Cross-evaluation of five slidemakers and three automated image analysis systems: The pitfalls of automation?

INTRODUCTION: Automated slidemakers and stainers and digital microscopes are coupled with haematology analysers to achieve better efficiency and cost-effectiveness. This study evaluates the integrated performance of slidemakers and digital microscopes commonly available on the market. METHODS: We compared the percentage of neutrophils for five slidemakers (two Siemens Advia Autoslides, a SysmexSP-10 and SP-50 and an Abbot Alinity hs) and a Horiba Hemaprep to the corresponding haematology analyser data (Siemens Advia 2120i, Sysmex XN and Abbot Alinity hq). Differential leucocyte counting (DLC) was performed on three different CellaVision digital microscopes (DM96, DM1200 and DI-60) and manually. The quality of the smears was assessed using a CellaVision SmearChecker. RESULTS: We observed a significant positive absolute bias (P < .05) for the percentage of neutrophils with the Autoslide and Alinity hs smears on the digital microscopes, but not when DLC was performed manually. The SP-10 and SP-50 showed no bias regardless of the DLC method. No bias was observed for the Hemaprep smears. All the smears had an acceptable monolayer quality, stain intensity and colour. All smears, except those from Sp-10, were of an acceptable length. CONCLUSION: Users should be aware of a potential lack of accuracy that can be encountered when using some slidemakers and digital microscopes. All laboratories should validate or verify the differential counts from slidemakers and digital microscope with automated cell differential counters. Manual count validation should only be considered if a significant proportion of clinically relevant abnormal cells are present. Otherwise, haematology analyser results should be favoured.

The value of seroprevalence data as surveillance tool for Lyme borreliosis in the general population: the experience of Belgium.

BACKGROUND: Serological surveillance, based on the measurement of the presence of specific antibodies in a given population, can be used in addition to traditional and routine disease surveillance methods. The added value of this has been largely documented for vaccine-preventable diseases, but to a lesser extent for vector-borne diseases. This study aimed to evaluate the utility of seroprevalence data as additional source of information on the epidemiology of Lyme borreliosis in Belgium. METHODS: In total, 3215 residual blood samples collected in 2013-2015 were analysed with Liaison® Borrelia IgG kit (DiaSorin S.p.A, Saluggia, Italy). Positive and equivocal results were further examined with immunoblotting (recomLine Borrelia IgG kit, Mikrogen, Neuried, Germany). Crude prevalence estimates of equivocal and seropositive results were calculated and further adjusted accounting for clustered sampling and standardized for age, sex and population per province, according to the Belgian population structure in 2014. The effect of age, sex and region on seropositivity was assessed using log-binomial regression. RESULTS: The overall weighted national seroprevalence for Borrelia burgdorferi sensu lato, adjusted for clustered sampling, age, sex and province was 1.06% (95%CI 0.67-1.67). Although not statistically significant, the highest prevalences were observed in men and in those younger than 15 years or older than 59 years of age. At provincial level, the seroprevalence estimates do not follow the geographical distribution of tick bites and diagnoses of Lyme borreliosis as detected through other surveillance systems. CONCLUSIONS: Although the use of residual samples for seroprevalence estimates has several advantages, it seems to be a limited tool for serological surveillance of Lyme borreliosis in Belgium, other than follow-up of trends if repeated over time. A population-based sampling strategy might provide a more representative nationwide sample, but would be very time intensive and expensive. Seroprevalence studies within risk groups or risk areas in Belgium could provide a useful alternative approach to complement routine surveillance data of Lyme borreliosis.

Chromosomal Amplification of the blaOXA-58 Carbapenemase Gene in a Proteus mirabilis Clinical Isolate.

Horizontal gene transfer may occur between distantly related bacteria, thus leading to genetic plasticity and in some cases to acquisition of novel resistance traits. Proteus mirabilis is an enterobacterial species responsible for human infections that may express various acquired ß-lactam resistance genes, including different classes of carbapenemase genes. Here we report a Proteus mirabilis clinical isolate (strain 1091) displaying resistance to penicillin, including temocillin, together with reduced susceptibility to carbapenems and susceptibility to expanded-spectrum cephalosporins. Using biochemical tests, significant carbapenem hydrolysis was detected in P. mirabilis 1091. Since PCR failed to detect acquired carbapenemase genes commonly found in Enterobacteriaceae, we used a whole-genome sequencing approach that revealed the presence of blaOXA-58 class D carbapenemase gene, so far identified only in Acinetobacter species. This gene was located on a 3.1-kb element coharboring a blaAmpC-like gene. Remarkably, these two genes were bracketed by putative XerC-XerD binding sites and inserted at a XerC-XerD site located between the terminase-like small- and large-subunit genes of a bacteriophage. Increased expression of the two bla genes resulted from a 6-time tandem amplification of the element as revealed by Southern blotting. This is the first isolation of a clinical P. mirabilis strain producing OXA-58, a class D carbapenemase, and the first description of a XerC-XerD-dependent insertion of antibiotic resistance genes within a bacteriophage. This study revealed a new role for the XerC-XerD recombinase in bacteriophage biology.

Quantification of darunavir and etravirine in human peripheral blood mononuclear cells using high performance liquid chromatography tandem mass spectrometry (LC-MS/MS), clinical application in a cohort of 110 HIV-1 infected patients and evidence of a potential drug-drug interaction.

OBJECTIVES: To describe the validation of a sensitive high performance liquid chromatography tandem mass spectrometry (LC-MS/MS) method allowing the simultaneous quantification of darunavir (DRV) and etravirine (ETR) in peripheral blood mononuclear cells (PBMCs) and its application in a cohort of HIV-1 infected patients. METHODS: Blood samples were obtained from 110 patients. PMBCs were isolated using density gradient centrifugation. Drug extraction from PBMCs was performed with a 60:40 methanol-water (MeOH-H2O) solution containing deuterated IS (DRV-d9 and ETR-d8). The chromatographic separation was performed on a RP18 XBridge™ column. RESULTS: The geometric mean (GM) of cell associated concentration ([DRV]CC) and plasmatic concentration ([DRV]plasma) were 360.5ng/mL (CI95%:294.5-441.2) and 1733ng/mL (CI95%:1486-2021), respectively. A geometric mean intracellular (IC)/plasma ratio (GMR) of 0.21 (CI95%:0.18-0.24) was calculated. Adjusted for dose/body surface area and post-intake time, a statistically significant correlation was observed between [DRV]Plasma and the eGFR (p=0.002) and between [DRV]Plasma and the concomitant use of ETR (p=0.038). For the 10 patients receiving ETR in addition to DRV, the GM of [ETR]Plasma (available for 8 out of 10 patients) and [ETR]CC were 492.3ng/mL and 2951ng/mL respectively. The GMR of ETR was 7.6 (CI95%: 3.61-13.83). CONCLUSIONS: A handy and sensitive high performance LC-MS/MS method allowing the simultaneous quantification of DRV and ETR in PBMCs has been described and successfully applied in the largest cohort of DRV-treated patients reported to date. ETR accumulates more efficiently in PBMCs compared to DRV. We have also highlighted a possible impact of ETR on DRV plasma concentrations requiring further investigations.

Anti-Müllerian hormone testing: Evaluation of a novel method allowing more automation.

The anti-Müllerian hormone (AMH), a 140 kDa homodimeric glycoprotein of the transforming growth factor ß (TGF-ß) superfamily, is a useful serum biomarker of ovarian reserve. As an indicator, it mainly allows for the evaluation of primary ovarian insufficiency, the diagnosis of polycystic ovary syndrome (PCOS) and the prediction of the outcome of in vitro fertilization. Despite its increasing clinical importance, the methods for measuring AMH concentrations still require various manual steps. In this regard, our data showed that a novel fully-automated AMH immunoassay has excellent analytical performances, as well as a significant relationship with a well-established AMH enzyme linked immunosorbent assay (ELISA). The within- and between-run coefficients of variation (CV) of the new Elecsys(®) AMH immunoassay were ≤ 2.5%. The limit of detection of this automated assay was 0.01 ng/mL. While the correlation between the Elecsys(®) AMH method and the Ansh(®) Labs Ultrasensitive AMH/MIS ELISA was excellent (r = 0.97, p < 0.0001), a bias was nonetheless observed. An automated assay format for AMH can certainly be advantageous in reducing the analysis turnaround time and in consolidating assay. However, AMH assays are not standardized and interchangeable, as confirmed by our study, and a transition to routine therefore requires a careful evaluation and close communication with physicians.