Diagnostic performance of four SARS-CoV-2 antibody assays in patients with COVID-19 or with bacterial and non-SARS-CoV-2 viral respiratory infections.

SARS-CoV-2 antibody assays are used for epidemiological studies and for the assessment of vaccine responses in highly vulnerable patients. So far, data on cross-reactivity of SARS-CoV-2 antibody assays is limited. Here, we compared four enzyme-linked immunosorbent assays (ELISAs; Vircell SARS-CoV-2 IgM/IgA and IgG, Euroimmun SARS-CoV-2 IgA and IgG) for detection of anti-SARS-CoV-2 antibodies in 207 patients with COVID-19, 178 patients with serological evidence of different bacterial infections, 107 patients with confirmed viral respiratory disease, and 80 controls from the pre-COVID-19 era. In COVID-19 patients, the assays showed highest sensitivity in week 3 (Vircell-IgM/A and Euroimmun-IgA: 78.9% each) and after week 7 (Vircell-IgG: 97.9%; Euroimmun-IgG: 92.1%). The antibody indices were higher in patients with fatal disease. In general, IgM/IgA assays had only limited or no benefit over IgG assays. In patients with non-SARS-CoV-2 respiratory infections, IgG assays were more specific than IgM/IgA assays, and bacterial infections were associated with more false-positive results than viral infections. The specificities in bacterial and viral infections were 68.0 and 81.3% (Vircell-IgM/IgA), 84.8 and 96.3% (Euroimmun-IgA), 97.8 and 86.0% (Vircell-IgG), and 97.8 and 99.1% (Euroimmun-IgG), respectively. Sera from patients positive for antibodies against Mycoplasma pneumoniae, Chlamydia psittaci, and Legionella pneumophila yielded particularly high rates of unspecific false-positive results in the IgM/IgA assays, which was revealed by applying a highly specific flow-cytometric assay using HEK 293 T cells expressing the SARS-CoV-2 spike protein. Positive results obtained with anti-SARS-CoV-2 IgM/IgA ELISAs require careful interpretation, especially if there is evidence for prior bacterial respiratory infections.

Evaluation of the Amplex eazyplex Loop-Mediated Isothermal Amplification Assay for Rapid Diagnosis of Pneumocystis jirovecii Pneumonia.

Quantitative PCR (qPCR) assays are the gold standard for diagnosis of Pneumocystis jirovecii pneumonia (PCP). However, they are laborious and require skilled personnel. Therefore, execution outside regular working hours of the molecular biology laboratory is limited. The eazyplex P. jirovecii assay (PJA) uses loop-mediated isothermal amplification for detection of P. jirovecii It is performed directly with respiratory specimens, without the need for special skills, and delivers a result within 3 to 25 min. The goal of our study was to compare the performance of the eazyplex PJA with that of established P. jirovecii qPCR assays. All archived bronchoalveolar lavage fluid (BALF) samples that had previously tested positive for P. jirovecii by qPCR assay and 50 control samples (retrospective part), as well as all BALF samples received for P. jirovecii analysis over a period of 4 months (prospective part), were tested. Forty-nine patients with proven PCP and 126 patients without PCP were included. The sensitivity and specificity of the eazyplex PJA (95.7% and 96.5%, respectively) were comparable to those for three different P. jirovecii qPCR assays. The detection limit of the eazyplex PJA was analogous to 103 copies of the major surface glycoprotein gene per 25 µl of BALF, corresponding to 10 to 20 P. jirovecii cells. The eazyplex PJA reliably discriminated patients with PCP from patients with P. jirovecii colonization. It delivered a positive result within a mean of 9 min 38 s and required a hands-on time of 2 min 45 s. In summary, the eazyplex PJA showed identical performance for the diagnosis of PCP, compared to qPCR assays. However, in terms of time to result, practicability, and robustness, the eazyplex PJA is clearly superior and allows for around-the-clock molecular testing.

Prolonged stability by cyclization: Macrocyclic phosphino dipeptide isostere inhibitors of beta-secretase (BACE1).

Cyclization of recently reported linear phosphino dipeptide isostere inhibitors of BACE1 via side chain olefin metathesis yielded macrocyclic BACE1 inhibitors. The most potent compound II-P1 (IC(50) of 47nM) and the corresponding linear analog I were tested for serum stability. The approach led to three times prolonged half life serum stability of 44min for the macrocyclic inhibitor II-P1 compared to the linear compound I.

Development and evaluation of peptidic ligands targeting tumour-associated urokinase plasminogen activator receptor (uPAR) for use in alpha-emitter therapy for disseminated ovarian cancer.

PURPOSE: Among gynecologic malignancies, ovarian cancer has the highest mortality due to rapid peritoneal dissemination. Treatment failure particularly arises from failure to eliminate disseminated cells. Our aim was to develop peptidic radioligands targeting tumour cell-associated urokinase receptor (uPAR, CD87) for alpha-emitter therapy for advanced ovarian cancer. METHODS: DOTA-conjugated, uPAR-directed ligands were synthesised on solid-phase. Binding of peptides to human cells expressing uPAR was assayed by flow cytofluorometry or, in case of (213)Bi-labelled peptides, by measuring cell-bound radioactivity. Bio-distribution of the (213)Bi-labelled peptide P-P4D was analysed in nude mice 28 days after intraperitoneal inoculation of OV-MZ-6 ovarian cancer cells in the absence or presence of the plasma expander gelofusine. RESULTS: uPAR-selective ligands were developed based on published high-affinity uPAR-binding peptides. For preparation of N-terminally cross-linked divalent ligands, a novel solid-phase procedure was developed. Specific binding of (213)Bi-labelled peptides to monocytoid U937 and OV-MZ-6 cells was demonstrated using the natural ligand of uPAR, pro-uPA, or a soluble form of uPAR, suPAR, as competitors. The pseudo-symmetrical covalent dimer (213)Bi-P-P4D displayed superior binding to OV-MZ-6 cells in vitro. Accumulation of (213)Bi-P-P4D in tumour tissue was demonstrated by bio-distribution analysis in nude mice bearing intraperitoneal OV-MZ-6-derived tumours. Gelofusine reduced kidney uptake of (213)Bi-P-P4D by half. CONCLUSION: Ovarian cancer cells overexpressing uPAR were specifically targeted in vitro and in vivo by (213)Bi-P-P4D. Kidney uptake of (213)Bi-P-P4D was distinctly reduced using gelofusine. Thus, this radiopeptide may represent a promising option for therapy for disseminated ovarian cancer.

Synthesis and biological evaluation of phosphino dipeptide isostere inhibitor of human beta-secretase (BACE1).

Phosphino dipeptide (PDP) isosteres are known to be useful analogues of the transition state of metalloprotease substrates. Here we describe the use of this unit for the design of aspartic protease inhibitors. A PDP analogue of OM00-3, a potent BACE1 inhibitor, was synthesized and exhibited high biological activity (IC50 approximately 12 nM).