Volume 48, Issue 2

Objectives Therapeutic drug monitoring of β-lactam antibiotics has become an important tool for treatment of severe infections, especially for critically ill patients who often exhibit altered PK/PD. Therapeutic targets are based on MIC, which refers to the active concentration of the drug. Cefazolin, a β-lactam agent used for treating of MSSA bacteraemia, has a protein binding of approximately 80 %. Therefore, a reliable determination of the active, non-protein-bound concentration is required to ensure optimal therapeutic outcome. Methods From seven critically ill patients who received an initial dose of 2 g cefazolin, followed by a continuous 24 h infusion, a total of 24 serum samples were obtained. The non-protein-bound concentration was directly measured after ultrafiltration and compared to prediction based total concentrations and protein binding values from the literature. For the analysis, a rapid and reliable LC-MS 3 based assay was established, offering maximum sensitivity and specificity. Results The measured non-protein-bound concentration varied over a wide range (7.6–118 mg/L), with 22 out of 24 samples exhibiting cefazolin levels above the therapeutic target values (8–16 mg/L). Additionally, the observed protein binding ranged from 29 to 78 % (median 66.8 %), which was significantly lower than that reported in the literature. When comparing the measurements to the predictive performance of total concentrations and protein binding values, poor results were obtained. Conclusions The results show a high variability in plasma protein binding of cefazolin in critically ill patients. Therefore, the “one-dose-fits-all” principle can no longer be considered up to date. For personalised cefazolin therapy based on therapeutic drug monitoring (TDM) it is recommended to determine the active, non-protein-bound drug concentration, as calculations from the total fraction yield poor results.

Objectives Detection of specific antinuclear antibodies is very important in term of diagnosis, prognosis and management of patients with systemic autoimmune rheumatic diseases. Chemiluminescence microarray immunoassay (CLMIA) is a microdot array-based method that allows simultaneous detection of multiple antinuclear antibodies, which received increasing attention. Methods A CLMIA method that can detect 14 kinds of antinuclear antibodies was established and optimized. Basic performance and diagnostic performance of CLMIA was evaluated by comparing it with line immunoassay (LIA) and indirect immunofluorescence (IIF). Results Through conditional exploration, the optimal blocking time and blocking temperature were determined to be 18 h and 25 °C, respectively. The enzyme-labeled secondary antibody reaction concentration was 0.1 μg/mL, the incubation temperature of serum and enzyme-labeled secondary antibody were 30 °C, and the incubation time of serum and enzyme-labeled secondary antibody were 40 min. After parameter optimization, CLMIA demonstrated high accuracy with a relative bias <15 %; high sensitivity with detection limits below 3 IU/mL for dsDNA and below 1 RU/mL for other ANAs; and high reproducibility with both intra-assay and inter-assay coefficients of variation (CV) <15 %.The CLMIA detection method established in this study was also demonstrated to have good clinical diagnostic performance, showing the highest area under curve (AUC=0.87, p=0.042 and p=0.03). The CLMIA and LIA revealed substantial to good agreements on specific antinuclear antibodies except anti-dsDNA, with the Cohen’s kappa from 0.72 to 0.89. Samples that produced discrepant results between the CLMIA and LIA methods were further analyzed. Upon additional testing, most of these samples were ultimately determined to have been correctly detected by the CLMIA assay rather than the LIA assay, suggesting that CLMIA also shows some superiority in diagnosing dsDNA. Conclusions The CLMIA could become a potential routine method for detecting ANAs with the advantages of good detection performance.

Objectives The Treponema pallidum particle agglutination (TPPA) has been used for decades for serological diagnostics of syphilis but is no longer available. Therefore, we evaluated the Treponema IgG ELISA (TpG) alone and in combination with the Treponema IgM ELISA (TpG+M, both from Euroimmun) as possible substitutes for TPPA as a confirmatory test in a two-tier syphilis screening algorithm. Furthermore, we investigated whether a TPPA titer of 5,120 which is used as cut-off for therapeutic decision in pregnant women in Germany can be transferred to an appropriate cut-off value of the TpG. Methods All serum samples with reactive syphilis screening test (CLIA, Diasorin) within a 13-months period were included (n=739). In addition to TPPA and rapid plasma reagin test both ELISA tests were done in all samples. Results Sensitivity, specificity, positive and negative predictive values were 92.2, 100, 100, and 74.5 % for TpG, and 93.2, 85.4, 96.6, and 74.1 % for TpG+M. By ROC analysis the cut-off of TpG corresponding to a TPPA titer ≥5,120 was calculated to be 54 RU/mL with a sensitivity of 99.6 % and a resulting specificity of 58.6 %. Conclusions TpG appears suitable to substitute TPPA as a confirmatory test for syphilis diagnostics but TpG-negative samples have to be evaluated by further tests like FTA-Abs or immunoblot. Treponema IgM determined in addition to TpG did not improve the test performance compared to the TPPA as a reference standard. Valid prediction of a TPPA titer ≥5,120 from TpG result appears not reasonable.

Objectives Adrenocorticotropic hormone (ACTH) is extremely unstable and can easily degrade at room temperature. The experts agreed that the samples should be transported in an ice bath. If it cannot be detected immediately, the plasma should be separated and frozen, which is difficult to carry out in routine practice. This study was performed to explore the preanalytical factors that influence the stability of adrenocorticotrophic hormone (ACTH) measurements. Methods ACTH levels in 21 EDTA whole-blood samples were measured immediately after 0 h and then divided into three equal groups according to the corresponding values (low, L; median, M; and high, H). Next, three sample processing methods (including seven subtypes) were used: whole blood was uncentrifuged (named the A method), stored at 4 °C or 22 °C, centrifuged but not subjected to plasma removal (the B method), stored at 4 °C or 22 °C, and centrifuged with the plasma removed and stored (the C method) at 4 °C, 22 °C, and −20 °C. Each subtype contained three samples, namely, L, M, and H; these samples were retested using a Siemens XP2000 at different times. The change bias was calculated at 0 h. Results Compared to that at 0 h, there was no significant change in ACTH up to 24 h when the sample was stored at 4 °C or 22 °C with the B method (p>0.05), while it significantly changed (up or down >10 %) at 4 °C/24 h (bias is expressed as the mean±SEM; 13.37±21 %, p<0.05) and 22 °C/12 h (9.13±7.68 %, p<0.05) with the A method; and 4 °C/24 h (8.93±5.54 %, p<0.05), 22 °C/12 h (9.5±4.47 %, p<0.05) and −20 °C/3 h (12.03±4.8 %, p<0.05) with the C method. Conclusions After ACTH samples were centrifuged, the presence of plasma without removal did not affect the detection value, and the sample was stored at 4 °C for up to 24 h. There was a significant difference in the detection of ATCH when the sample was stored at −20 °C and thawed again (p<0.05).

Objectives To evaluate the ovarian reserve (OR) in women with antiphospholipid syndrome (APS), systemic lupus erythematosus (SLE), especially SLE-associated APS, and to determine the association between OR and clinical and laboratory parameters. Methods We compared the antral follicle count (AFC), anticardiolipin antibody, and anti-Müllerian hormone (AMH), inhibin B (INHB), antiphospholipid (aPL) antibody, follicle-stimulating hormone (FSH), progesterone (P), testosterone (T), and estradiol (E2) among patients with primary APS (PAPS), SLE-APS, and SLE who were treated at Jinhua Central Hospital between 2017 and 2020. We conducted correlations and logistic regression analyses to identify the risk factors of OR failure in women with APS. Results Serum AMH were positively correlated with AFC and INHB in APS patients, and low AMH was independent risk factor for OR decline in APS patients. The ROC curve showed a high accuracy for AMH in the prediction of OR failure. Compared to healthy subjects (HS), patients with PAPS, SLE-APS, and SLE exhibited lower serum AMH, AFC, INHB, and E2 levels and higher FSH and levels (p<0.05). Of all the patients, those with SLE-APS manifested the lowest serum AMH, AFC, INHB, and E2 levels and the highest FSH levels (p<0.05). Conclusions APS and SLE patients showed lower indications of OR, including AFC and AMH, compared to HS. SLE-APS patients also appeared to have a lower OR than either SLE or PAPS patients.

Objectives The GeneSoC ® that launched recently enables the quantitative detection of target genes (in approximately 15 min) using microfluidic thermal cycling technology. Here, we compared the diagnostic performance of the “GeneSoC ® SARS-CoV-2 N2 Detection Kit” (Kyorin assay) and conventional severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection assays to verify the clinical usefulness of the Kyorin assay. Methods Two hundred samples (100 nasopharyngeal and 100 saliva specimens) were collected from patients with suspected SARS-CoV-2 infection between May 2020 and August 2021. Conventional SARS-CoV-2 detection assays were performed using the Japanese National Institute of Infectious Diseases (NIID) assay, Ampdirect™ 2019-nCoV Detection Kit (SHIMADZU assay), and Lumipulse Presto SARS-CoV-2 Ag (FUJIREBIO assay), according to each manufacturer’s instructions. Results Using the NIID and SHIMADZU assays as references, the positive and negative concordance rates and the kappa coefficient in the Kyorin assay were 96.9–97.9 %, 99.0–100.0 %, and 0.96–0.98, respectively. The positivity rate of the FUJIREBIO assay was slightly lower than that of the reference assay (p<0.05). Conclusions The Kyorin assay showed a favorable concordance rate with conventional SARS-CoV-2 detection assays, making it a useful, rapid, and high-performance assay for detecting SARS-CoV-2, which may lead to early diagnosis and appropriate treatment.