Multicenter evaluation of the Toxoplasma gondii Real-TM (Sacace) kit performance for the molecular diagnosis of toxoplasmosis.

The molecular detection of Toxoplasma gondii DNA is a key tool for the diagnosis of disseminated and congenital toxoplasmosis. This multicentric study from the Molecular Biology Pole of the French National Reference Center for toxoplasmosis aimed to evaluate Toxoplasma gondii Real-TM PCR kit (Sacace). The study compared the analytical and clinical performances of this PCR assay with the reference PCRs used in proficient laboratories. PCR efficiencies varied from 90% to 112%; linearity zone extended over four log units (R2 > 0.99) and limit of detection varied from 0.01 to ≤1 Tg/mL depending on the center. Determined on 173 cryopreserved DNAs from a large range of clinical specimens, clinical sensitivity was 100% [106/106; 95 confidence interval (CI): 96.5%-100%] and specificity was 100% (67/67; 95 CI: 94.6%-100%). The study revealed two potential limitations of the Sacace PCR assay: the first was the inconsistency of the internal control (IC) when added to the PCR mixture. This point was not found under routine conditions when the IC was added during the extraction step. The second is a lack of practicality, as the mixture is distributed over several vials, requiring numerous pipetting operations. Overall, this study provides useful information for the molecular diagnosis of toxoplasmosis; the analytical and clinical performances of the Sacace PCR kit were satisfactory, the kit having sensitivity and specificity similar to those of expert center methods and being able to detect low parasite loads, at levels where multiplicative analysis gives inconsistently positive results. Finally, the study recommends multiplicative analysis in particular for amniotic fluids, aqueous humor, and other single specimens.

Automatic patient-level recognition of four Plasmodium species on thin blood smear by a real-time detection transformer (RT-DETR) object detection algorithm: a proof-of-concept and evaluation.

Malaria remains a global health problem, with 247 million cases and 619,000 deaths in 2021. Diagnosis of Plasmodium species is important for administering the appropriate treatment. The gold-standard diagnosis for accurate species identification remains the thin blood smear. Nevertheless, this method is time-consuming and requires highly skilled and trained microscopists. To overcome these issues, new diagnostic tools based on deep learning are emerging. This study aimed to evaluate the performances of a real-time detection transformer (RT-DETR) object detection algorithm to discriminate Plasmodium species on thin blood smear images. The algorithm was trained and validated on a data set consisting in 24,720 images from 475 thin blood smears corresponding to 2,002,597 labels. Performances were calculated with a test data set of 4,508 images from 170 smears corresponding to 358,825 labels coming from six French university hospitals. At the patient level, the RT-DETR algorithm exhibited an overall accuracy of 79.4% (135/170) with a recall of 74% (40/54) and 81.9% (95/116) for negative and positive smears, respectively. Among Plasmodium-positive smears, the global accuracy was 82.7% (91/110) with a recall of 90% (38/42), 81.8% (18/22), and 76.1% (35/46) for P. falciparum, P. malariae, and P. ovale/vivax, respectively. The RT-DETR model achieved a World Health Organization (WHO) competence level 2 for species identification. Besides, the RT-DETR algorithm may be run in real-time on low-cost devices such as a smartphone and could be suitable for deployment in low-resource setting areas lacking microscopy experts.IMPORTANCEMalaria remains a global health problem, with 247 million cases and 619,000 deaths in 2021. Diagnosis of Plasmodium species is important for administering the appropriate treatment. The gold-standard diagnosis for accurate species identification remains the thin blood smear. Nevertheless, this method is time-consuming and requires highly skilled and trained microscopists. To overcome these issues, new diagnostic tools based on deep learning are emerging. This study aimed to evaluate the performances of a real-time detection transformer (RT-DETR) object detection algorithm to discriminate Plasmodium species on thin blood smear images. Performances were calculated with a test data set of 4,508 images from 170 smears coming from six French university hospitals. The RT-DETR model achieved a World Health Organization (WHO) competence level 2 for species identification. Besides, the RT-DETR algorithm may be run in real-time on low-cost devices and could be suitable for deployment in low-resource setting areas.

Which sub-compartments of fat mass and fat-free mass are related to blood viscosity factors?

The size of body compartments is a determinant of several factors of blood viscosity. Red cell aggregation is proportional to fat mass while hematocrit is proportional to both fat-free mass and abdominal adiposity, but which parts of these body components are involved in this relationship is not known. Segmental bioelectrical impedance analysis (sBIA) provides a possibility to delineate the relationships more precisely between various subdivisions of the body and blood viscosity factors, going farther than preceding studies using non segmental BIA. In this study we investigated in 38 subjects undergoing a standardized breakfast test with mathematical modelling of glucose homeostasis and a segmental bioelectrical impedance analysis (sBIA) the relationships between the various compartments of the body and viscosity factors. Blood and plasma viscosity were measured with the Anton Paar rheometer and analyzed with Quemada's model. The parameters better correlated to hematocrit are fat free mass (r = 0.562) and its two components muscle mass (r = 0.516) and non-muscular fat-free mass (r = 0.452), and also trunk fat mass (r = 0.383) and waist-to hip ratio (r = 0.394). Red cell aggregation measurements were correlated with both truncal and appendicular fat mass (r ranging between 0.603 and 0.728). Weaker correlations of M and M1 are found with waist circumference and hip circumference. This study shows that the correlation between lean mass and hematocrit involves both muscle and non-muscle moieties of lean mass, and that both central and appendicular fat are determinants of red cell aggregation.

Assessment of Droplet Digital PCR for the Detection and Absolute Quantification of Toxoplasma gondii: A Comparative Retrospective Study.

Accurate tools for Toxoplasma gondii detection and quantification can be valuable for the early and effective management of toxoplasmosis. Droplet digital PCR (ddPCR) is a next-generation end-point PCR technique with high performance. The objective of the study was to evaluate the performance of ddPCR for the detection and absolute quantification of T. gondii. From January 2019 to October 2020, DNA samples collected at the Laboratory of Parasitology and Mycology of Pitié-Salpêtrière Hospital in Paris were retrospectively analyzed by ddPCR and real-time quantitative PCR (qPCR). To detect T. gondii with the best sensitivity possible, the REP-529 multicopy target was used. For absolute quantification of T. gondii, a specific single-copy target of α-tubulin was designed. T. gondii detection by ddPCR and qPCR was strongly correlated (R2 = 0.93), with a total concordance of 96.7% (n = 145/150). Quantification of T. gondii using ddPCR was successful for 15 of 35 samples showing a parasite load ≥170 copies/mL of DNA eluate using the α-tubulin target. The qPCR REP-529 quantification based on a standard curve was approximate and dependent on the strain genotype, which led to an estimate of parasite copy number 14- to 160-fold superior to the ddPCR result. In total, ddPCR is an effective molecular method for T. gondii detection that shows equivalent performance to qPCR. For robust T. gondii quantification, ddPCR is clearly more accurate than semiquantitative qPCR methods.

Molecular Diagnosis of Toxoplasmosis: Multicenter Evaluation of the Toxoplasma RealCycler Universal PCR Assay on 168 Characterized Human Samples.

Real-time PCR plays a crucial role in the diagnosis of toxoplasmosis. In this multicenter study, the Toxoplasma RealCycler Universal assay was assessed for the diagnosis of toxoplasmosis by eight reference laboratories. DNAs from diverse clinical samples were included: 141 characterized samples from patients with different clinical forms of proven toxoplasmosis and 27 from patients without toxoplasmosis were tested in duplicate with the commercial assay. Final diagnosis was affirmed by each center by analysis of clinical settings and biological follow-up. Calibrated Toxoplasma gondii standards and 11 external quality control samples were also included. Discrepant results observed after the first run of commercial PCR were controlled by both reference and commercial PCR assays. Using the commercial assay, the detection threshold varied from 0.01 to 1 tachyzoites/mL, depending on the center. The relationship between crossing point and DNA concentration was linear over 4 log units (r2 > 0.99), and PCR efficiencies were satisfactory (89% to 104%). The results of the 11 external quality control samples were concordant after one retesting, but those for 3 clinical samples remained discrepant. Sensitivity and specificity were calculated at 97.8% (95% CI, 97.8%-100%) and 100% (95% CI, 87.2%-100%), respectively. Provided that PCRs are performed at least in duplicate to detect low parasitic loads, Toxoplasma RealCycler Universal PCR showed suitable performances to diagnose the different forms of toxoplasmosis.

Beyond the Calorie Paradigm: Taking into Account in Practice the Balance of Fat and Carbohydrate Oxidation during Exercise?

Recent literature shows that exercise is not simply a way to generate a calorie deficit as an add-on to restrictive diets but exerts powerful additional biological effects via its impact on mitochondrial function, the release of chemical messengers induced by muscular activity, and its ability to reverse epigenetic alterations. This review aims to summarize the current literature dealing with the hypothesis that some of these effects of exercise unexplained by an energy deficit are related to the balance of substrates used as fuel by the exercising muscle. This balance of substrates can be measured with reliable techniques, which provide information about metabolic disturbances associated with sedentarity and obesity, as well as adaptations of fuel metabolism in trained individuals. The exercise intensity that elicits maximal oxidation of lipids, termed LIPOXmax, FATOXmax, or FATmax, provides a marker of the mitochondrial ability to oxidize fatty acids and predicts how much fat will be oxidized over 45-60 min of low- to moderate-intensity training performed at the corresponding intensity. LIPOXmax is a reproducible parameter that can be modified by many physiological and lifestyle influences (exercise, diet, gender, age, hormones such as catecholamines, and the growth hormone-Insulin-like growth factor I axis). Individuals told to select an exercise intensity to maintain for 45 min or more spontaneously select a level close to this intensity. There is increasing evidence that training targeted at this level is efficient for reducing fat mass, sparing muscle mass, increasing the ability to oxidize lipids during exercise, lowering blood pressure and low-grade inflammation, improving insulin secretion and insulin sensitivity, reducing blood glucose and HbA1c in type 2 diabetes, and decreasing the circulating cholesterol level. Training protocols based on this concept are easy to implement and accept in very sedentary patients and have shown an unexpected efficacy over the long term. They also represent a useful add-on to bariatric surgery in order to maintain and improve its weight-lowering effect. Additional studies are required to confirm and more precisely analyze the determinants of LIPOXmax and the long-term effects of training at this level on body composition, metabolism, and health.

Multicenter Comparative Assessment of the TIB MolBiol Toxoplasma gondii Detection Kit and Four Laboratory-Developed PCR Assays for Molecular Diagnosis of Toxoplasmosis.

Toxoplasmosis can be a life-threatening infection, particularly during pregnancy and in immunocompromised patients. The biological diagnosis of toxoplasmosis is challenging and has been revolutionized by molecular detection methods. This article summarizes the data of a multicenter study involving four centers to assess the performances of a commercial PCR assay as compared with four in-house PCR assays using Toxoplasma gondii standards, 20 external quality control specimens, and 133 clinical samples. This clinical cohort includes well-characterized clinical samples corresponding to different clinical situations: confirmed congenital toxoplasmosis (44 samples), toxoplasmosis in immunocompromised patients (25 samples), and chorioretinitis (5 samples). Furthermore, 59 samples from patients without toxoplasmosis were included as negative controls. The analytical sensitivities of the five methods tested were very similar; and the limit of Toxoplasma DNA detection was around 0.01 T. gondii genome per reaction for all the methods. The overall concordance between the commercial PCR and the four in-house PCR assays was 97.7% (130/133). The clinical sensitivity and specificity were >98% and could be increased for the commercial kit when PCR was performed in multiplicate to detect low parasitic loads. In conclusion, the commercial PCR assay shows suitable performances to diagnose the different clinical forms of toxoplasmosis.

Coupling Complete Blood Count and Steroidomics to Track Low Doses Administration of Recombinant Growth Hormone: An Anti-Doping Perspective.

Growth Hormone (GH) under its human recombinant homologue (rhGH), may be abused by athletes to take advantage of its well-known anabolic and lipolytic properties; hence it is prohibited in sports by the World Anti-Doping Agency. Due to the rapid turnover of rhGH, anti-doping screening tests have turned to monitor two endocrine biomarkers (IGF-I and P-III-NP), but unfortunately, they show population-wise variability, limiting the identification rate of rhGH users. Previous studies have evidenced the numerous effects of GH on human physiology, especially in hematopoiesis and steroidogenesis. In this work, aiming to discover novel physiological rhGH biomarkers, we analyzed the complete blood count and the steroidomics profile of healthy, physically active, young males treated either with EPO + rhGH or EPO + placebo. The time-trends of these two physiological routes have been analyzed through geometric trajectory analysis (GTA) and OPLS-DA. Individuals supplemented with micro-doses of rhGH exhibited different leukopoietic and steroidal profiles compared to the control population, suggesting a role of the rhGH in both pathways. In the article, hypotheses on the observed differences are discussed according to the most recent literature and compared to results in animal models. The use of leukopoietic and steroidal biomarkers together with endocrine biomarkers (IGF-1 and P-III-NP) allows to correctly classify over 98% of samples with no false positives, miss-classifying only one single sample (false negative) over a total of 56; a promising result, if compared to the current rhGH detection strategies.

Limitations of PCR detection of filarial DNA in human stools from subjects non-infected with soil-transmitted helminths.

The standard techniques for diagnosis of human filariasis are the microscopic examination of blood smears or skin biopsies, which are relatively invasive and poorly sensitive at low levels of infection. Recently, filarial DNA has been detected in fecal samples from non-human primates in Central Africa. The aim of this study was to demonstrate proof-of-concept of a non-invasive molecular diagnosis technique for human filariasis by targeting fragments of 12S rDNA, Cox1, ITS1 and LL20-15kDa ladder antigen-gene by conventional PCR in DNA extracted from stool samples of 52 people infected with Mansonella perstans and/or Loa loa. Of these, 10 patients were infected with soil-transmitted helminths (Trichuris trichiura and/or Ascaris lumbricoides), and none were positive for Necator americanus. Interestingly, no filarial gene fragments were detected in the stools of any of the 52 patients. Future studies should evaluate whether a co-infection with soil-transmitted helminths causing gastrointestinal bleeding and likely allowing (micro)filaria exit into the digestive tract, may facilitate the molecular detection of filarial DNA fragments in stool samples.

TITLE: Limites de la détection par PCR d'ADN de filaires dans les selles humaines de sujets non-infectés par les géohelminthes. ABSTRACT: Les techniques standards de diagnostic des filarioses humaines (examen microscopique de gouttes épaisses ou de biopsies cutanées) sont relativement invasives et peu sensibles à de faibles niveaux d'infection. De l'ADN de filaires a été récemment détecté dans des échantillons de fèces de primates non-humains en Afrique centrale. L'objectif de cette étude était de démontrer la preuve de concept d'un diagnostic moléculaire non invasif des filarioses chez l'homme en ciblant des fragments d'ADNr 12S, Cox1, ITS1 et l'antigène LL20-15kDa par PCR classique. L'ADN a été extrait d'échantillons de selles de 52 personnes infectées par Mansonella perstans et/ou Loa loa. Parmi ces patients, dix étaient infectés par des géohelminthes (Trichuris trichiura et/ou Ascaris lumbricoides) et aucun n'était positif pour Necator americanus. De manière intéressante, aucun fragment de gène de filaires n'a été détecté dans les selles des 52 patients. Des études futures devraient être menées pour évaluer si une coinfection avec des géohelminthes (provoquant des hémorragies gastro-intestinales et permettant probablement l'effraction de (micro)filaires dans le tube digestif) facilite la détection moléculaire de fragments d'ADN de filaires dans les selles.

Impact of pre-analytic step duration on molecular diagnosis of toxoplasmosis for five types of biological samples.

INTRODUCTION: Toxoplasma-PCR is essential to diagnose ocular, cerebral, disseminated and congenital toxoplasmosis. This multicenter study evaluated the impact of sample storage duration at +4°C on PCR assay performances in order to propose guidelines for the storage of samples during shipment or/and before PCR. MATERIALS AND METHODS: Five matrices, amniotic (AF), cerebrospinal (CSF), and bronchoalveolar lavage fluids (BALF), whole blood (WB) and buffy coat (BC), were artificially spiked with different amounts of Toxoplasma gondii (20, 100, 500 tachyzoites per mL of sample) or with previously infected THP1 cells. DNA extractions were performed at day 0 and after 2, 4 and 7 days of storage at +4°C. Each extract was amplified at least twice by real-time PCR. RESULTS: A total of 252 spiked samples was studied. No increase of crossing point was observed and all samples were positive for AF, BALF, BC and infected THP1-spiked WB after up to 7 days at 4°C. For CSF spiked with 20 parasites/mL, only 50% of PCR reactions were positive at D7 (p<0.05). For WB spiked with type II parasites, all reactions remained positive at D7 but amplifications were significantly delayed from D2; and for WB spiked with RH strain, the proportion of positive reactions decreased at D7. CONCLUSION: The storage of clinical samples at +4°C is compatible with the molecular detection of T. gondii parasites. Provided that PCR assays are performed in duplicate, storage of samples is possible up to 7 days. However, from the fifth day onwards, and for samples susceptible to contain low parasitic loads, we recommend to perform the PCR in multiplicate.

Metabolic Influences Modulating Erythrocyte Deformability and Eryptosis.

Many factors in the surrounding environment have been reported to influence erythrocyte deformability. It is likely that some influences represent reversible changes in erythrocyte rigidity that may be involved in physiological regulation, while others represent the early stages of eryptosis, i.e., the red cell self-programmed death. For example, erythrocyte rigidification during exercise is probably a reversible physiological mechanism, while the alterations of red blood cells (RBCs) observed in pathological conditions (inflammation, type 2 diabetes, and sickle-cell disease) are more likely to lead to eryptosis. The splenic clearance of rigid erythrocytes is the major regulator of RBC deformability. The physicochemical characteristics of the surrounding environment (thermal injury, pH, osmolality, oxidative stress, and plasma protein profile) also play a major role. However, there are many other factors that influence RBC deformability and eryptosis. In this comprehensive review, we discuss the various elements and circulating molecules that might influence RBCs and modify their deformability: purinergic signaling, gasotransmitters such as nitric oxide (NO), divalent cations (magnesium, zinc, and Fe2+), lactate, ketone bodies, blood lipids, and several circulating hormones. Meal composition (caloric and carbohydrate intake) also modifies RBC deformability. Therefore, RBC deformability appears to be under the influence of many factors. This suggests that several homeostatic regulatory loops adapt the red cell rigidity to the physiological conditions in order to cope with the need for oxygen or fuel delivery to tissues. Furthermore, many conditions appear to irreversibly damage red cells, resulting in their destruction and removal from the blood. These two categories of modifications to erythrocyte deformability should thus be differentiated.

Rapid diagnostic tests failing to detect infections by Plasmodium falciparum encoding pfhrp2 and pfhrp3 genes in a non-endemic setting.

BACKGROUND: Rapid diagnostic tests (RDTs) detecting the histidine-rich protein 2 (PfHRP2) have a central position for the management of Plasmodium falciparum infections. Yet, variable detection of certain targeted motifs, low parasitaemia, but also deletion of pfhrp2 gene or its homologue pfhrp3, may result in false-negative RDT leading to misdiagnosis and delayed treatment. This study aimed at investigating the prevalence, and understanding the possible causes, of P. falciparum RDT-negative infections at Montpellier Academic Hospital, France. METHODS: The prevalence of falsely-negative RDT results reported before and after the introduction of a loop-mediated isothermal amplification (LAMP) assay, as part as the malaria screening strategy in January 2017, was analysed. Negative P. falciparum RDT infections were screened for pfhrp2 or pfhrp3 deletion; and exons 2 were sequenced to show a putative genetic diversity impairing PfHRP2 detection. RESULTS: The overall prevalence of P. falciparum negative RDTs from January 2006 to December 2018 was low (3/446). Whereas no cases were reported from 2006 to 2016 (0/373), period during which the malaria diagnostic screen was based on microscopy and RDT, prevalence increased up to 4.1% (3/73) between 2017 and 2018, when molecular detection was implemented for primary screening. Neither pfhrp2/3 deletion nor major variation in the frequency of repetitive epitopes could explain these false-negative RDT results. CONCLUSION: This paper demonstrates the presence of pfhrp2 and pfhrp3 genes in three P. falciparum RDT-negative infections and reviews the possible reasons for non-detection of HRP2/3 antigens in a non-endemic setting. It highlights the emergence of falsely negative rapid diagnostic tests in a non-endemic setting and draws attention on the risk of missing malaria cases with low parasitaemia infections using the RDT plus microscopy-based strategy currently recommended by French authorities. The relevance of a novel diagnostic scheme based upon a LAMP assay is discussed.

Inhibition of polymerase chain reaction: Pathogen-specific controls are better than human gene amplification.

PCR inhibition is frequent in medical microbiology routine practice and may lead to false-negative results; however there is no consensus on how to detect it. Pathogen-specific and human gene amplifications are widely used to detect PCR inhibition. We aimed at comparing the value of PCR inhibitor detection using these two methods. We analysed Cp shifts (ΔCp) obtained from qPCRs targeting either the albumin gene or the pathogen-specific sequence used in two laboratory-developed microbiological qPCR assays. 3152 samples including various matrixes were included. Pathogen-specific amplification and albumin qPCR identified 62/3152 samples (2.0%), and 409/3152 (13.0%) samples, respectively, as inhibited. Only 16 samples were detected using both methods. In addition, the use of the Youden's index failed to determine adequate Cp thresholds for albumin qPCR, even when we distinguished among the different sample matrixes. qPCR targeting the albumin gene therefore appears not adequate to identify the presence of PCR inhibitors in microbiological PCR assays. Our data may be extrapolated to other heterologous targets and should discourage their use to assess the presence of PCR inhibition in microbiological PCR assays.

Combined administration of microdoses of growth hormone and erythropoietin: Effects on performance and evaluation of GH detection capability using anti-doping methods.

The combination of growth hormone (GH) and recombinant erythropoietin (rEPO) is thought to be used particularly in endurance sports. Our objective was to reproduce a 2-week administration of rEPO microdoses alone or in combination with GH microdoses (three times a week) on healthy and athletic male subjects and to evaluate if GH had any additional effects compared to EPO treatment alone. The effects of the treatments on hematological parameters and VO2max were studied as well as the detection of GH in serum. While the rEPO microdose regimen was associated with a significant increase in reticulocytes, no clear elevation in hemoglobin concentration (HGB) was observed. Using a correction by plasma volume did not reveal more effects of EPO on HGB. Our results did not show any additional effect when the GH microdoses were co-administered. In addition, no clear increase in VO2max was observed after treatment, with an elevation in only half the subjects in both groups (EPO and EPO+GH). A clear effect of GH on insulin-like growth factor I (IGF-I) was seen but it was lower on procollagen III amino-terminal propeptide (P-III-NP). GH detection using the direct isoform test identified only one subject 24 hours after receiving GH. The GH biomarker test combining IGF-I and P-III-NP was not able to detect the GH administration. However, a longitudinal follow-up of the intraindividual variations showed a significant increase in IGF-I 24 and 48 hours after GH administration in most subjects, while the effect of GH microdoses on P-III-NP was less straightforward.

Shear-dependency of the predicted ideal hematocrit.

 The ideal hematocrit is the hematocrit (Hct) value resulting in the highest value of Hct/viscosity (h/η) ratio and can thus be predicted from viscometric measurements with the use of equations such as Quemada's one which yield the determination of the bell-shaped curve of h/η as a function of Hct. In a series of recent papers we applied this approach to various populations, using viscometry at high shear rate (1000 s-1). However the shape of this curve has been reported to be dependent on the shear rate, resulting in a right-shift in this top value when Hct increase. We present here in 11 young recreative athletes the evolution of the predicted top of the h/η curve and optimal theoretical Hct and the discrepancy between theoretical and optimal values over the range of shear rates 1 to 6000 s-1. Results show that the predicted optimal value of both h/η and Hct increases when shear rate increases and that the discrepancy between predicted laquooptimalraquo and actual values decreases and becomes almost asymptotic at very high shear (500 s-1). It is minimal at 2720 s-1. The correlation between predicted laquooptimalraquo and actual values of both parameters describes the same evolution. Therefore, it is better for assessing h/η and its agreement with theoretical values, and for determining the theoretical ideal hematocrit, to measure blood viscosity at shear rates equal or superior to 500 s-1.

Fetal growth retardation and hemorheological predictors of oxygen delivery in hypertensive vs normotensive pregnant women.

Physiological modifications of blood rheology during pregnancy and their alterations in pregnant hypertensive women have been extensively studied in the 1980's. Since vascular resistance is higher in hypertensive pregnant women whose newborns are small-for gestational-age (SGA), we investigated in a personal database if growth retardation of newborns is related to the oxygen delivery index (ratio hematocrit/blood viscosity) and to the difference between hematocrit (Hct) and the prediction of its optimal valued based on Quemada's equation. A sample of 38 hypertensive pregnant women (age 29 yr±1) was compared with 64 controls matched for age and gestational age, studied at 35±1 weeks gestation, extracted from a larger series of 162 pregnant women. On the whole the hypertensive group gave birth to smaller children (p = 0.014). Plasma viscosity correlated with blood pressure (BP) only in hypertensive women (r = 0.403 p < 0.05). The bell-shaped curve of predicted optimal Hct of non hypertensive pregnant women was similar to that of non-pregnant women, but in hypertensive women it was shifted toward higher values (p = 0.07), and the predicted optimal Hct (but not the actual one) was correlated with systolic blood pressure (SBP) (r = 0.349 p < 0.001) and diastolic blood pressure (DBP) (r = 0.218 p < 0.05). The predicted optimal Hct/viscosity (h/η) ratio was higher in hypertensive women whose newborns exhibited a low birth weight (p = 0.03), resulting in a higher discrepancy between actual and model-predicted «ideal¼ values of h/η ratio (p = 0.03) and Hct (p = 0.02) compared with the subgroup with no growth retardation. Therefore, in hypertensive women whose newborns exhibited a low birth weight, hemorheological parameters predicting oxygen supply are shifted to lower values than predicted by the model.

Leg electrical resistance predicts venous blood viscosity and hematocrit.

 We previously reported that whole body bioelectrical impedance analysis (BIA) measurements are correlated to some hemorheologic factors, suggesting a relationship between viscosity factors and electric properties of flowing blood not only in vitro but also in vivo. Recently we reported that with segmental BIA (analyzing the body considered as composed of 5 cylinders) predictive equations for various determinants of blood viscosity were closer than for the wole body. Another widely used BIA technique uses leg-to-leg impedance measurements so that two cylinders (the two legs) are analyzed. We investigated whether impedance measured with this technique (Tanita TBF-300) is also a predictor of blood viscosity factors. From viscometric measurements performed on venous blood drawn in recreative athletes over the range of shear rates 1 to 6000 s-1 (RHEOMETRE Anton Paar CP 50-1), we found a correlation between leg-leg resistance at 50 kHz (Rx[50 kHz]) and blood viscosity at 1000 s-1 (η1000= 0.0051 Rx[50 kHz] + 1.3265; r = 0.521 p = 0.028 yielding a prediction of η1000 (Bland Altman plot: bias 0.05 [RANGE - 0.24; 0.34]. Neither plasma viscosity nor the red cell rheology index «k¼ of Quemada's model are correlated with Rx[50 kHz], but hematocrit (Hct) does (Hct (%) = 0.0217 Rx[50 kHz] + 33.783; r = 0.480 p = 0.044) yielding a prediction of Hct (Bland Altman plot: bias - 0.11, [range - 1.67; 1.45]. The discrepancy between actual and predicted Hct is also correlated with resistance at 50 kHz (r = 0.575 p = 0.031) as does the discrepancy between actual and predicted Hct/viscosity ratio (r = -0.651 p = 0.006). Therefore, as other previously studied methods, leg to leg BIA predicts viscosity, suggesting that blood rheology may influence the passage of an electric current in the legs.

Evolution of Toxoplasma-PCR methods and practices: a French national survey and proposal for technical guidelines.

The molecular diagnosis of toxoplasmosis lacks standardisation due to the use of numerous methods with variable performance. This diversity of methods also impairs robust performance comparisons between laboratories. The harmonisation of practices by diffusion of technical guidelines is a useful way to improve these performances. The knowledge of methods and practices used for this molecular diagnosis is an essential step to provide guidelines for Toxoplasma-PCR. In the present study, we aimed (i) to describe the methods and practices of Toxoplasma-PCR used by clinical microbiology laboratories in France and (ii) to propose technical guidelines to improve molecular diagnosis of toxoplasmosis. To do so, a yearly self-administered questionnaire-based survey was undertaken in proficient French laboratories from 2008 to 2015, and guidelines were proposed based on the results of those as well as previously published work. This period saw the progressive abandonment of conventional PCR methods, of Toxoplasma-PCR targeting the B1 gene and of the use of two concomitant molecular methods for this diagnosis. The diversity of practices persisted during the study, in spite of the increasing use of commercial kits such as PCR kits, DNA extraction controls and PCR inhibition controls. We also observed a tendency towards the automation of DNA extraction. The evolution of practices did not always go together with an improvement in those, as reported notably by the declining use of Uracil-DNA Glycosylase to avoid carry-over contamination. We here propose technical recommendations which correspond to items explored during the survey, with respect to DNA extraction, Toxoplasma-PCR and good PCR practices.

Seeking the optimal hematocrit: May hemorheological modelling provide a solution?

Hematocrit increases during exercise and is usually decreased after regular training. However the interpretation of these facts is ambiguous since hematocrit is both a determinant of oxygen supply and the major determinant of blood viscosity. Classically hematocrit was assumed to impair blood flow, but it has been evidenced to exert a biphasic effect on it. In order to cope with these two apparently opposite effects of hematocrit, hemorheologists have proposed the concept hematocrit/viscosity ratio (h/η). This h/η ratio is related to tissue oxygenation in vascular diseases (eg, POAD) but not in healthy subjects. h/η displays a bell-shaped curve as a function of hematocrit and the hematocrit value corresponding to the maximal h/η can be assumed to be a theoretically optimal hematocrit. We propose to analyse exercise-related alterations in hematocrit according to this theoretical approach, viscosity at high shear rate being reconstructed with Quemada's equation from actual plasma viscosity and red cell rigidity at various hematocrit levels. While theoretical and actual h/η are fairly correlated in athletes both before and after exercise, actual hematocrit is lower at rest and higher after exercise compared to the theoretical one. The main statistic correlate of these discrepancies between actual and predicted hematocrit is red cell rigidity. Submaximal exercise acutely decreases the h/η ratio (despite increasing both hematocrit and viscosity). This change is well predicted by the model and there is a strong correlation between predicted and actual h/η ratio. Endurance training tends to increase h/η and to reduce the discrepancy between predicted and actual hematocrit. Accordingly trained athletes have a higher h/η (both model-predicted and actual) than sedentary subjects, and a lower hematocrit, this lowering being rather correlated to training volume than to fitness improvement. On the whole, this approach suggests that homeostatic "viscoregulation" in athletes results in a fine tuning of h/η which seems to be a closely regulated parameter. Hematocrit alterations in this context are an adaptation involved in this regulation.