Validation of in vitro-produced and freeze-dried whole cell lysate antigens for ELISA Trypanosoma evansi antibody detection in camels.

Trypanosoma evansi is a blood parasite responsible for surra in mammals, with a high impact in camels and horses. The WOAH-recommended reference method for detecting immunoglobulin G directed against T. evansi is ELISA, using whole cell lysate antigens (WCLAs). WCLAs are prepared with T. evansi produced in laboratory rodents, separated from blood cells using DE-cellulose anion exchange chromatography. As parasite lysates are fragile, antigens are preserved frozen pending use. For these reasons and others, T. evansi WCLAs are not commercially available. They are produced in small quantities, in a limited number of specialized laboratories, and they require a reliable and expensive cold chain for their shipment. In this study, we assessed and validated in vitro production of T. evansi and lyophilization of WCLAs in comparison with the reference method using frozen WCLAs prepared with parasites produced in rodents. Using a set of 400 samples monthly collected from 12 naturally infected camels followed-up for 1384 days, and two batches of referenced serum samples (infected, n = 12; non-infected, n = 15), statistical studies on qualitative and semi-quantitative results of the ELISAs did not show any significant difference when comparing the four combinations of parasites produced in vivo or in vitro, and frozen or freeze-dried WCLSAs. A repeatability study (28 repeats in 9 serum samples) was fully satisfying (p-value = 0.055). With the more convenient in vitro-produced freeze-dried WCLAs it was possible to: (i) avoid the ethical concern of in vivo production, (ii) improve the standardization of antigen production, (iii) secure antigen preservation during shipment and (iv) save a considerable amount of money (DE52-cellulose and dry-ice cold chain being avoided). Additional studies with other Trypanosoma spp are required for further extending ELISA to regional laboratories in enzootic areas, especially in view of the current progress in the "Progressive Control Pathway" (PCP) for trypanosomes in Africa.

No more cold-chain failures, using dehydrated reagents in ELISA antibody-detection against animal trypanosomes of African origin.

Animal trypanosomoses due to trypanosomes of African origin (ATAO), mainly caused by Trypanosoma congolense type Savannah (TCS), T. brucei brucei (TBB), T. vivax (TV), and T. evansi, are widespread diseases that affect domestic and wild mammals and have a huge economic impact. ATAO clinical suspicions are usually confirmed by parasitological and molecular methods, while sero-epidemiological surveys are generally carried out using the OIE-recommended ELISA method based on whole cell lysate soluble antigens (WCLSA) from purified trypanosomes; this reagent is usually stored frozen. With a view to expanding this ELISA test, we assessed, standardized, and validated the use of dehydrated rather than frozen WCLSA and serum samples. For the three ELISA assays (TV, TCS & TBB), a repeatability study revealed no significant difference between repeats. The results obtained using frozen rather than freeze-dried antigen and serum strongly correlated for Pearson's correlation values (>0.93) and Lin's measure ("very good" to "excellent"). Reproducibility was robust, with Pearson's correlation values >0.97 for inter technician effects, and 0.87 (TV) to 0.97 (TBB & TCS) for inter-laboratory tests; their combination was "very satisfactory" to "excellent" according to Lin's measure and there was no impact on qualitative test results. Dehydrated reagents offer the advantage of shipment at room temperature, allowing the secured provision of reagents to regional laboratories. Together with a compendium of standard diagnostic protocols for ATAO (/OIE), dehydrated reagents will enable the serological diagnosis of ATAO at regional level in endemic countries. This very welcome improvement in the context of the Progressive Control Pathway for trypanosomes, recently launched by African countries, will possibly be extended to Latin America in the near future.

Early detection of postoperative acute kidney injury by Doppler renal resistive index in cardiac surgery with cardiopulmonary bypass.

BACKGROUND: Acute kidney injury (AKI) is common after cardiac surgery, affecting outcome. Early detection of an AKI marker is likely to speed diagnosis and implementation of measures to preserve renal function. In septic shock and unselected ventilated subjects, an increased Doppler renal resistive index (RRI) is a predictor of AKI. This study aims to determine whether RRI would act similarly in the postoperative setting of cardiac surgery. METHODS: This study included 65 subjects aged more than 60 yr undergoing elective heart surgery with cardiopulmonary bypass (CPB) and at risk of AKI. All presented at least one AKI risk factor [arteritis, diabetes, or serum creatinine (sCr) clearance of 30-60 ml min(-1)] and were haemodynamically stable without arrhythmia. Doppler RRI was measured in the immediate postoperative period (POP) while subjects were ventilated and sedated. AKI was assessed when sCr increased 30% above the preoperative baseline. RESULTS: Eighteen subjects developed AKI between days 1 and 4, with six requiring dialysis. RRI in the POP was increased in AKI [RRI: 0.79 (0.08) with AKI vs 0.68 (0.06) without AKI, P<0.001], correlating to AKI severity [0.68 (0.06) without AKI, 0.77 (0.08) with AKI but no dialysis, and 0.84 (0.03) with AKI and dialysis, P<0.001]. RRI was similar in subjects receiving catecholamines. RRI >0.74 in the POP predicted delayed AKI with high sensitivity and specificity (0.85 and 0.94, respectively). Multivariate analysis showed that AKI was associated with increased RRI and transfusion. CONCLUSIONS: RRI used in the immediate POP after cardiac surgery with CPB enabled prediction of delayed AKI and anticipation of its severity.

[One case of nosocomial A(H1N1)v2009 influenza in Réunion Island]. / Un cas de grippe A(H1N1)v2009 nosocomiale à la Réunion.

A 19-year-old patient admitted in an oncology unit for an autograft (Hodgkin disease), developed on day 20 a fatal acute respiratory failure and multiple organ failure due to an infection of the A(H1N1)v2009 virus, which was acquired in the hospital, despite partial preventive measures. At that time, the specific vaccine was not available in Réunion. We discuss the nosocomial origin of the infection. Following the epidemic wave, the vaccination rate of the general population and the hospital employees remains very low.

[Severe cases of A(H1N1)v2009 infection in Réunion Island in 2009 and 2010]. / Formes graves d'infection par le virus A(H1N1)v2009 à La Réunion en 2009 et en 2010.

In the Southern hemisphere, Réunion Island acts as a sentinel for infections preferentially occurring during the austral winter that are likely to reach the Northern hemisphere a few months later. We relate the main features concerning patients that were admitted during years 2009 and 2010 in our intensive care unit with an A(H1N1)v2009 infection, mainly for acute respiratory distress. Demographic, clinical, and biological data as well as given medications and outcome were prospectively collected among all PCR-confirmed influenza-infected patients. In 2009 and 2010, 25 patients met the criteria. Patients' median age was 40.4 (±17.4) years. Most of them (22/25) had comorbidities such as: chronic diseases, overweight, obesity, pregnancy, and Down syndrome. Maximum bed-occupation rate was 10 days per million inhabitants. Main diagnosis for ICU admission was virus-related pneumonia. Twenty-two out of 25 patients needed mechanical ventilation, some required rescue therapies such as extracorporeal membranous oxygenation (ECMO) or hi-frequency oscillation ventilation (HFOV), both only available in few French hospitals. Within the study period, 12 patients died (48%) mainly of multi-organ failure. Through 2009 and 2010 autumn and winter periods, for several weeks, the A(H1N1)v2009 virus infection resulted in a significant increase of workload in Réunion Island ICUs. In 2010, the failure of the mass immunization campaign, particularly among the at-risk groups, led to severe cases of A(H1N1)v2009 infections, particularly among patients with comorbidities. Our data may contribute toward better management of influenza virus pandemics in the future.

[Medical paediatric simulation: a European survey]. / Enquête européenne sur la pratique de la simulation médicale pédiatrique.

OBJECTIVE: To present the effective applications of paediatric medical simulation in terms of education, evaluation, density, development, goals and constraints. STUDY DESIGN: Survey realized within 38 paediatric simulation centres (PSC) in Europe, identified by Web search and through the Society in Europe for Simulation Applied to Medicine (SESAM. RESULTS: Twenty centers answered the questionnaire (52%). Ninety-four percent of the PSC had beforehand acquired an experience of adult medical simulation, 94.6% of the PSC were created before 2006. Ninety percent of the PSC owned at least one high-fidelity pediatric simulator. The 80% of the PSC indicated multiple funding sources. Eighty percent of the PSC had at least one specific instructor for the paediatric simulation (average=2.7 paediatric instructors per centre). The PCS reported to get onto various topics: neonatology (25% of the PCS), prehospital medicine (36.8%), paediatric anaesthesiology (74%) and paediatric intensive care (89%). Simulation allowed 70% of the centers to lead some research project. Ninety-five percent of the centers agreed about an European collaboration on research projects or about the mutualization of the created scenarios. CONCLUSION: The material, financial and human means of the interviewed centres are consequential but heterogeneous in Europe. MSP offers numerous and various application fields and generates some research activity.

First outbreak of Trypanosoma evansi in camels in metropolitan France.

The first outbreak of trypanosomosis caused by Trypanosoma evansi in camels in France was reported on a farm in the Aveyron Department. Five camels were imported from the Canary Islands to the farm in early July 2006, and trypanosomes were observed on a stained blood smear from one of them, which died in October. On further investigations, trypanosomes were observed in the blood of five camels, three of them indigenous to the farm and two that had been imported. On the basis of microscopical examination (morphological criteria and measurements) and serological results based on the card agglutination T evansi test and PCR typing, the parasites were identified as T evansi. After treatment with melarsomine, the infected camels rapidly became negative by parasitological tests and were negative two to four months later by serological tests. The parasite was probably transmitted by tabanids and Stomoxys calcitrans, which were abundant in July to September 2006. No parasites were observed in other animals on the farm or on neighbouring farms, but some of the sheep on these farms were positive by PCR or serology.

Pharmacokinetics of antiviral polyoxometalates in rats.

Polyoxometalates are soluble mineral compounds formed principally of oxide anions and early transition metal cations. The polyoxometalates K12H2[P2W12O48].24H2O (JM 1591), K10[P2W18Zn4(H2O)2O68].20H2O (JM 1596), and [(CH3)3NH]8[Si2W18Nb6O77] (JM 2820) demonstrate potent antiviral activity against human immunodeficiency virus types 1 and 2, herpes simplex virus, and cytomegalovirus in vitro. The preclinical pharmacokinetics of these three compounds were characterized after single-dose intravenous administration of 50 mg/kg to rats. Plasma, urine, and feces were collected for 168 h, and polyoxometalate concentrations were determined by atomic emission. Serum protein binding was measured by equilibrium dialysis. All three compounds were highly bound to serum proteins in a concentration-dependent manner. Total and unbound concentrations of the three compounds in plasma declined in a triexponential manner with terminal half-lives of 246.0 +/- 127.0, 438.4 +/- 129.4, and 32.2 +/- 5.37 h (mean +/- standard deviation) for JM 1591, JM 1596, and JM 2820, respectively. Systemic clearances based on total concentrations in plasma were low, averaging 0.016 +/- 0.002, 0.015 +/- 0.002, and 0.018 +/- 0.003 liter/h/kg for JM 1591, JM 1596, and JM 2820, respectively. The clearances of unbound compounds from plasma averaged 0.966 +/- 0.136, 0.050 +/- 0.005, and 0.901 +/- 0.165 liter/h/kg for JM 1591, JM 1596, and JM 2820, respectively. For JM 1596, the clearance of unbound compound from the kidneys was lower than the glomerular filtration rate (0.086 liter/h/kg), suggesting this polyoxometalate underwent renal tubular reabsorption. However, JM 1591 and JM 2820 appeared to undergo tubular secretion. The fraction of the dose recovered in urine was 11.5, 46.8, and 10.6% for JM 1591, JM 1596, and JM 2820, respectively. Approximately 5% of the dose of each polyoxometalate was recovered in feces. The steady-state volume of distribution based on total concentrations averaged 1.44 liters/kg for JM 1591, 2.39 liters/kg for JM 1596, and 0.59 liter/kg for JM 2820, indicating moderate to wide distribution throughout the body. All three compounds were detected in various tissues 1 week after single-dose administrations, with the highest levels found in the kidneys and liver. The results of this study indicate that the disposition of polyoxometalates is highly dependent on their molecular structure.

Mechanism of alterations in isolated rat liver mitochondrial function induced by gold complexes of bidentate phosphines.

Au(DPPE)+2 (bis[1,2-bis(diphenylphosphino)ethane] gold(I] is an organo-gold antineoplastic agent that has anti-tumor activity in a variety of in vitro cell lines and in vivo rodent tumor models. Preliminary studies suggested that this compound represented a novel class of inhibitors of mitochondrial function. The purpose of this study was, therefore, to determine the mechanism of mitochondrial dysfunction induced by Au(DPPE)+2. Au(DPPE)+2 induced a rapid, dose-related collapse of the inner mitochondrial membrane potential (EC50 = 28.0 microM) that was not potentiated by Ca2+ preloading. Au(DPPE)+2-induced dissipation of mitochondrial membrane potential was accompanied by an efflux of Ca2+ from mitochondria upon exposure to Au(DPPE)+2. Ca2+ efflux in these experiments was via a reversal of the Ca2+ uniporter as efflux could be inhibited with ruthenium red. Au(DPPE)+2 did not increase the permeability of mitochondria to oxalacetate, indicating that the collapse of membrane potential may not be a result of gross increased inner membrane permeability. However, Au(DPPE)+2 may mediate an increased permeability of the inner membrane to cations and protons. Au(DPPE)+2 caused passive swelling in potassium acetate buffer in the absence of valinomycin, suggesting Au(DPPE)+2 facilitated the exchange of H+ and K+. Ca2+ cycling was not extensive and did not contribute to the decrease in membrane potential. These data suggest that one possible mechanism of Au(DPPE+2-induced uncoupling of mitochondrial oxidative phosphorylation is via increased permeability of the inner mitochondrial membrane to cations. The disruption of mitochondrial function may be a key process leading to hepatocyte cell injury by this drug.