Beyond Seek and Destroy: how to Generate Allelic Series Using Genome Editing Tools.

Genome editing tools have greatly facilitated the functional analysis of genes of interest by targeted mutagenesis. Many usable genome editing tools, including different site-specific nucleases and editor databases that allow single-nucleotide polymorphisms (SNPs) to be introduced at a given site, are now available. These tools can be used to generate high allelic diversity at a given locus to facilitate gene function studies, including examining the role of a specific protein domain or a single amino acid. We compared the effects, efficiencies and mutation types generated by our LbCPF1, SpCAS9 and base editor (BECAS9) constructs for the OsCAO1 gene. SpCAS9 and LbCPF1 have similar efficiencies in generating mutations but differ in the types of mutations induced, with the majority of changes being single-nucleotide insertions and short deletions for SpCAS9 and LbCPF1, respectively. The proportions of heterozygotes also differed, representing a majority in our LbCPF1, while with SpCAS9, we obtained a large number of biallelic mutants. Finally, we demonstrated that it is possible to specifically introduce stop codons using the BECAS9 with an acceptable efficiency of approximately 20%. Based on these results, a rational choice among these three alternatives may be made depending on the type of mutation that one wishes to introduce, the three systems being complementary. SpCAS9 remains the best choice to generate KO mutations in primary transformants, while if the desired gene mutation interferes with regeneration or viability, the use of our LbCPF1 construction will be preferred, because it produces mainly heterozygotes. LbCPF1 has been described in other studies as being as effective as SpCAS9 in generating homozygous and biallelic mutations. It will remain to be clarified in the future, whether the different LbCFP1 constructions have different efficiencies and determine the origin of these differences. Finally, if one wishes to specifically introduce stop codons, BECAS9 is a viable and efficient alternative, although it has a lower efficiency than SpCAS9 and LbCPF1 for creating KO mutations.

Treatment for Uncomplicated Plasmodium falciparum Malaria in French Soldiers Deployed in Sub-Saharan Africa: Gaps Between Policy and Field Practice.

BACKGROUND: Malaria prevention and treatment are big challenges for the French forces deployed in sub-Saharan Africa. Since December 2013, 1,800 French soldiers have been deployed at any one time in the Central African Republic in the framework of "Operation Sangaris" and European Union Force (EUFOR). Over the 2014-2015 period, about 500 cases of malaria were notified in these troops during the operation or after their return (annual incidence: 13.4 p.100 person-year). The recommendation to use dihydroartemisinin-piperaquine (DHA-PQ) as the first-line treatment for French soldiers suffering from uncomplicated Plasmodium falciparum malaria in endemic areas is not always followed in practice in the field by French military general practitioners (GPs). METHODS: We conduced a retrospective Knowledge-Attitude-Practice study by self-administered questionnaire, to all military French doctors who were in mission in Central African Republic from January 2014 to July 2015 to try to understand what were the reasons for the GP not to prescribe DHA-PQ on the field. FINDINGS: Thirty-six GPs (53%) answered to the questionnaire. Eighty-three percent of them knew about the recommendation to use DHA-PQ for un uncomplicated Pf malaria. Fifty-eight percent had a favorable attitude toward DHA-PQ. The factors associated with the prescription of another drug (Atovaquone-proguanil) were: the habit (odds ratio [OR] 0.1, confidence interval (CI) 0-0.6], the fact that Atovaquone-proguanil is more practical to use [OR 0.01, CI 0-0.1]. In practice, only 37.5% prescribed DHA-PQ the most of the time during their mission. Factors associated with a non-favorable attitude toward DHA-PQ were: the necessity to calculate a QTc interval during the treatment [OR 0.2, confidence interval 0-0.9], and the fact that DHA-PQ must be taken on an empty stomach [OR 0.3, CI 0.1-0.8]. GP who received a formation before their mission about malaria and treatment had a favorable attitude toward DHA-PQ. DISCUSSION: There is very satisfactory knowledge by the military GPs stationed in the Central African Republic on both the recommendations and prescription of antimalarial drugs. The present study highlights some difficulties in implementing the recommendations in an operational context, notably factors limiting the prescription of DHA-PQ during military deployment (need for ECG monitoring, empty stomach, and lack of habit). Proposals can be made to improve the efficacy, tolerance, and practicability of malaria treatment in the field. The main focus should be a more flexible application of the French DHA-PQ risk management plan in the field, specific training and communication about DHA-PQ use, the generalization of ECG printing equipment in the field, and the switch from DHA-PQ to an alternative artemisinin-based combination therapy during deployments in malaria-endemic areas.

[The study of social representations of alcohol within French Navy]. / Représentations sociales de l'alcool au sein de la Marine nationale.

INTRODUCTION: The misuse of alcohol has harmful social and health impacts within a population. The French military must be particularly vigilant with alcohol, due to specificities linked with weaponry. This study was designed to explore social representations of alcohol based on a sample of the French Navy in order to prioritize prevention focuses on alcohol misuses in a military environment. METHODS: A semi-directed interview-based exploratory study was conducted in the south of France at the end of 2015. Data was submitted to manual categorical analysis and textual analysis (Iramuteq software). RESULTS: Sixty-two marines from the French Navy were interviewed including 30 Navy and 32 Marine Firefighters. Data analysis indicated that the motivation to consume alcohol comes more from a social influence (extrinsic motivations) than for pleasure induced by alcohol effects (intrinsic motivations). For Navy personnel, the sense of belonging to the Navy is strengthened by drinking habits with a strong social cohesion. In contrast, the Marine Firefighters had a negative perception of alcohol because of a possible degradation of their professional image for the civilian population. CONCLUSION: The identification of social representations of alcohol allows for a better apprehension of drinking behavior within a French military population, particularly by focusing on content and organization of discourses about alcohol. Following this survey, a quantitative study is currently being drawn up among military personnel from the French Navy in order to clarify some of the elements identified and presented within this article.

Monitoring of returnees from Ebola-affected areas: lessons learned based on the experience of French armed forces deployed in Guinea, 2015.

Introduction: During the 2014-15 Ebola virus disease (EVD) outbreak in West Africa, French armed forces were involved in the treatment and management of Ebola patients in Guinea. The constant flow of military personnel returning from their deployment posed a risk of secondary dissemination of the Ebola virus. Our objective was to describe the follow-up of returning service members that was implemented to prevent this risk of dissemination in France. Method: For the French military, a specific complementary follow-up was added to the national monitoring to take into account the need for a detailed record for follow-up of returning military personnel and to keep the military chain of command informed. Results: All the 410 service members deployed in Guinea underwent monitoring among whom 22 were suspected of EVD. Three of them were considered as possible EVD cases but none of them was tested positive for EVD. Conclusion: The monitoring organized for French service members deployed in Guinea made it possible to follow all exposed military personnel after their return, know their health status on a near real-time basis and be aware of all alerts. To reach this goal the collaboration with French national health agencies was necessary and should be improved in the future.

A Dual Role for the OsK5.2 Ion Channel in Stomatal Movements and K+ Loading into Xylem Sap.

The roles of potassium channels from the Shaker family in stomatal movements have been investigated by reverse genetics analyses in Arabidopsis (Arabidopsis thaliana), but corresponding information is lacking outside this model species. Rice (Oryza sativa) and other cereals possess stomata that are more complex than those of Arabidopsis. We examined the role of the outward Shaker K+ channel gene OsK5.2. Expression of the OsK5.2 gene (GUS reporter strategy) was observed in the whole stomatal complex (guard cells and subsidiary cells), root vasculature, and root cortex. In stomata, loss of OsK5.2 functional expression resulted in lack of time-dependent outward potassium currents in guard cells, higher rates of water loss through transpiration, and severe slowdown of stomatal closure. In line with the expression of OsK5.2 in the plant vasculature, mutant plants displayed a reduced K+ translocation from the root system toward the leaves via the xylem. The comparison between rice and Arabidopsis show that despite the strong conservation of Shaker family in plants, substantial differences can exist between the physiological roles of seemingly orthologous genes, as xylem loading depends on SKOR and stomatal closure on GORK in Arabidopsis, whereas both functions are executed by the single OsK5.2 Shaker in rice.

Assessment of the French National Health Insurance Information System as a tool for epidemiological surveillance of malaria.

OBJECTIVE: Epidemiological surveillance of malaria in France is based on a hospital laboratory sentinel surveillance network. There is no comprehensive population surveillance. The objective of this study was to assess the ability of the French National Health Insurance Information System to support nationwide malaria surveillance in continental France. MATERIALS AND METHODS: A case identification algorithm was built in a 2-step process. First, inclusion rules giving priority to sensitivity were defined. Then, based on data description, exclusion rules to increase specificity were applied. To validate our results, we compared them to data from the French National Reference Center for Malaria on case counts, distribution within subgroups, and disease onset date trends. RESULTS: We built a reusable automatized tool. From July 1, 2013, to June 30, 2014, we identified 4077 incident malaria cases that occurred in continental France. Our algorithm provided data for hospitalized patients, patients treated by private physicians, and outpatients for the entire population. Our results were similar to those of the National Reference Center for Malaria for each of the outcome criteria. DISCUSSION: We provided a reliable algorithm for implementing epidemiological surveillance of malaria based on the French National Health Insurance Information System. Our method allowed us to work on the entire population living in continental France, including subpopulations poorly covered by existing surveillance methods. CONCLUSION: Traditional epidemiological surveillance and the approach presented in this paper are complementary, but a formal validation framework for case identification algorithms is necessary.

Do alcohol-based hand rubs reduce the incidence of acute diarrhea during military deployments? A prospective randomized trial.

BACKGROUND: Acute diarrhea remains a public health concern in armed forces deployed in tropical areas where access to water and soap is limited. This study aims to assess the effectiveness of alcohol-based hand rubs (ABHR) on incidence of diarrhea in poor hygiene conditions. METHOD: A prospective randomized trial was conducted between November 2014 and January 2015 among French military troops deployed in Africa to compare a group of soldiers receiving usual hand hygiene recommendations (control group), to a group of soldiers who received ABHR in addition to usual hand hygiene recommendations (intervention group). Data on diarrhea and hygiene behaviors were collected using self-questionnaires. The incidence rate of diarrhea episodes in groups was compared. RESULTS: Participation rate was 59% (236/400). The proportion of individuals who used ABHR was 97% in the intervention group and 62% in the control group. The overall incidence rate of diarrheal episodes was observed to be in the region of 60 per 100 persons-month without any significant difference between groups after adjustment on confounding factors (p = 0.93). Handwashing with soap was used on average 4 times a day in the control group and twice a day in the intervention group (p = 0.93). It was the only significant protective factor for diarrhea (p < 10-3). CONCLUSION: Our results support that supplying soap and good quality water should be a priority on the field.

Acute acoustic trauma in the French armed forces during 2007-2014.

CONTEXT: Despite existing preventive measures, the number of acute acoustic trauma (AAT) cases reported to the French Military Epidemiological Surveillance System (MESS) remains high. AIMS: The objective of this study was to describe AAT and the preventive measures already implemented. SUBJECTS AND METHODS: We conducted a descriptive cross-sectional analysis of AAT using data from the MESS for the period 2007-2014. In addition, we reviewed the current prevention measures that exist in the French armed forces. STATISTICAL ANALYSIS USED: Comparisons between different incidence rates were made by Poisson and quasi-Poisson regression. RESULTS: Between 2007 and 2014, 10,487 AAT cases were reported to the MESS, with a significant decrease in 2013 (P < 0.001). AAT incidence rates were the highest among those aged <25 years - 14.3 per 1000 person-years (PYs) (P < 0.001), and those in the army; with 8.1 per 1000 PYs (P < 0.001), and men had almost twice the risk of women (P < 0.001). AAT mainly occurred in training schools or at camps during exercises. The main prevention actions identified were the following: official regulations, education, making hearing protection devices (HPDs) available for all service members, and regular hearing monitoring. A working group has been set up and has proposed an informative chapter in the weapon handling instruction book, an AAT simulator, and a new HPD, the 3M® earplug, with an information brochure. CONCLUSIONS: AAT rates decreased from 2007 to 2014 in the French armed forces. Further analysis is needed to identify the underlying factors involved to improve the prevention actions proposed. The MESS and targeted surveys will assess the impact of the different prevention measures implemented.

High third-generation cephalosporin resistant Enterobacteriaceae prevalence rate among neonatal infections in Dakar, Senegal.

BACKGROUND: Neonatal infection constitutes one of Senegal's most important public health problems, with a mortality rate of 41 deaths per 1,000 live births. METHODS: Between January 2007 and March 2008, 242 neonates with suspected infection were recruited at three neonatal intensive care units in three major tertiary care centers in Dakar, the capital of Senegal. Neonatal infections were confirmed by positive bacterial blood or cerebrospinal fluid culture. The microbiological pattern of neonatal infections and the antibiotic susceptibility of the isolates were characterized. In addition, the genetic basis for antibiotic resistance and the genetic background of third-generation cephalosporin-resistant (3GC-R) Enterobacteriaceae were studied. RESULTS: A bacteriological infection was confirmed in 36.4 % (88/242) of neonates: 22.7 % (30/132) during the early-onset and 52.7 % (58/110) during the late-onset periods (p > 0.20). Group B streptococci accounted for 6.8 % of the 88 collected bacterial isolates, while most of them were Enterobacteriaceae (n = 69, 78.4 %). Of these, 55/69 (79.7 %) were 3GC-R. The bla CTX-M-15 allele, the bla SHV and the bla TEM were highly prevalent (63.5, 65.4 and 53.8 %, respectively), usually associated with qnr genes (65.4 %). Clonally related strains of 3GC-R Klebsiella pneumoniae and 3GC-R Enterobacter cloacae, the two most commonly recovered 3GC-R Enterobacteriaceae (48/55), were detected at the three hospitals, underlining the role of cross-transmission in their spread. The overall case fatality rate was 18.6 %. CONCLUSIONS: Measures should be taken to prevent nosocomial infections and the selection of resistant bacteria.

Factors associated with malaria chemoprophylaxis compliance among French service members deployed in Central African Republic.

BACKGROUND: Malaria is a public health concern in the French armed forces, with 400-800 cases reported every year and three deaths in the past 2 years. However, lack of chemoprophylaxis (CP) compliance is often reported among service members. The aim of this study was to explore factors associated with CP compliance. METHODS: A retrospective study (1296 service members) was carried out among troops deployed in Central African Republic. Determinants of CP were collected by self-questionnaire. Socio-demographic variables, behavioural characteristics, belief variables, operational determinants such as troops in contact (TIC) and number of nights worked per week and peer-to-peer reinforcement were studied. Relationships between covariates and compliance were explored using logistic regressions (outcome: compliance as a dummy variable). RESULTS: Chemoprophylaxis compliance was associated with other individual preventive measures against mosquito bites (bed net use, OR (odds ratio) = 1.41 (95% CI [1.08-1.84]), and insecticide on clothing, OR = 1.90 ([1.43-2.51]) and malaria-related behaviours (taking chemoprophylaxis at the same time every day, OR = 2.37 ([1.17-4.78]) and taking chemoprophylaxis with food, OR = 1.45 ([1.11-1.89])). High perceived risk of contracting malaria, OR = 1.59 ([1.02-2.50]), positive perception of CP effectiveness, OR = 1.62 ([1.09-2.40]) and the practice of peer-to-peer reinforcement, OR = 1.38 ([1.05-1.82]) were also associated with better compliance. No association was found with TIC and number of nights worked. CONCLUSIONS: This study, which shows a positive relationship between peer-to-peer reinforcement and CP compliance, also suggests the existence of two main personality profiles among service members: those who seek risks and those who are health-conscious. Health education should be expanded beyond knowledge, know-how and motivational factors by using a comprehensive approach based on identification of health determinants, development of psychosocial skills and peer-to-peer reinforcement.

Prevention of combat-related infections: antimicrobial therapy in battlefield and barrier measures in French military medical treatment facilities.

Infection is a major complication associated with combat-related injuries. Beside immobilization, wound irrigation, surgical debridement and delayed coverage, post-injury antimicrobials contribute to reduce combat-related infections, particularly those caused by bacteria of the early contamination flora. In modern warfare, bacteria involved in combat-related infections are mainly Gram-negative bacteria belonging to the late contamination flora. These bacteria are frequently resistant or multiresistant to antibiotics and spread through the deployed chain of care. This article exposes the principles of war wounds antimicrobial prophylaxis recommended in the French Armed Forces and highlights the need for high compliance to hygiene standard precautions, adapted contact precautions and judicious use of antibiotics in French deployed military medical treatment facilities (MTF).

The 2009 A(H1N1) influenza pandemic in the French Armed Forces: epidemiological surveillance and operational management.

OBJECTIVE: The main objective of this study was to evaluate the contribution of a newly implemented daily surveillance system to the management of the 2009 A(H1N1) influenza pandemic by the military decision-makers at different levels in the French Department of Defence. METHODS: The study sample included all medical advisors in the Ministry of Defence and the French Armed Forces Staff and also the members of the specific committee dedicated to flu pandemic control. The variables studied were mental representation of epidemiology, relevance, usefulness, and real-time use of surveillance data using quantitative questionnaires and qualitative face-to-face semistructured interviews. RESULTS: Among the risk managers of the flu pandemic in the Armed Forces, 84% responded. The data generated by epidemiological surveillance were considered relevant and useful, and were reported as effectively used. On the basis of the information produced, concrete actions were planned and implemented in the French Armed Forces. CONCLUSION: In a pandemic situation involving low mortality, the daily monitoring of the disease did not target public health issues, but it was mainly used to assess the availability of the Armed Forces in real time. For the military staff, epidemiological surveillance represents an essential information tool for the conduct of operations.

Is serving in the armed forces associated with tobacco or cannabis initiation? A study of onset sequences before and after joining the French armed forces.

The aim of this study was to evaluate whether joining the military could constitute a predictor of subsequent tobacco or cannabis use. Data from four cross-sectional surveys conducted between 2005 and 2009 in the French armed forces were used to constitute a retrospective cohort (n=4208). A Markov multi-state model was fitted, estimating the probabilities at 1year for all possible pathways between entry into the military profession, tobacco initiation and cannabis initiation. The model was adjusted for current substance use status. Substance initiation appeared to mainly occur before entering the services than during the service period (3.0 times greater likelihood for tobacco and 2.5 times greater likelihood for cannabis). Probabilities of primo-initiation (i.e. starting with that substance) before joining the services were similar for tobacco (3.6% at one year) and cannabis (3.5%). When the study was restrained to current users, although differences were not statistically significant, probabilities of substance primo-initiation during the service period seemed to be greater than before. While the substance use itinerary in the French general population most often begins by tobacco, military seem to have a higher propensity to cannabis primo-initiation, which could traduce more sensation-seeking behaviors. If certain results in our study could also evoke a propensity to substance initiation during the service period, we did not find evidence to verify the hypothesis of a military social inducing effect. Further research is needed to identify behavioral characteristics which could explain increased use during the service period.

Spread of Influenza A(H1N1) oseltamivir-resistant viruses in Africa in 2008 confirmed by multiple introductions in Senegal.

BACKGROUND: Among Influenza neuraminidase inhibitors (NAIs), oseltamivir corresponds to the most widely used agent to treat influenza disease. However since 2001, several cases of resistance to NAIs have been reported for circulating seasonal A(H1N1) Influenza viruses. A direct resistance mechanism may be invoked, involving critical mutations in the viral NA gene that prevent the drug binding to its target. Same phenomenon is reported for adamantanes drugs and mutations in the M2 channel protein gene of Influenza viruses. METHODS: Reverse-Transcription/Restriction Fragment Length Polymorphism (RT-PCR/RFLP) method, phenotypic testing for oseltamivir resistance, and sequencing of NA, HA and M2 genes were used in this study. Phylogenetic analyses were performed using BioEdit and Mega 5 softwares for alignment of sequences and phylogenetic trees building respectively. RESULTS: Using a simple RT-PCR/RFLP method, we found that the 86 seasonal A(H1N1) isolates from 2008 bear the oseltamivir resistance-associated mutation (H274Y) in the NA gene. In contrast all isolates isolated in Senegal in 2007 were sensitive to oseltamivir. These results were first confirmed by finding high IC50 values using a phenotypic testing for oseltamivir resistance, and secondly by sequencing the whole NA gene. Regarding M2 gene, no mutation associated to adamantanes resistance was characterized of the isolates. CONCLUSIONS: The present work provides evidence of circulation of drug-resistant seasonal A(H1N1) viruses during the 2008 influenza season (July to September) in Senegal. The results are in favor of multiple introductions of oseltamivir resistant viruses (ORV) A(H1N1) in Senegal.Phylogenetic analyses of isolates with complete sequences of N1 and HA1 genes showed that they belong to clade 2B and suggest sequential introductions in Africa.

The accuracy of self-reported data concerning recent cannabis use in the French armed forces.

BACKGROUND: The aims were to evaluate the accuracy of self-report of past-month cannabis use in a representative sample of French military staff members and to evaluate the scale of the prevarication bias. METHOD: Data from three cross-sectional surveys conducted between 2005 and 2008 (n = 3493) were used. The characteristics of self-report (sensitivity, specificity, positive predictive value and negative predictive value) were computed using tetrahydrocannabinol detection in urine as the reference. RESULTS: The prevalence for past-month cannabis use was 16.1% and for positive testing was 13.4%. The discriminant power of self-report was good, with an area under the receiver operating characteristics curve 0.90. Specificity (94.5%) and negative predictive values (97.8%) were good, but sensitivity (85.7%) and positive predictive values (70.4%) were lower. The lowest sensitivity values were observed in the higher categories of personnel and in the Navy, which could reflect some prevarication in these sub-populations who might believe they were more exposed to sanctions if detected. CONCLUSIONS: Despite certain limitations of urine analysis as a reference, because of its poor detection of occasional users, our study is in favour of good accuracy of self-reported data on cannabis use, even among the military. However, our results, derived from a population study, do not enable any assumptions on the validity of self-reported data collected during individual testing procedures for the purpose of improving occupational safety.

Determinants of compliance with anti-vectorial protective measures among non-immune travellers during missions to tropical Africa.

BACKGROUND: The effectiveness of anti-vectorial malaria protective measures in travellers and expatriates is hampered by incorrect compliance. The objective of the present study was to identify the determinants of compliance with anti-vectorial protective measures (AVPMs) in this population that is particularly at risk because of their lack of immunity. METHODS: Compliance with wearing long clothing, sleeping under insecticide-impregnated bed nets (IIBNs) and using insect repellent was estimated and analysed by questionnaires administered to 2,205 French military travellers from 20 groups before and after short-term missions (approximately four months) in six tropical African countries (Senegal, Ivory Coast, Chad, Central African Republic, Gabon and Djibouti). For each AVPM, the association of "correct compliance" with individual and collective variables was investigated using random-effect mixed logistic regression models to take into account the clustered design of the study. RESULTS: The correct compliance rates were 48.6%, 50.6% and 18.5% for wearing long clothing, sleeping under bed nets and using repellents, respectively. Depending on the AVPM, correct compliance was significantly associated with the following factors: country, older than 24 years of age, management responsibilities, the perception of a personal malaria risk greater than that of other travellers, the occurrence of life events, early bedtime (i.e., before midnight), the type of stay (field operation compared to training), the absence of medical history of malaria, the absence of previous travel in malaria-endemic areas and the absence of tobacco consumption.There was no competition between compliance with the different AVPMs or between compliance with any AVPM and malaria chemoprophylaxis. CONCLUSION: Interventions aimed at improving compliance with AVPMs should target young people without management responsibilities who are scheduled for non-operational activities in countries with high risk of clinical malaria. Weak associations between compliance and history of clinical malaria or variables that pertain to threat perception suggest that cognition-based interventions referencing a "bad experience" with clinical malaria could have only a slight impact on the improvement of compliance. Further studies should focus on the cognitive and behavioural predictors of compliance with AVPMs.