Splenic infarction associated with transient anti-prothrombin antibodies is a rare manifestation of acute Mycoplasma pneumoniae infection.

We report the first paediatric case of splenic infarction following acute Mycoplasma pneumoniae infection with induction of anti-prothrombin (aPT) antibodies. A 12-year-old boy was admitted to the paediatric emergency department for a left pleuropneumonia and a splenic infarction. aPT antibodies were transitorily detected. The patient recovered fully after antibiotic therapy and a 3-month course of anticoagulation treatment. Antiphospholipid (aPL) antibodies induced by acute infections have already been reported but cases of clinically relevant thrombosis remain rare. The pathogenicity of aPT antibodies is discussed here. We hypothesize that these antibodies were involved in this symptomatic hypercoagulable state.

Effect of double-filtration plasmapheresis for antibody-mediated rejection on hemostasis parameters and thrombin generation.

INTRODUCTION: Donor-specific alloantibodies (DSAs) cause kidney-allograft loss in chronic antibody-mediated rejection (CAMR). Treatment relies on blocking antibody-producing cells and removing DSAs by apheresis: e.g., double-filtration plasmapheresis (DFPP). MATERIALS AND METHODS: To determine the impact of DFPP (6 or 8 sessions/patient) on clotting factors and natural anticoagulants, and on thrombin generation, we performed a prospective and observational study in five CAMR kidney-transplant patients who received DFPP plus rituximab therapy. Thrombin generation was performed in poor platelet plasma (PPP) with 5 pM tissue factor without and with 2 nM recombinant human thrombomodulin. RESULTS: After the first DFPP session, median levels of high molecular-weight proteins (fibrinogen, FV, FVIII, FXI, FXIII, von Willebrand factors and α2-MG) decreased significantly to <50% of baseline values, whereas levels of low molecular-weight factors (<100 kDa) were not significantly modified, except for protein S and TFPI. Of note, binding-protein (BP) S, i.e., C4BP, was significantly decreased. Over the course of successive DFPP sessions, both high and lower molecular-weight proteins (<100 kDa) with longer half-lives (>2 days, prothrombin and factor XII) were significantly decreased. DFPP also highly affected thrombin generation in the absence of thrombomodulin but not significantly in the presence of thrombomodulin. After the first DFPP session, mean endogenous thrombin potential (ETP) and peak thrombin (PH) significantly decreased when the thrombin generation assay was performed without thrombomodulin (respectively, 1084 nM·min for ETP and 210 nM for PH after the first DFPP session compared to 1616 nM·min and 264 nM at baseline). In the presence of thrombomodulin, there was only a slight decrease in ETP and PH (respectively 748 nM·min, and 172 nM after the first DFPP session compared to 822 nM·min and 179 nM at baseline). After the last session, median ETP and PH decreased respectively to 646 nM·min and 143 nM without thrombomodulin, and, to 490 nM·min and 117 nM with thrombomodulin. CONCLUSIONS: DFPP significantly removed high molecular-weight proteins from the haemostatic system and profoundly decreased levels of protein S and TFPI. Overall thrombin-generation balance was only moderately affected in the presence of thrombomodulin. Nevertheless, high depletion of fibrinogen, FXIII and Von Willebrand Factor may expose patients to an increased risk of bleeding.

Impact of four direct oral anticoagulants on rotational thromboelastometry (ROTEM).

INTRODUCTION: Rotational Thromboelastometry (ROTEM) is a point of care method used to monitor coagulation during surgery and to guide transfusion strategies in patients presenting with severe bleeding. The aim of our study was to determine the impact of four direct oral anticoagulants (DOACs) on 3 commonly used ROTEM tests. METHODS: Whole blood samples from 20 healthy donors were spiked in vitro with apixaban, edoxaban, rivaroxaban or dabigatran at 5 different plasma concentrations (0-1000 ng/mL). EXTEM, INTEM and FIBTEM tests were systematically performed. RESULTS: There was a linear relationship between the increase in clotting time (CT) and plasma DOAC concentrations in both the EXTEM and INTEM tests. We found that the DOAC concentration required to double EXTEM CT was 1042 ± 225 ng/mL for apixaban, 134 ± 38 ng/mL for edoxaban, 176 ± 26 ng/mL for rivaroxaban and 284 ± 73 ng/mL for dabigatran. INTEM CT was less sensitive than EXTEM CT whatever the anticoagulant. EXTEM CT was above the normal range for 5 of 5 spiked samples when the plasma concentrations were ~1000 ng/mL for apixaban, ~100 ng/mL for edoxaban, ~200 ng/mL for rivaroxaban and ~200 ng/mL for dabigatran. Maximum Clot Firmness in EXTEM, INTEM and FIBTEM tests was not affected whatever the DOAC or its concentration. CONCLUSION: This study found a DOAC dose-dependent increase in ROTEM CTs. ROTEM tests were only poorly impacted by low levels of edoxaban, rivaroxaban or dabigatran. Apixaban had only a low effect even at high concentrations.

High-yield production of secreted active proteins by the Pseudomonas aeruginosa type III secretion system.

The Escherichia coli system is the system of choice for recombinant protein production because it is possible to obtain a high protein yield in inexpensive media. The accumulation of protein in an insoluble form in inclusion bodies remains a major disadvantage. Use of the Pseudomonas aeruginosa type III secretion system can avoid this problem, allowing the production of soluble secreted proteins.

Adapting a conventional PCR assay for Toxoplasma gondii detection to real-time quantitative PCR including a competitive internal control.

We have developed a quantitative PCR assay (LightCycler* using the pair of primers JW58 and JW59 for the detection of the 35-fold repeated B gene of oxoplasma gondii. This real-time PCR, using fluorescence resonance energy transfert (FRET) hybridization probes, allows the quantification of . gondii with several technical requirements not previously described: i) an internal amplification control (co-amplified in a single tube with the same primers), ii) Uracil-N-Glycosylase and iii) a standard curve corresponding to a serial dilution from a calibrated suspension of T. gondii ranging from 40 to 4.106( )parasites in one ml of amniotic fluid (1 to 105( ) . gondii/PCR). In artificial samples, one parasite could be detected if at least three reactions were performed.