[High-throughput sequencing for infectious disease diagnoses: Example of shotgun metagenomics in central nervous system infections]. / Séquençage à haut débit pour le diagnostic en maladies infectieuses : exemple de la métagénomique shotgun dans les infections du système nerveux central.

The advent of high-throughput sequencing in clinical microbiology is opening the way to new diagnostic and prognostic approaches in infectious diseases. Detection, identification and characterisation of pathogenic microorganisms are essential steps in diagnosis and implementation of appropriate antimicrobial therapy. However, standard methods of microbiological diagnosis are failing in some cases. In addition, the emergence of new infections, facilitated by international travel and global warming, requires the implementation of innovative diagnostic methods. Among the different strategies used in clinical microbiology and reviewed in this article, shotgun metagenomics is the only technique that allows today a panpathogenic and unbiased detection of all microorganisms potentially responsible for an infectious disease, including those still unknown. The aims of this article are to present the different possible strategies of high-throughput sequencing used in the microbiological diagnosis of infectious diseases and to highlight the diagnostic contribution of shotgun metagenomics in the field of central nervous system infections.

Targeting lung macrophages for fungal and parasitic pulmonary infections with innovative amphotericin B dry powder inhalers.

The incidence of fungal pulmonary infections is known to be on the increase, and yet there is an alarming gap in terms of marketed antifungal therapies that are available for pulmonary administration. Amphotericin B (AmB) is a highly efficient broad-spectrum antifungal only marketed as an intravenous formulation. Based on the lack of effective antifungal and antiparasitic pulmonary treatments, the aim of this study was to develop a carbohydrate-based AmB dry powder inhaler (DPI) formulation, prepared by spray drying. Amorphous AmB microparticles were developed by combining 39.7 % AmB with 39.7 % γ-cyclodextrin, 8.1 % mannose and 12.5 % leucine. An increase in the mannose concentration from 8.1 to 29.8 %, led to partial drug crystallisation. Both formulations showed good in vitro lung deposition characteristics (80 % FPF < 5 µm and MMAD < 3 µm) at different air flow rates (60 and 30 L/min) when used with a DPI, but also during nebulisation upon reconstitution in water.

[Drug-induced acute angle closure glaucoma]. / Quels sont les médicaments à risque de précipiter un glaucome aigu par fermeture de l'angle ?

Acute angle closure glaucoma is an ophthalmic emergency that can lead to blindness in some cases. The presenting signs are often suggestive, like ocular pain and blurred vision accompanied by headache, nausea and vomiting. These symptoms must be recognized as soon as possible, and the patient must be addressed, urgently, to an ophthalmologist for treatment. Many drugs may lead to an acute angle closure glaucoma in patients with risk factors. This article aims to remind the anatomical risk factors as well as the drugs that may induce an acute angle closure glaucoma. For a better understanding, this article will provide a brief reminder of the pathophysiological mechanism of acute angle closure glaucoma.

Le glaucome aigu par fermeture de l'angle, appelé communément crise de glaucome, est une urgence ophtalmologique pouvant potentiellement conduire à la cécité. Les symptômes sont assez bruyants, les manifestations les plus fréquentes sont une douleur oculaire et une vision floue accompagnées de céphalées, de nausées et de vomissements. Ces symptômes doivent être reconnus le plus rapidement possible afin de référer le patient chez un ophtalmologue pour une prise en charge urgente. Plusieurs médicaments peuvent précipiter la survenue d'une crise chez un patient prédisposé. Il convient donc de rappeler les facteurs de risque anatomiques ainsi que les médicaments pouvant précipiter un glaucome aigu par fermeture de l'angle. Pour une meilleure compréhension, cet article fait un rappel succinct des mécanismes physiopathologiques impliqués dans la survenue d'un glaucome aigu par fermeture de l'angle.

Modelled Target Attainment after Temocillin Treatment in Severe Pneumonia: Systemic and Epithelial Lining Fluid Pharmacokinetics of Continuous versus Intermittent Infusions.

The objective of this article is to describe the population pharmacokinetics (PK) of temocillin administered via continuous infusion (CI) versus intermittent infusion (II) in critically ill patients with pneumonia. Secondary objectives included characterization of epithelial lining fluid (ELF)/plasma penetration ratios and determination of the probability of target attainment (PTA) for a range of MICs. Thirty-two mechanically ventilated patients who were treated for pneumonia with 6 g of temocillin daily for in vitro sensitive pathogens were assigned to either the II (2 g every 8 h over 0.5 h) or the CI (6 g over 24 h after a loading dose of 2 g) group. A population pharmacokinetic model was developed using unbound plasma, and total ELF concentrations of temocillin and related Monte Carlo simulations were performed to assess PTAs. The area under the concentration-time curve from 0 to 24 h (AUC0-24) ELF/plasma penetration ratio was 0.73, at steady state, for both modes of infusion and whatever the level of creatinine clearance. Monte Carlo simulations showed that for the minimal pharmacodynamic (PD) targets of 50% T > 1× MIC (II group) and 100% T > 1× MIC (CI group), PK/PD breakpoints were 4 mg/L in plasma and 2 mg/L in ELF and 4 mg/L in plasma and ELF, respectively. The breakpoint was 8 mg/L in ELF for both modes of infusion in patients with creatinine clearance (CLCR) < 60 mL/min/1.73 m2. While CI provides better PKPD indexes, the latter remain below available recommendations for systemic infections, except in the case of moderate renal impairment, thereby warranting future clinical studies in order to determine the efficacy of temocillin in severe pneumonia.

Severity of parechovirus infections in infants under 3 months of age and comparison with enterovirus infections: A French retrospective study.

While enteroviruses (EV) are a well-recognized cause of aseptic meningitis in children, human parechoviruses (HPeV), especially genotype 3, have been increasingly reported as a frequent cause of sepsis-like illness and meningitis among young infants. The aim of this study was to describe the epidemiological, clinical, and laboratory characteristics of HPeV infections in infants and to compare them with those of well-known EV infections. This monocentric retrospective study was carried out at the pediatric unit of Nantes University Hospital from January 2015 to August 2018. All patients under 18 years of age with diagnosis codes referring to fever, for whom viral infection was suspected and cerebrospinal fluid (CSF) specimens were collected, were included. All CSF specimens were screened by duplex real-time polymerase chain reaction (PCR) assay that allows for the simultaneous detection of EV and HPeV in clinical samples. During the study period, 1373 CSF specimens from patients under 18 were included. A total of 312 CSF samples were positive for HPeV (n=34) or EV (n=278). Among the 34 HPeV-positive patients, 97% (33/34) were under 3 months of age, whereas the rate was 54% (149/278) for EV-positive patients (P<0.001); thus, patients under 3 months of age were defined as the study population for the rest of this work. A review of the medical records was carried out for the positive cases. In this population, the HPeV detection rate was 5.6% versus 25.3% (P<0.001) for EV. All but one of the HPeV samples available for genotyping were HPeV-3. No seasonality was observed for HPeV infections. Length of hospital stay tended to be longer for children infected with HPeV compared with those infected by EV (3 days vs. 2 days, P=0.05). Clinicians reported more severe illness presentations among HPeV-infected infants, with more frequent administration of fluid bolus (P<0.02). Regarding laboratory characteristics, a significant lack of cellular reaction in the CSF (P=0.004) as well as lower C-reactive protein (CRP) levels (P=0.006) and neutrophil counts (P<0.001) were noted for HPeV infections compared with EV infections. Our results confirm the early onset of HPeV infections (more than 95% of patients aged under 3 months). The clinical presentation and laboratory characteristics of the two infections was similar. However, some higher clinical severity criteria and a lack of CSF pleocytosis were regularly observed in patients with HPeV infections. Considering the significant proportion (5.6%; 95% CI, 3.7-7.5) of all CSF samples in our series, HPeV detection should be systematically included in the microbiological diagnosis of febrile children under 3 months of age.

Sequential Time-Kill, a Simple Experimental Trick To Discriminate between Pharmacokinetics/Pharmacodynamics Models with Distinct Heterogeneous Subpopulations versus Homogenous Population with Adaptive Resistance.

Experiments were conducted with polymyxin B and two Klebsiella pneumonia isogenic strains (the wild type, KP_WT, and its transconjugant carrying the mobile colistin resistance gene, KP_MCR-1) to demonstrate that conducting two consecutive time-kill experiments (sequential TK) represents a simple approach to discriminate between pharmacokinetics/pharmacodynamics models with two heterogeneous subpopulations or adaptive resistance.

Pain Modulation Mechanisms in ASD Adults.

We tested endogenous pain modulation mechanisms in adults with autism spectrum disorders (ASD). Nineteen ASD adults without intellectual disabilities were included, matched with 19 healthy volunteers on the basis of sex and chronological age. An experimental pain model was used to measure excitatory and inhibitory pain mechanisms in a single session. Statistical analyses indicated that endogenous pain modulation mechanisms in ASD group did not differ significantly from those of healthy adults. The pain scores were very disparate in ASD group with a greater range of extreme scores than in control group. Unlike schizophrenic patients, there was no systematic dysfunction of endogenous excitatory pain modulation mechanisms, but the high variability requires to be wise to interpret the results and formulate conclusion.

Semi-mechanistic PK/PD modelling of combined polymyxin B and minocycline against a polymyxin-resistant strain of Acinetobacter baumannii.

OBJECTIVES: To expand on previous reports of synergy between polymyxin B (PMB) and minocycline (MIN) against Acinetobacter baumannii; and to gain insight into the qualitative and quantitative determinants of their synergy. METHODS: A semi-mechanistic pharmacokinetic/pharmacodynamic (PK/PD) model was developed on the basis of data from in vitro time-kill experiments with determination of resistant bacterial count to describe the effects of PMB and MIN alone and in combination. The model was enriched by complementary experiments providing information on the characteristics of the resistant subpopulation. RESULTS: The model successfully described the data and made possible quantification of the strength of interaction between the two drugs and formulation of hypotheses about the mechanisms of the observed interaction. The effect of the combination was driven by MIN, with PMB acting as an helper drug; simulations at clinically achievable concentrations showed that 1.5 mg/L MIN +0.2 mg/L PMB is expected to produce sustained killing over 30 hours, while 0.3 mg/L MIN +1 mg/L PMB is met by bacterial regrowth. Interaction equations showed that maximal synergy is reached for PMB concentrations ≥0.1 mg/L and MIN concentrations ≥1 mg/L. CONCLUSIONS: Semi-mechanistic PK/PD modelling was used to investigate the quantitative determinants of synergy between PMB and MIN on a PMB-resistant A. baumannii strain. The developed model, improving on usual study techniques, showed asymmetry in the drug interaction, as PMB acted mostly as a helper to MIN, and provided simulations as a tool for future studies.

Pharmacodynamic modelling of ß-lactam/ß-lactamase inhibitor checkerboard data: illustration with aztreonam-avibactam.

OBJECTIVES: Checkerboard experiments followed by fractional inhibitory concentration (FIC) index determinations are commonly used to assess in vitro pharmacodynamic interactions between combined antibiotics, but FIC index cannot be determined in case of antibiotic/non-active compound combinations. The aim of this study was to use a simple modelling approach to quantify the in vitro activity of aztreonam-avibactam, a new ß-lactam-ß-lactamase inhibitor combination. METHODS: MIC checkerboard experiments were performed with 12 Enterobacteriaceae with diverse ß-lactamases profiles. Aztreonam MICs in the absence and presence of avibactam at different concentrations (ranging from 0.0625 to 4 mg/L) were determined. Aztreonam MIC versus avibactam concentrations were fitted by an inhibitory Emax model with a baseline effect parameter. RESULTS: A concentration-dependent relationship was observed with a steep initial reduction of aztreonam MIC at low avibactam concentrations and reaching a maximum at higher avibactam concentrations that was adequately fitted by the model. Maximum avibactam effect was characterized by the ratio of aztreonam MICs in the absence of avibactam (MIC0) and when avibactam concentration tends toward infinity (MIC∞), and this ratio ranged between 90 and 10 068 depending on the strain. Avibactam potency was characterized by avibactam concentrations corresponding to 50% of the maximum effect (IC50 values between 0.00022 and 0.053 mg/L). CONCLUSIONS: An inhibitory Emax model with a baseline effect could quantify maximum avibactam effect and potency among various strains. This simple modelling approach can be used to compare the activity of other combinations of antibiotics with non-antibiotic drugs when FIC index is inappropriate.

The role of infection models and PK/PD modelling for optimising care of critically ill patients with severe infections.

Critically ill patients with severe infections are at high risk of suboptimal antimicrobial dosing. The pharmacokinetics (PK) and pharmacodynamics (PD) of antimicrobials in these patients differ significantly from the patient groups from whose data the conventional dosing regimens were developed. Use of such regimens often results in inadequate antimicrobial concentrations at the site of infection and is associated with poor patient outcomes. In this article, we describe the potential of in vitro and in vivo infection models, clinical pharmacokinetic data and pharmacokinetic/pharmacodynamic models to guide the design of more effective antimicrobial dosing regimens. Individualised dosing, based on population PK models and patient factors (e.g. renal function and weight) known to influence antimicrobial PK, increases the probability of achieving therapeutic drug exposures while at the same time avoiding toxic concentrations. When therapeutic drug monitoring (TDM) is applied, early dose adaptation to the needs of the individual patient is possible. TDM is likely to be of particular importance for infected critically ill patients, where profound PK changes are present and prompt appropriate antibiotic therapy is crucial. In the light of the continued high mortality rates in critically ill patients with severe infections, a paradigm shift to refined dosing strategies for antimicrobials is warranted to enhance the probability of achieving drug concentrations that increase the likelihood of clinical success.

[Post-licensure safety surveillance for Prevenar 13® in France]. / Bilan français des effets indésirables du vaccin Prévenar 13®.

OBJECTIVE: To describe the profile and the incidence of adverse events (AEs) reported with Prevenar 13® since its commercialization. METHOD: Analysis of all adverse events reported with Prevenar 13® in France between 1st July 2010 and 31 October 2014. RESULTS: In 4 years and 4 months, 376 AEs, including 252 severe (67%), were recorded, 83 of which occurred following an injection of Prevenar 13® alone: 39 cutaneous AEs, 16 neurological AEs, four cases of collapse or shock, nine cases of fever, and one of thrombocytopenia. For the serious AEs, the outcome was favorable in 88% of cases and none of the 12 reported deaths were attributed to a side effect of vaccination. Fifty-nine cases of pneumococcal disease that suggest an ineffective vaccine were reported, but only 16 can be considered as a real failure of the vaccination. DISCUSSION: In many cases, Prevenar 13® was administered on the same day as a hexavalent vaccine with which the AEs reported were expected. The profile of AEs reported following Prevenar 13® alone is similar to that seen with Prevenar 7®. CONCLUSION: Since its release in 2010, the Prevenar 13® pharmacovigilance survey, which includes more than 11,800,000 distributed doses, did not show any new information in terms of tolerance safety.

Population Pharmacokinetics of Colistin Methanesulfonate and Colistin in Critically Ill Patients with Acute Renal Failure Requiring Intermittent Hemodialysis.

Colistin is increasingly used as a last option for the treatment of severe infections due to Gram-negative bacteria in critically ill patients requiring intermittent hemodialysis (HD) for acute renal failure. Our objective was to characterize the pharmacokinetics (PK) of colistin and its prodrug colistin methanesulfonate (CMS) in this population and to suggest dosing regimen recommendations. Eight intensive care unit (ICU) patients who were under intermittent HD and who were treated by CMS (Colimycine) were included. Blood samples were collected between two consecutive HD sessions. CMS and colistin concentrations were measured by a specific chromatographic assay and were analyzed using a PK population approach (Monolix software). Monte Carlo simulations were conducted to predict the probability of target attainment (PTA). CMS nonrenal clearance was increased in ICU-HD patients. Compared with that of ICU patients included in the same clinical trial but with preserved renal function, colistin exposure was increased by 3-fold in ICU-HD patients. This is probably because a greater fraction of the CMS converted into colistin. To maintain colistin plasma concentrations high enough (>3 mg/liter) for high PTA values (area under the concentration-time curve for the free, unbound fraction of a drug [fAUC]/MIC of >10 and fAUC/MIC of >50 for systemic and lung infections, respectively), at least for MICs lower than 1.5 mg/liter (nonpulmonary infection) or 0.5 mg/liter (pulmonary infection), the dosing regimen of CMS should be 1.5 million international units (MIU) twice daily on non-HD days. HD should be conducted at the end of a dosing interval, and a supplemental dose of 1.5 MIU should be administered after the HD session (i.e., total of 4.5 MIU for HD days). This study has confirmed and complemented previously published data and suggests an a priori clear and easy to follow dosing strategy for CMS in ICU-HD patients.

Relationship between adaptation and cardiovascular response to tonic cold and heat pain Adaptability to tonic pain and cardiovascular responses.

BACKGROUND: The mechanisms of adaptation to tonic pain are not elucidated. We hypothesized that the adaptability to tonic pain is related to the cardiovascular system. METHODS: Twenty-six subjects received over two sessions in a random order: tonic cold (7 ± 0.2 °C) and heat pain (47.5 ± 0.5 °C) on the hand for 5 min. Pain intensity, blood pressure (BP), and heart rate (HR) were continuously monitored. RESULTS: Pain experience during the heat (HIT) and cold (CIT) immersion tests exhibited different average time courses, being approximated with a linear and cubic function, respectively. In each test, two groups of participants could be identified based on the time course of their tonic thermal pain: one-third of participants were pain adaptive and two-thirds non adaptive. The adaptive group exhibited higher initial pain, lower last pain, and shorter latency to peak pain than the non-adaptive one. Interestingly, some participants were adaptive to both pain stimuli, most were not. HIT as well as CIT produced a stable elevation of BP. However, BP was higher during CIT than HIT (p = 0.034). HR was also increased during CIT and HIT, but the two tests differed with respect to the time course of responses. Finally, the intensity and time course of pain rating to both HIT and CIT correlated with neither BP nor HR responses. CONCLUSIONS: These results suggest that individual sensitivity and adaptability to tonic thermal pain is related to the intensity of initial pain rating and the latency to peak pain but not to cardiovascular responses.

Co-purification of arrestin like proteins with alpha-enolase from bovine myocardial tissues and the possible role in heart diseases as an autoantigen.

AIM: Previously, we reported that visual arrestin co-purified with glycolytic enzymes. The aim of this study was to analyze the co-purification of arrestin like proteins (ALP) in bovine cardiac tissues with enolases. METHODS: The soluble extract of bovine myocardial tissues from different regions such as left and right atriums and ventricles of the bovine heart (n = 3) was analyzed by ACA-34 gel filtration, immuno-affinity column, SDS-PAGE, ELISA, western blot and a sandwich immune assay for quantification of ALP and sequence analysis. RESULTS: We observed that; 1) The cardiac muscle contained a 50 kDa ALP at a concentration of 751 pg/mg of soluble protein extract, 2) ALP purified, by immunoaffinity, contained alpha-enolase of 48 kDa confirmed by protein sequence analysis; 3) Cardiomyocyte cells exposed to anti arrestin and anti enolase monoclonal antibodies showed decreased proliferation in vitro, 4) High level of autoantibodies were detected by ELISA (3.57% for arrestin and 9.12% for α-enolase) in serum of patients with infarcted heart disease. CONCLUSION: We suggest a possible interaction between ALP and alpha-enolases yielding a complex that may be involved in the induction of cardiac autoimmune diseases.

[Prophylaxis of mother-to-infant transmission of hepatitis B virus]. / Prévention de la transmission materno-infantile du virus de l'hépatite B.

Hepatitis B virus (HBV) infection is a worldwide health problem and mother-to-infant (or vertical) transmission is the main source of chronic infection in Asian countries. Administration of HBV vaccine to the infant at birth, with or without concurrent specific immunoglobulin, efficiently prevents such transmission (efficacy>90%). In France, testing Ag HBs is mandatory during pregnancy in all pregnant women. Infants born to Ag HBs-positive mothers should receive the first injection of vaccine and one injection of specific immunoglobulins at birth. Vaccination should thereafter be completed according to a three-injection protocol (at 1 and 6 months) or a four-injection protocol in case of prematurity. Failure of immunoprophylaxis can be observed when the viral load is very high in the mother during pregnancy (HBV-DNA levels>200,000 IU/mL). In such women, antiviral therapy with analogs (lamivudine, telbivudine, or tenofovir) during the third trimester of pregnancy and 1 month post-partum, in association with accurate immunoprophylaxis, may prevent vertical transmission. The optimal cut-off value of maternal viral load for antiviral therapy in late pregnancy and post-partum to prevent vertical transmission is still under debate.

Recommendations on practice of conditioned pain modulation (CPM) testing.

Protocols for testing conditioned pain modulation (CPM) vary between different labs/clinics. In order to promote research and clinical application of this tool, we summarize the recommendations of interested researchers consensus meeting regarding the practice of CPM and report of its results.

New colistin population pharmacokinetic data in critically ill patients suggesting an alternative loading dose rationale.

Colistin is an old antibiotic that has recently gained a considerable renewal of interest as the last-line defense therapy against multidrug-resistant Gram-negative bacteria. It is administered as colistin methanesulfonate (CMS), an inactive prodrug, and it was shown that due to slow CMS conversion, colistin plasma concentrations increase very slowly after treatment initiation, which constitutes the rationale for a loading dose in critically ill patients. However, faster CMS conversion was observed in healthy volunteers but using a different CMS brand, which may also have a major impact on colistin pharmacokinetics. Seventy-three critically ill patients not undergoing dialysis received multiple doses of CMS. The CMS concentrations were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and a pharmacokinetic analysis was conducted using a population approach. We confirmed that CMS renal clearance and colistin concentrations at steady state are mostly governed by creatinine clearance, but we predict a typical maximum concentration of drug in serum (Cmax) of colistin close to 2 mg/liter, occurring 3 h after an initial dose of 2 million international units (MIU) of CMS. Accordingly, the estimated colistin half-life (t1/2) was relatively short (3.1 h), with rapid attainment of steady state. Our results are only partially consistent with other recently published results. We confirm that the CMS maintenance dose should be adjusted according to renal function in critically ill patients. However, much higher than expected colistin concentrations were observed after the initial CMS dose, with rapid steady-state achievement. These discrepancies challenge the pharmacokinetic rationale for a loading dose, which may still be appropriate for rapid bacterial eradication and an improved clinical cure rate.

[Catch-up vaccination of worldwide newcoming (adopted, refugee or migrant) children in France]. / Mise à jour des vaccinations de l'enfant arrivant de l'étranger (adopté, réfugié ou migrant) en France.

In France, international adoption includes around to 90,000 children since 1980 and near 300,000 immigrant children were counted in 2008. This population is heterogeneous, according to age and country of origin, and its large number. It is not easy to completely and surely assess the vaccine status of the child. Due to a great variability of individual situations, it is not possible to have systematic and unchangeable rules. This article aims to give an update of catch-up vaccination of internationally adopted or refugee or migrant children in France. The vaccination status of a child who recently arrived in France is complex and has to be adapted to his country of origin. Some of them were never vaccinated whereas the vaccine status of others is uncertain or unknown. Three parameters have to be considered: the age of the child, the country of origin, and sometimes serology in the case of doubts of his vaccine status. Catch-up vaccination of foreign children has to be adapted to French vaccine recommendations, as a reference, and to vaccines already administered to the child.

Transcutaneous electrical nerve stimulation and conditioned pain modulation influence the perception of pain in humans.

BACKGROUND: Research in animal models suggests that transcutaneous electrical nerve stimulation (TENS) and conditioned pain modulation (CPM) produce analgesia via two different supraspinal pathways. No known studies have examined whether TENS and CPM applied simultaneously in human subjects will enhance the analgesic effect of either treatment alone. The purpose of the current study was to investigate whether the simultaneous application of TENS and CPM will enhance the analgesic effect of that produced by either treatment alone. METHODS: Sixty healthy adults were randomly allocated into two groups: (1) CPM plus active TENS; (2) CPM plus placebo TENS. Pain threshold for heat (HPT) and pressure (PPT) were recorded from subject's left forearm at baseline, during CPM, during active or placebo TENS, and during CPM plus active or placebo TENS. CPM was induced by placing the subjects' contralateral arm in a hot water bath (46.5 °C) for 2 min. TENS (100 µs, 100 Hz) was applied to the forearm for 20 min at a strong but comfortable intensity. RESULTS: Active TENS alone increased PPT (but not HPT) more than placebo TENS alone (p = 0.011). Combining CPM and active TENS did not significantly increase PPT (p = 0.232) or HPT (p = 0.423) beyond CPM plus placebo TENS. There was a significant positive association between PPT during CPM and during active TENS (r(2) = 0.46; p = 0.003). CONCLUSIONS: TENS application increases PPT; however, combining CPM and TENS does not increase the CPM's hypoalgesic response. CPM effect on PPT is associated with the effects of TENS on PPT.