Therapeutic plasma exchange decreases plasma anti-SARS-CoV-2 spike IgG without increasing the proximate incidence of COVID-19.

BACKGROUND: Therapeutic plasma exchange (TPE) removes both pathologic and protective immunoglobulins (Ig). SARS-CoV-2 immunity is partially mediated by anti-SARS-CoV-2 spike antibodies (SAb), which impair viral host-cell invasion. Nonetheless, the systematic effect of TPE on SAb concentration and SARS-CoV-2 immunity is unknown. METHODS: Paired plasma waste specimens from the first (first-TPE) and last (last-TPE) TPE treatment were collected from 9 patients between July 21, 2021 and March 1, 2022. The effects of TPE on Ig levels were assessed by quantitatively comparing the SAb, total IgG, and total IgM levels first-/last-TPE treatment. Complementary qualitative assessment for these changes was achieved via protein electrophoresis (PEP) and immunofixation (IFE). A retrospective review was performed to investigate the incidence of new SARS-CoV-2 infections following TPE v. other treatment at the same outpatient apheresis/infusion center during the same time frame. RESULTS: Median SAb levels between the first- and last-TPE waste specimens decreased significantly from 424.6 AU/mL to 17.0 AU/mL (P = 0.004). Concordantly, PEP and IFE analysis demonstrated broad Ig decreases. Cumulative incidence of subsequent COVID-19 diagnosis at 30, 90, and 180 days post-procedure did not differ between the TPE v. other treatment groups (n = 709 total patients). CONCLUSIONS: TPE significantly reduced SAb levels, a marker of SARS-CoV-2 immunity, but did not appear to provoke increased incidence of COVID-19 infections. Further investigation of the kinetics of TPE-mediated SAb decrease and post-TPE recovery are warranted.

Taking a step back from testing: Preanalytical considerations in molecular infectious disease diagnostics.

Recent studies evaluating the preanalytical factors that impact the outcome of nucleic-acid based methods for the confirmation of SARS-CoV-2 have illuminated the importance of identifying variables that promoted accurate testing, while using scarce resources efficiently. The majority of laboratory errors occur in the preanalytical phase. While there are many resources identifying and describing mechanisms for main laboratory testing on automated platforms, there are fewer comprehensive resources for understanding important preanalytical and environmental factors that affect accurate molecular diagnostic testing of infectious diseases. This review identifies evidence-based factors that have been documented to impact the outcome of nucleic acid-based molecular techniques for the diagnosis of infectious diseases.

Comparative evaluation of blood collection tubes for clinical chemistry analysis.

BACKGROUND: Routine chemistry testing is typically performed using serum or plasma to assess a patient's clinical status. At our institution, serum is the specimen type used. To reduce processing times, evaluation of plasma-based and rapid serum gel separator tubes was performed. METHODS: We compared the results of routine chemistry analytes collected in serum gel separator tubes (SST), plasma gel separator tubes (PST), rapid serum gel separator tubes (RST), and plasma tubes without gel separators (DGT). Result concordance was assessed at baseline (immediate testing after processing) and up to one week of refrigerated storage. Other parameters assessed were the susceptibility to hemolysis and lipemia interference, and changes in results after re-centrifugation. Percent changes were compared against the SST and evaluated according to established bias thresholds. RESULTS: Total protein and potassium results at baseline in plasma-based tubes had percent changes from the SST that exceeded acceptability thresholds. Stability was significantly shortened for glucose, potassium, aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) when collected in the PST as compared to the SST. The RST was the least susceptible to hemolysis and lipemia interferents. Re-centrifugation affected the serum-based analysis of potassium. CONCLUSIONS: Plasma may reduce processing time at the expense of shortened sample stability and may require specimen source-specific reference intervals for potassium and total protein. The RST provides an alternate option to reduce processing time, while maintaining storage stability.

Evaluation of Serological SARS-CoV-2 Lateral Flow Assays for Rapid Point-of-Care Testing.

Rapid point-of-care tests (POCTs) for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific antibodies vary in performance. A critical need exists to perform head-to-head comparisons of these assays. The performances of 15 different lateral flow POCTs for the detection of SARS-CoV-2-specific antibodies were compared on a well-characterized set of 100 samples. Of these, 40 samples from known SARS-CoV-2-infected, convalescent individuals (collected an average of 45 days after symptom onset) were used to assess sensitivity. Sixty samples from the prepandemic era (negative control) that were known to represent infections with other respiratory viruses (rhinoviruses A, B, and C and/or coronavirus 229E, HKU1, and NL63 OC43) were used to assess specificity. The timing of seroconversion was assessed using five lateral flow assays (LFAs) and a panel of 272 longitudinal samples from 47 patients for whom the time since symptom onset was known. Among the assays that were evaluated, the sensitivity and specificity for any reactive band ranged from 55% to 97% and from 78% to 100%, respectively. Assessing the performance of the IgM and the IgG bands alone, sensitivity and specificity ranged from 0% to 88% and 80% to 100% for IgM and from 25% to 95% and 90% to 100% for IgG, respectively. Longitudinal testing revealed that the median times after symptom onset to a positive result were 7 days (interquartile range [IQR], 5.4 to 9.8) for IgM and 8.2 days (IQR, 6.3 to 11.3) for IgG. The testing performances differed widely among LFAs, with greatest amount of variation related to the sensitivity of the assays. The IgM band was the band most likely to misclassify prepandemic samples. The appearances of IgM and IgG bands occurred almost simultaneously.

Evaluation of Serological SARS-CoV-2 Lateral Flow Assays for Rapid Point of Care Testing.

BACKGROUND: Rapid point-of-care tests (POCTs) for SARS-CoV-2-specific antibodies vary in performance. A critical need exists to perform head-to-head comparison of these assays. METHODS: Performance of fifteen different lateral flow POCTs for the detection of SARS-CoV-2-specific antibodies was performed on a well characterized set of 100 samples. Of these, 40 samples from known SARS-CoV-2-infected, convalescent individuals (average of 45 days post symptom onset) were used to assess sensitivity. Sixty samples from the pre-pandemic era (negative control), that were known to have been infected with other respiratory viruses (rhinoviruses A, B, C and/or coronavirus 229E, HKU1, NL63 OC43) were used to assess specificity. The timing of seroconversion was assessed on five POCTs on a panel of 272 longitudinal samples from 47 patients of known time since symptom onset. RESULTS: For the assays that were evaluated, the sensitivity and specificity for any reactive band ranged from 55%-97% and 78%-100%, respectively. When assessing the performance of the IgM and the IgG bands alone, sensitivity and specificity ranged from 0%-88% and 80%-100% for IgM and 25%-95% and 90%-100% for IgG. Longitudinal testing revealed that median time post symptom onset to a positive result was 7 days (IQR 5.4, 9.8) for IgM and 8.2 days (IQR 6.3 to 11.3). CONCLUSION: The testing performance varied widely among POCTs with most variation related to the sensitivity of the assays. The IgM band was most likely to misclassify pre-pandemic samples. The appearance of IgM and IgG bands occurred almost simultaneously.

Advancements in the gold standard: Measuring steroid sex hormones by mass spectrometry.

Sex hormones, such as testosterone and estrogens, play an essential role in regulating physiological and reproductive development throughout the lifetime of the individual. Although variation in levels of these hormones are observed throughout the distinct stages in life, significant deviations from reference ranges can result in detrimental effects to the individual. Alterations, by either an increase or decrease, in hormone levels are associated with physiological changes, decreased reproductive capabilities, and increased risk for diseases. Hormone therapies (HTs) and assisted reproductive technologies (ARTs) are commonly used to address these factors. In addition to these treatments, gender-affirming therapies, an iteration of HTs, are also a prominent treatment for transgender individuals. Considering that the effectiveness of these treatments relies on achieving therapeutic hormone levels, monitoring of hormones has served as a way of assessing therapeutic efficay. The need for reliable methods to achieve this task has led to great advancements in methods for evaluating hormone concentrations in biological matrices. Although immunoassays are the more widely used method, mass spectrometry (MS)-based methods have proven to be more sensitive, specific, and reliable. Advances in MS technology and its applications for therapeutic hormone monitoring have been significant, hence integration of these methods in the clinical setting is desired. Here, we provide a general overview of HT and ART, and the immunoassay and MS-based methods currently utilized for monitoring sex hormones. Additionally, we highlight recent advances in MS-based methods and discuss future applications and considerations for MS-based hormone assays.

A lytic polysaccharide monooxygenase-like protein functions in fungal copper import and meningitis.

Infection by the fungal pathogen Cryptococcus neoformans causes lethal meningitis, primarily in immune-compromised individuals. Colonization of the brain by C. neoformans is dependent on copper (Cu) acquisition from the host, which drives critical virulence mechanisms. While C. neoformans Cu+ import and virulence are dependent on the Ctr1 and Ctr4 proteins, little is known concerning extracellular Cu ligands that participate in this process. We identified a C. neoformans gene, BIM1, that is strongly induced during Cu limitation and which encodes a protein related to lytic polysaccharide monooxygenases (LPMOs). Surprisingly, bim1 mutants are Cu deficient, and Bim1 function in Cu accumulation depends on Cu2+ coordination and cell-surface association via a glycophosphatidyl inositol anchor. Bim1 participates in Cu uptake in concert with Ctr1 and expression of this pathway drives brain colonization in mouse infection models. These studies demonstrate a role for LPMO-like proteins as a critical factor for Cu acquisition in fungal meningitis.

Specific Histidine Residues Confer Histatin Peptides with Copper-Dependent Activity against Candida albicans.

The histidine-rich salivary peptides of the histatin family are known to bind copper (Cu) and other metal ions in vitro; however, the details of these interactions are poorly understood, and their implications for in vivo antifungal activity have not been established. Here, we show that the availability of Cu during exposure of Candida albicans to histatin-5 (Hist-5) modulates its antifungal activity. Antifungal susceptibility testing revealed that co-treatment of Hist-5 with Cu improved the EC50 from ∼5 to ∼1 µM, whereas co-treatment with a high-affinity Cu-specific chelator abrogated antifungal activity. Spectrophotometric titrations revealed two previously unrecognized Cu(I)-binding sites with apparent Kd values at pH 7.4, ∼20 nM, and confirmed a high-affinity Cu(II)-binding site at the Hist-5 N-terminus with an apparent Kd of ∼8 pM. Evaluation of a series of His-to-Ala full-length and truncated Hist-5 peptides identified adjacent His residues (bis-His) as critical anchors for Cu(I) binding, with the presence of a third ligand revealed by X-ray absorption spectroscopy. On their own, the truncated peptides were ineffective at inhibiting the growth of C. albicans, but treatment with supplemental Cu resulted in EC50 values down to ∼5 µM, approaching that of full-length Hist-5. The efficacy of the peptides depended on an intact bis-His site and correlated with Cu(I) affinity. Together, these results establish new structure-function relationships linking specific histidine residues with Cu binding affinity and antifungal activity and provide further evidence of the involvement of metals in modulating the biological activity of these antifungal peptides.

Unusual Synergism of Transferrin and Citrate in the Regulation of Ti(IV) Speciation, Transport, and Toxicity.

Human serum transferrin (sTf) is a protein that mediates the transport of iron from blood to cells. Assisted by the synergistic anion carbonate, sTf transports Fe(III) by binding the metal ion in a closed conformation. Previous studies suggest sTf's role as a potential transporter of other metals such as titanium. Ti is a widely used metal in colorants, foods, and implants. A substantial amount of Ti is leached into blood from these implants. However, the fate of the leached Ti and its transport into the cells is not known. Understanding Ti interaction with sTf assumes a greater significance with our ever increasing exposure to Ti in the form of implants. On the basis of in vitro studies, it was speculated that transferrin can bind Ti(IV) assisted by a synergistic anion. However, the role and identity of the synergistic anion(s) and the conformational state in which sTf binds Ti(IV) are not known. Here we have solved the first X-ray crystal structure of a Ti(IV)-bound sTf. We find that sTf binds Ti(IV) in an open conformation with both carbonate and citrate as synergistic anions at the metal binding sites, an unprecedented role for citrate. Studies with cell lines suggest that Ti(IV)-sTf is transported into cells and that sTf and citrate regulate the metal's blood speciation and attenuate its cytotoxic property. Our results provide the first glimpse into the citrate-transferrin synergism in the regulation of Ti(IV) bioactivity and offers insight into the future design of Ti(IV)-based anticancer drugs.

Sequence proximity between Cu(II) and Cu(I) binding sites of human copper transporter 1 model peptides defines reactivity with ascorbate and O2.

The critical nature of the copper transporter 1 (Ctr1) in human health has spurred investigation of Ctr1 structure and function. Ctr1 specifically transports Cu(I), the reduced form of copper, across the plasma membrane. Thus, extracellular Cu(II) must be reduced prior to transport. Unlike yeast Ctr1, mammalian Ctr1 does not rely on any known mammalian reductase. Previous spectroscopic studies of model peptides indicate that human Ctr1 could serve as both copper reductase and transporter. Ctr1 peptides bind Cu(II) at an amino terminal high-affinity Cu(II), Ni(II) ATCUN site. Ascorbate-dependent reduction of the Cu(II)-ATCUN complex is possible by virtue of an adjacent HH (bis-His), as this bis-His motif and one methionine ligand constitute a high affinity Ctr1 Cu(I) binding site. Here, we synthetically varied the distance between the ATCUN and bis-His motifs in a series of peptides based on the human Ctr1 amino terminal, with the general sequence MDHAnHHMGMSYMDS, where n=0-4. We tested the ability of each peptide to reduce Cu(II) with ascorbate and stabilize Cu(I) under ambient conditions (20% O2). This study reveals that significant differences in coordination structure and chemical behavior with ascorbate and O2 result from changes in the sequence proximity of ATCUN and bis-His. Peptides that deviate from the native Ctr1 pattern were less effective at forming stable Cu(I)-peptide complexes and/or resulted in O2-dependent oxidative damage to the peptide.