Karin De Ridder

Trypanosoma brucei Modifies the Tsetse Salivary Composition, Altering the Fly Feeding Behavior That Favors Parasite Transmission

Tsetse flies are the notorious transmitters of African trypanosomiasis, a disease caused by the Trypanosoma parasite that affects humans and livestock on the African continent. Metacyclic infection rates in natural tsetse populations with Trypanosoma brucei, including the two human-pathogenic subspecies, are very low, even in epidemic situations. Therefore, the infected fly/host contact frequency is a key determinant of the transmission dynamics. As an obligate blood feeder, tsetse flies rely on their complex salivary potion to inhibit host haemostatic reactions ensuring an efficient feeding. The results of this experimental study suggest that the parasite might promote its transmission through manipulation of the tsetse feeding behavior by modifying the saliva composition. Indeed, salivary gland Trypanosoma brucei-infected flies display a significantly prolonged feeding time, thereby enhancing the likelihood of infecting multiple hosts during the process of a single blood meal cycle. Comparison of the two major anti-haemostatic activities i.e. anti-platelet aggregation and anti-coagulation activity in these flies versus non-infected tsetse flies demonstrates a significant suppression of these activities as a result of the trypanosome-infection status. This effect was mainly related to the parasite-induced reduction in salivary gland gene transcription, resulting in a strong decrease in protein content and related biological activities. Additionally, the anti-thrombin activity and inhibition of thrombin-induced coagulation was even more severely hampered as a result of the trypanosome infection. Indeed, while naive tsetse saliva strongly inhibited human thrombin activity and thrombin-induced blood coagulation, saliva from T. brucei-infected flies showed a significantly enhanced thrombinase activity resulting in a far less potent anti-coagulation activity. These data clearly provide evidence for a trypanosome-mediated modification of the tsetse salivary composition that results in a drastically reduced anti-haemostatic potential and a hampered feeding performance which could lead to an increase of the vector/host contact and parasite transmission in field conditions.

Identification of a tsetse fly salivary protein with dual inhibitory action on human platelet aggregation.

Background Tsetse flies (Glossina sp.), the African trypanosome vectors, rely on anti-hemostatic compounds for efficient blood feeding. Despite their medical importance, very few salivary proteins have been characterized and functionally annotated. Methodology/Principal Findings Here we report on the functional characterisation of a 5′nucleotidase-related (5′Nuc) saliva protein of the tsetse fly Glossina morsitans morsitans. This protein is encoded by a 1668 bp cDNA corresponding at the genomic level with a single-copy 4 kb gene that is exclusively transcribed in the tsetse salivary gland tissue. The encoded 5′Nuc protein is a soluble 65 kDa glycosylated compound of tsetse saliva with a dual anti-hemostatic action that relies on its combined apyrase activity and fibrinogen receptor (GPIIb/IIIa) antagonistic properties. Experimental evidence is based on the biochemical and functional characterization of recombinant protein and on the successful silencing of the 5′nuc translation in the salivary gland by RNA interference (RNAi). Refolding of a 5′Nuc/SUMO-fusion protein yielded an active apyrase enzyme with Km and Vmax values of 43±4 µM and 684±49 nmol Pi/min×mg for ATPase and 49±11 µM and 177±37 nmol Pi/min×mg for the ADPase activity. In addition, recombinant 5′Nuc was found to bind to GPIIb/IIIa with an apparent KD of 92±25 nM. Consistent with these features, 5′Nuc potently inhibited ADP-induced thrombocyte aggregation and even caused disaggregation of ADP-triggered human platelets. The importance of 5′Nuc for the tsetse fly hematophagy was further illustrated by specific RNAi that reduced the anti-thrombotic activities in saliva by approximately 50% resulting in a disturbed blood feeding process. Conclusions/Significance These data show that this 5′nucleotidase-related apyrase exhibits GPIIb/IIIa antagonistic properties and represents a key thromboregulatory compound of tsetse fly saliva.

Blood and charcoal added to acidified agar media promote the growth of Mycobacterium genavense

Ten different agar media were tested for the in vitro growth of Mycobacterium genavense in primary cultures and in subcultures from BACTEC vials. These agar media were based on Middlebrook 7H9, 7H10 and 7H11, and supplemented with additives: mycobactin J, yeast extract, charcoal, or defibrinated sheep blood. Some media were acidified with phosphoric acid to a final pH of 6.2 +/- 0.2. Fourteen M. genavense strains from nude mouse organs as well as one decontaminated clinical specimen (from a bird) were tested. The optimal medium for primary cultures of M. genavense was Middlebrook 7H11 acidified to pH 6.2 +/- 0.2 and supplemented with charcoal and sheep blood: on this medium, all strains produced colonies within 6-12 weeks of incubation in numbers approaching the number of bacilli inoculated. It was also the only medium to support the growth of the decontaminated clinical specimen. Added blood and charcoal appeared not as essential for subcultures as for primary cultures. Three media supported the growth of all strains within 1 month incubation: they were acidified, and were supplemented with yeast extract or pancreatic digest of casein, and with either blood or charcoal.

Tsetse Salivary Gland Proteins 1 and 2 Are High Affinity Nucleic Acid Binding Proteins with Residual Nuclease Activity

Analysis of the tsetse fly salivary gland EST database revealed the presence of a highly enriched cluster of putative endonuclease genes, including tsal1 and tsal2. Tsal proteins are the major components of tsetse fly (G. morsitans morsitans) saliva where they are present as monomers as well as high molecular weight complexes with other saliva proteins. We demonstrate that the recombinant tsetse salivary gland proteins 1&2 (Tsal1&2) display DNA/RNA non-specific, high affinity nucleic acid binding with KD values in the low nanomolar range and a non-exclusive preference for duplex. These Tsal proteins exert only a residual nuclease activity with a preference for dsDNA in a broad pH range. Knockdown of Tsal expression by in vivo RNA interference in the tsetse fly revealed a partially impaired blood digestion phenotype as evidenced by higher gut nucleic acid, hematin and protein contents.

Intake of Fat-Soluble Vitamins in the Belgian Population: Adequacy and Contribution of Foods, Fortified Foods and Supplements

A key challenge of public health nutrition is to provide the majority of the population with a sufficient level of micronutrients while preventing high-consumers from exceeding the tolerable upper intake level. Data of the 2014 Belgian food consumption survey (n = 3200) were used to assess fat-soluble vitamin (vitamins A, D, E and K) intake from the consumption of foods, fortified foods and supplements. This study revealed inadequate intakes for vitamin A, from all sources, in the entire Belgian population and possible inadequacies for vitamin D. The prevalence of inadequate intake of vitamin A was lowest in children aged 3–6 (6–7%) and highest in adolescents (girls, 26%; boys, 34–37%). Except for women aged 60–64 years, more than 95% of the subjects had vitamin D intake from all sources below the adequate intake (AI) of 15 μg/day. The risk for inadequate intake of vitamins K and E was low (median > AI). Belgian fortification and supplementation practices are currently inadequate to eradicate suboptimal intakes of vitamins A and D, but increase median vitamin E intake close to the adequate intake. For vitamin A, a small proportion (1–4%) of young children were at risk of exceeding the upper intake level (UL), while for vitamin D, inclusion of supplements slightly increased the risk for excessive intakes (% > UL) in adult women and young children. The results may guide health authorities when developing population health interventions and regulations to ensure adequate intake of fat-soluble vitamins in Belgium.

Fat-soluble vitamin intake from the consumption of food, fortified food and supplements : design and methods of the Belgian VITADEK study

BackgroundThe adequacy of micronutrient intake is a public health concern, as both insufficient and excessive intake levels may result in adverse health effects. Data on dietary intake are needed to evaluate potential problems regarding inadequate intake at population level and to formulate effective public health and food safety recommendations. Assessing the intake of micronutrients in population subgroups such as infants, toddlers, pregnant and lactating women is challenging and requires specific approaches. This paper describes the Belgian VITADEK study, developed to assess fat-soluble vitamin intake from the consumption of food, fortified foods and supplements in four vulnerable groups namely infants, toddlers, pregnant and lactating women.MethodsSubjects were selected according to a multi-stage stratified sampling design with a selection of clusters proportionate to the population size. Recruitment occurred in collaboration with Belgian child health consultation centres and obstetric clinics. Participants were asked to complete a self-administered online food frequency questionnaire (FFQ) or to answer the questionnaire by phone if online participation was not possible.The questionnaire was tailored to the specific diet of the different target populations. In order to capture vitamin intake from the consumption of foods, fortified foods and supplements, a market study was conducted to take an inventory of the fortified foods and supplements available on the Belgian market. The food list of the FFQ was based on both this inventory and the top 90% food groups that contribute to fat-soluble vitamin intake. Since fortification differs at brand level, food groups and subgroups were split up to the level of the brand of foods. Brand pictures were used as mnemonics to facilitate the recall of the consumed food items and portion pictures were used to facilitate the reproduction of the consumed portion sizes. Finally a composition table was compiled allowing for the computation of vitamin intake from all sources providing as such more accurate estimates of fat-soluble vitamin intake.DiscussionThe results will allow assessing inadequate micronutrient intake by comparison of vitamin intake with dietary reference values. The data will further allow describing the most contributing food groups as well as the contribution of fortified foods and supplements to total vitamin intake. The data will enable evaluating whether infants, toddlers, pregnant and/or lactating women are reached by the actual Belgian fortification and supplementation programmes. Finally the retrieved data will reveal the potential for voluntary fortification and the need for future fortification and supplementation programmes.

Treatment for substance use disorders: the Belgian Treatment Demand Indicator registration protocol.

BackgroundRegistration of patients with substance use disorders is of key importance to get insights and to study trends in patients characteristics and substance use patterns. The Treatment Demand Indicator (TDI) is gathering this information at European level since 2000. In Belgium, this registration started at national level in 2011 and an increasing number of facilities of different types are participating in this data collection since then.Methods/DesignThis surveillance register collects information on every treatment episode started by patients for their substance use disorder. Information is collected on socio-demographic characteristics of the patient, treatment history and substance use patterns. Patients are identified uniquely using their national identification number in order identify multiple episodes followed by a same person. A large range of treatment facilities have the possibility to participate in this registration to allow a wide coverage of the population.DiscussionThe objective of the paper is to facilitate the use of data by authorities or researchers by correctly describing all aspects of the indicator. The case definition, the variables collected and the way data should be reported are of key importance to use and interpret the data correctly. An overview of the data registered in 2014 gives also an idea of the content of the database. The article also pictures the strengths and limitations of the register and foresees some future improvements.

Do current fortification and supplementation programs assure adequate intake of fat-soluble vitamins in Belgian infants, toddlers, pregnant women, and lactating women?

Adequate intakes of fat-soluble vitamins are essential to support the growth and development of the foetus, the neonate, and the young child. By means of an online self-administered frequency questionnaire, this study aimed to evaluate the intake of vitamins A, D, E, and K in Belgian infants (n = 455), toddlers (n = 265), pregnant women (n = 161), and lactating women (n = 165). The contribution of foods, fortified foods, and supplements on the total intake was quantified. 5% of toddlers, 16% of pregnant women, and 35% of lactating women had an inadequate vitamin A intake. Conversely, excessive vitamin A intakes were associated with consumption of liver (products). Furthermore, 22% of infants were at risk for inadequate vitamin D intake due to the lack of prophylaxis, while consumption of highly dosed supplements posed a risk for excessive intakes in 6%–26% of infants. Vitamin D intake in pregnant women and lactating women was inadequate (median of 51%, respectively, 60% of the adequate intake). In all groups, the risk for inadequate intake of vitamin E and K was low. Contribution of fortified foods to vitamin A, D, E, and K intake was minor, except in toddlers. National fortification strategies should be investigated as an alternative or additional strategy to prevent vitamin D and A deficiency. There is a need to revise and set uniform supplement recommendations. Finally, non-users of vitamin D prophylaxis need to be identified for targeted treatment.

Longitudinal pharmacoepidemiological and health services research for substance users in treatment: protocol of the Belgian TDI-IMA linkage

BackgroundNot much is known about the health seeking behavior of people with substance use disorders before they enter specialized treatment and afterwards. This paper explains in detail the protocol that has been followed to establish the Belgian TDI-IMA-database, which is linking two separate databases: the Treatment Demand Indicator (TDI) and the database of the Intermutualistic Agency (IMA). The Treatment Demand Indicator is measuring incidence of people with substance use disorders entering drug treatment. The IMA-database covers data, collected in the framework of the compulsory Belgian health care and benefits insurance program, on reimbursed medication and the use of reimbursed health services. The linkage results in pharmacoepidemiological and health service data for people who were in treatment for substance use disorders and for a group of comparators.MethodsThe TDI-database was linked to the IMA-database for the period between 01/01/2008 and 31/12/2017, based on the national identification number of patients who have been in alcohol or drug treatment between 01/01/2011 and 31/12/2014. Through this linkage, pharmacoepidemiological and health service data became available for at least 3 years before the first registered episode in the TDI-database till at least 3 years after the first episode. For each person in TDI four comparators, who were not in specialized treatment, were matched on age, sex and place of residence.DiscussionThe TDI-IMA-database allows for an analysis of health seeking behavior and health care pathways of people before and after they entered specialized alcohol and drug treatment. The presented protocol could be used in other European countries to establish a linkage between existing health databases. This will allow for a better understanding of the health care needs of patients with substance use disorders.

Health care trajectories and medication consumption of substance users in treatment: linking TDI and IMA databases (Belgium)

Background As problem substance use has a relative low prevalence and is often socially stigmatised, it is rather difficult to study health care trajectories and medication consumption among substance users. In Belgium, the Treatment Demand Indicator register (TDI) is a major information source for drug epidemiology. However, it should be considered as an incidence register as only new drug-related treatment episodes are registered. Linkage with national compulsory health insurance data (IMA) will create a data source in which longitudinal and case-control studies can be performed.