clinical performance of a previously described PCR-based assay for the diagnosis of bacterial vaginosis (BV). A total of 1579 patients were enrolled in 5 locations; samples were classified as BV positive (n=538) or negative (n=1,041) based on an algorithm utilizing quantitative Gram-stain analysis of vaginal discharge and clinical evaluation (Amsel criteria); a next-generation sequencing (NGS) approach to determining diversity of vaginal microbiota was used to resolve discordant results between BV-PCR and Nugent/Amsel. BV-PCR demonstrated a sensitivity of 96.

Trueness, precision and stability of the LIAISON 1-84 parathyroid hormone (PTH) third-generation assay: comparison to existing intact PTH assays.

Background: Over the past few decades, parathyroid hormone (PTH) immunoassays have progressed through successive generations resulting in increased specificity and accuracy for detecting circulating PTH. With the introduction of third-generation assays, in which the biologically active PTH(1-84) is specifically targeted, the PTH(7-84) and other fragments are not detected. The specific recognition of only PTH(1-84) whole molecule allows for more reliable standardization and calibration than with the existing assays.

Development and Implementation of a Coagulation Factor Testing Method Utilizing Autoverification in a High-volume Clinical Reference Laboratory Environment.

Background: Testing coagulation factor activities requires that multiple dilutions be assayed and analyzed to produce a single result. The slope of the line created by plotting measured factor concentration against sample dilution is evaluated to discern the presence of inhibitors giving rise to nonparallelism. Moreover, samples producing results on initial dilution falling outside the analytic measurement range of the assay must be tested at additional dilutions to produce reportable results.